Specific binding members for TGFbeta1
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&bgr;1 and are useful in treating conditions associated with excess TGF&bgr;1 activity, such as fibrosis, immune responses and tumor progression
[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&bgr;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&bgr;1 and human antibodies specific for human TGF&bgr;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&bgr;1,
[0005] (ii) bind TGF&bgr;1 preferentially with respect to TGF&bgr;3, and
[0006] (iii) neutralise TGF&bgr;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&bgr;1. They do not bind to, or neutralise, TGF&bgr;2 or TGF&bgr;3.
[0008] Antibodies of the present invention strongly neutralise active TGF&bgr;1. The epitope for these antibodies lies in the C-terminal region of TGF&bgr;1 (residues 83-112) and includes the loop consisting of residues 92-98 of TGF&bgr;1, also known as finger 2, a region which has been identified as interacting with the receptor for TGF&bgr;. The antibodies bind preferentially to active TGF&bgr;1 with respect to latent TGF&bgr;1
[0009] Variants of SL15S that strongly neutralise TGF&bgr;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&bgr;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. 1 TABLE 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). 2 TABLE 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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;1. Preferred embodiments lack significant cross-reactivity with TGF&bgr;2 and/or TGF&bgr;3, preferably TGF&bgr;2 and TGF&bgr;3.
[0022] Preferred embodiments strongly neutralise TGF&bgr;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&bgr;1 preferentially to latent TGF&bgr;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&bgr;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&bgr;1 but not TGF&bgr;2 or &bgr;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&bgr;1, &bgr;2 or &bgr;3-induced inhibition of proliferation of TF1 is shown.
[0030] FIG. 2: Inhibition of [125I]TGF&bgr;1 binding to A549 cells by anti-TGF&bgr;1 scFv. ScFv preparations of SL15S, JT183 and CS37 were titrated 2-fold and tested for their ability to inhibit [125I]TGF&bgr;1 binding to A549 cells. SL15S and JT182 were used as his-preps, CS37 was fplc-purified.
[0031] FIG. 3: Inhibition of [125I]TGF&bgr;1 binding to A549 cells by anti-TGF&bgr;1 antibodies. SL15S scFv, SL15S IgG4 and SL15A IgG4 were compared with Genzyme mAb in their ability to inhibit [125I]TGF&bgr;1 binding to A549 cells Data are the average of 3 experiments, using a three-fold dilution series.
[0032] FIG. 4: Inhibition of [125I]TGF&bgr;1 binding to A549 cells by scFv in the presence of latent TGF&bgr;1. SL15S scFv and SL15A IgG4 were tested for their ability to inhibit [125I]TGF&bgr;1 binding to A549 cells. To investigate whether SL15S recognised latent TGF&bgr;1, the standard experiment was performed in the presence of 0.1 nM latent TGF&bgr;&bgr;1, active TGF&bgr;1 or acid-activated latent TGF&bgr;1. Data are expressed as % max for each set of conditions.
[0033] FIG. 5: Neutralisation of TGF&bgr;1-induced inhibition of TF1 cell proliferation by scFv and IgG in the presence of latent TGF&bgr;1. The ability of SL15S scFv and SL15A IgG4 to neutralise the growth inhibition induced by latent TGF&bgr;1, active TGF&bgr;1 or acid-activated TGF&bgr;1 was compared in the TF1 assay. In (a) varying concentrations of TGF&bgr;1 formats were compared where as in (b) scFv and IgG were compared against 20 pM TGF&bgr;1 formats. Data expressed as % control (growth in the absence of TGF&bgr;1). The inhibition induced by latent TGF&bgr;1 is due to the small amount of active TGF&bgr;1 present in the latent preparation.
[0034] FIG. 6: Inhibition of binding of CS37 scFv displayed on phage to TGF&bgr;1 using chimaeric TGF&bgr;s. Binding of CS37 scFv displayed on phage to TGF&bgr;1 was assayed by ELISA in the presence of: the TGF&bgr;1 isoform (TGF&bgr;1); the TGF&bgr;2 isoform (TGF&bgr;2); TGF&bgr;1/&bgr;2 (83-112) (1-1-2); TGF&bgr;2/&bgr;1 83-112 (2-2-1); TGF&bgr;1-&bgr;2 (40-112) (1-2-2); or TGF&bgr;1-&bgr;2(92-98) (92-98)
[0035] FIG. 7: Inhibition of binding of SL15S (Kylie scFv) displayed on phage to TGF&bgr;1 using chimaeric TGF&bgr;s. Binding of SL15S (Kylie) scFv displayed on phage to TGF&bgr;1 was assayed by ELISA in the presence of the TGF&bgr;1 isoform (TGF&bgr;1); the TGF&bgr;2 isoform (TGF&bgr;2); TGF&bgr;1/&bgr;2 (83-112) (1-1-2); TGF&bgr;2/&bgr;1 83-112 (2-2-1); TGF&bgr;1-&bgr;2 (40-112) (1-2-2); or TGF&bgr;1-&bgr;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&bgr;1). Null isotype-matched antibody (10&mgr;g), TGF&bgr;1 (1 ng) or CAT192 (10 &mgr;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&bgr;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 &mgr;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 &mgr;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&bgr;1 formed by VH and VL domains as disclosed in this application and the other binding site being for TGF&bgr;2 The TGF&bgr;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 104 individual members upwards, for example from 106 to 108 or 1010 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 &bgr;-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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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&kgr; or C&lgr; chains, preferably C&lgr; 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 131I or 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&bgr;1 have been shown to be effective in animal models for the treatment of fibrotic diseases and other diseases where TGF&bgr;1 is overexpressed such as rheumatoid arthritis or cancer. Antibodies against TGF&bgr;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&bgr;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&bgr;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&bgr;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&bgr; also inhibits endothelial proliferation so anti-TGF&bgr; antibodies may be used to stabilise atherosclerotic plaques and speed healing of vascular anastomoses. TGF&bgr; may stimulate smooth muscle proliferation so anti-TGF&bgr; 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&bgr; 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&bgr;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&bgr; binding molecule and using a bronchial explant model, it has been shown that allergen exposure of asthmatic mucosal tissue results in activation of TGF&bgr; 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&bgr; 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&bgr;2 by damaged epithelial cells and a marked enhancement of collagen III gene expression when conditioned medium is added to myofibroblast cultures. As TGF&bgr; isoforms are potent inhibitors of epithelial cell proliferation, excessive production of TGF&bgr; 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&bgr; 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&bgr;1 also modulates immune and inflammatory responses, for instance in response to malignancy and infection. An antibody against TGF&bgr;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&bgr;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&bgr;1 may be used as an adjuvant for immunotherapy.
[0094] An antibody against TGF&bgr;1 may be used for inhibition of angiogenesis, for instance in treatment of tumors The majority of tumor cells express detectable levels of TGF&bgr;1 (Wojtowicz-Praga 1997, J. Immunother. 20 (3): 165-77). Furthermore, cells that produce higher levels of TGF&bgr;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&bgr;1 plasma levels are correlated with disease progression. Thus the source of TGF&bgr;1 can be the tumor cells as well as surrounding tissue.
[0095] TGF&bgr; 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&bgr; as a result of abnormalities in the type II TGF&bgr; 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 mm3 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&bgr;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&bgr;1. Serum VEGF level is enhanced in patients with breast cancer and these levels directly correlate to serum TGF&bgr;1 levels. Thus, TGF&bgr;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&bgr;1 enhanced tumor growth in vivo. This group transected CHO cells with the TGF&bgr;1 gene resulting in overexpression of TGF&bgr;1. TGF&bgr;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&bgr;1-transected cells; vascularisation was diminished in the non-transected cells. In addition, an anti-TGF&bgr;1 neutralising antibody was able to inhibit both growth and angiogenesis in the tumors derived from TGF&bgr;1-transected cells. Thus, overproduction of TGF&bgr;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&bgr;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&bgr;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&bgr;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&bgr;1 in mice is prevented with 2G7 (repeated i.p. does), an anti-TGF&bgr;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&bgr; 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 &mgr;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&bgr; antibody (500 &mgr;g i.p. every other day) in combination with IL-2 (10000U i. p. 2× 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&bgr;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&bgr;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&bgr;1 antibodies used in these studies were small (10 ng and 1 &mgr;g respectively). Thus, combination of anti-TGF&bgr; 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&bgr;1 antibodies (treatment from day 3, every 3-4 days with 100 &mgr;g s.c. at the tumor site) The authors suggested that the antibodies reversed TGF&bgr;1-induced immunosuppression leading to inhibition of tumor growth and metastasis.
[0103] Previous work has also demonstrated that anti-TGF&bgr; 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&bgr;1-induced immunosuppression permitted metastasis of these tumor xenografts. However, a recent paper suggests that TGF&bgr;1 may enhance the invasive and metastatic potential of cells directly (Hojo et al., 1999, Nature 397:530-534). Cyclosporin dose-dependently induces TGF&bgr;1 release from human pulmonary adenocarcinoma cells in culture, however, the mechanism of TGF&bgr; production by cyclosporin is not known. Treatment of adenocarcinoma cells with cyclosporin (or TGF&bgr;1) results in membrane ruffling, pseudopodia formation, anchorage-independent (invasive) growth and motility. An anti-TGF&bgr;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&bgr;1 2 3 neutralising antibody, 1D11.16 (200 &mgr;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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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 &mgr;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&bgr;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&bgr;1 may be determined. Quantitation may be related to the amount of TGF&bgr;1 in a test sample, which may be of diagnostic interest, which may be of diagnostic interest, for example, measurement of TGF&bgr;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&bgr;1 is mixed with unlabelled TGF&bgr;1 (the test sample) and allowed to bind to the antibody. Bound TGF&bgr;1 is physically separated from unbound TGF&bgr;1 and the amount of radioactive TGF&bgr;1 bound to the antibody determined. The more TGF&bgr;1 there is in the test sample the less radioactive TGF&bgr;1 will bind to the antibody. A competitive binding assay may also be used with non-radioactive TGF&bgr;1, using TGF&bgr;1 or an analogue of TGF&bgr;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&bgr;1 levels in a competition assay, that is to say a method of measuring the level of TGF&bgr;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&bgr;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&bgr;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&bgr;1 with any specific binding member which both binds TGF&bgr;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&bgr;1 ELISA as described in Example 1.
[0127] Preferred specific binding members for TGF&bgr;1 compete for binding to TGF&bgr;1 with CAT 191, CAT 192 and/or CAT 193
[0128] Preferred embodiments strongly neutralise TGF&bgr;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&bgr;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&bgr;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 Plückthun, 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&bgr;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&bgr;1. ELISA plates (96 well; Falcon) were uncoated, or coated with recombinant TGF&bgr;1 (0.2 &mgr;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 125I-TGF&bgr;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 IC50 of 100 pM which is at least 100 fold better than CS37. The complete specificity of SL15 scFv for the TGF&bgr;1 isoform has been confirmed in the TF1 assay where no interaction with TGF&bgr;2 or TGF&bgr;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, &kgr; 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&ggr;4.
[0165] Light Chain Expression Vector
[0166] The V&kgr; 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&kgr; or pKylie&kgr;.
[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&ggr;4, was digested with BamHI and NotI and the 4497 bp fragment containing H chain DNA purified. The light chain vector, pCS37V&kgr; or pKylie&kgr;, 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&ggr;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 &mgr;g of pKylieg4&ggr;s were linearised by digestion with PvuI, ethanol precipitated and dissolved in 100 &mgr;l water. 107 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 &mgr;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 &mgr;l aliquots distributed into 6×96-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 &mgr;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× PBST and 100 &mgr;l OPD substrate added. Reactions were stopped after 5-10 min by the addition of 50 &mgr;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 &mgr;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&bgr;1 was measured for SL15S scFv (Kylie scFv) and its derivatives using a radioreceptor assay and a cell proliferation assay (TF1).
[0176] Materials
[0177] [125I]TGF&bgr;1 was supplied by Amersham (specific activity range 800-2200Ci/mmol). Recombinant human TGF&bgr;1, &bgr;2,&bgr;3, latent TGF&bgr;1 GM-CSF and IL-5 were obtained from R&D Systems (Minneapolis USA). Genzyme murine mAb against TGF&bgr;1, &bgr;2 and &bgr;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 2×105 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 [125I]TGF&bgr;1 in assay buffer.
[0181] After 1 hour at room temperature, 0.5 ml of this antibody/[125I]TGF&bgr;1 mixture was added in duplicate to the cells (already in 0.5 ml assay buffer) and incubated for 1 hour at 37° C. Final concentration of [125I]TGF&bgr;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× 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&bgr;1, % maximum was calculated for each set of conditions (eg in the presence of latent TGF&bgr;1, acid-activated latent TGF&bgr;1 or active TGF&bgr;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 4×105 cells/ml in fresh medium supplemented with 4 ng/ml IL-5 either with or without TGF&bgr;1, &bgr;2 or &bgr;3 (each at 50 pM) and 100 &mgr;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 &mgr;l added to cells in duplicate. Controls were cells with TGF&bgr; only (no antibody, maximum inhibition of growth) and cells with no TGF&bgr; and no antibody (minimum inhibition). Cells were incubated for 48 hours at 37° C.
[0189] At the end of the assay cell number was assayed using CellTiter96 (Promega) and data expressed as % neutralisation i.e. 1 % neutralisation = (test value - maximum inhibition) (minimum inhibition - maximum inhibition) × 100
[0190] In the study of latent TGF&bgr;1 data were expressed as % of control (growth in the absence of TGF&bgr;1) as the amount of active TGF&bgr;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&bgr;1 but not TGF&bgr;2 or &bgr;3 in the TF1 assay was investigated.
[0197] SL15S scFV (also known as Kylie scFV) neutralised the growth inhibition induced by TGF&bgr;1 but not that induced by TGF&bgr;2 or TGF&bgr;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&bgr;1, TGF&bgr;2 and TGF&bgr;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&bgr;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&bgr;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&bgr;1 and neutralise binding of TGF&bgr;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&bgr;1, with IC50 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&bgr;1[0203] The experiments described in this example demonstrated that SL15S scFv and SL15A IgG4 bind to and neutralise active, but not latent, TGF&bgr;1.
[0204] Latent TGF&bgr;1 is the biologically inactive form in which TGF&bgr;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&bgr;1 dimer (consisting of two 112 amino acid monomers) Latent TGF&bgr;1 is not recognised by cell surface TGF&bgr; receptors Active TGF&bgr;1 is thought to be released by the action of proteases in vivo which can be mimicked by acidification in vitro.
[0205] Latent TGF&bgr;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&bgr;1 has a reported ED50 in a proliferation assay of approximately 2 pM Presumably the effect of latent TGF&bgr;1 before acidification is due to the presence of some active TGF&bgr;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&bgr;1, acid-activated TGF&bgr;1 and active TGF&bgr;1. If the antibodies recognise latent TGF&bgr;1, their potency should be reduced. Interpretation of both assays is complicated by the fact that any active TGF&bgr;1 present in the latent preparation will compete with [125I]TGF&bgr;1 for binding and have biological activity on the cells.
[0207] Latent TGF&bgr;1 was acid-activated by the addition of 2 &mgr;l 0.5M HCl to 1 ml latent TGF&bgr;1 for 15 min at room temperature then neutralised with 43 &mgr;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&bgr;1 (0.1 nM) on the potency of SL15S scFv (Kylie scFv) or SL15A IgG4 (Kylie IgG), whereas acid-activation of the latent TGF&bgr;1, or an equivalent concentration of active TGF&bgr;1 reduces the ability of the antibody to neutralise [125I]TGF&bgr;1 binding (FIG. 4).
[0209] The TF1 proliferation assay was very sensitive to the activity of the active TGF&bgr;1 in the latent TGF&bgr;1 preparation. Nevertheless, the SL15S scFv or SL15A IgG4 were found to be able to neutralise the small proportion of active TGF&bgr;1 of the latent preparation and when the latent TGF&bgr;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&bgr;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&bgr;1 to which the antibodies CS37 and SL15S binds was determined.
[0212] TGF&bgr;1 and TGF&bgr;2 have similar structures but differ in their binding affinity for the TGF&bgr; 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&bgr;1 to the type II receptor. This has been done by making a series of chimeric molecules between TGF&bgr;1 and TGF&bgr;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&bgr;1 was identified as sufficient to retain efficient receptor binding.
[0213] Comparison of the sequences of TGF&bgr;1 and TGF&bgr;2 identified several differences including a loop consisting of residues 92-98, which includes 4 amino acid differences between TGF&bgr;1 and TGF&bgr;2. When these residues from TGF&bgr;2 were introduced into a TGF&bgr;1 backbone, receptor binding was greatly reduced, identifying these as key residues in the interaction of TGF&bgr;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&bgr;1. TGF&bgr;1/&bgr;2 (83-112) corresponds to the N-terminal and central region of TGF&bgr;1 from residues 1-82, fused with the C-terminal region of TGF&bgr;2 from residues 83-112. TGF&bgr;2/&bgr;183-112 corresponds to the N-terminal and central region of TGF&bgr;2 from residues 1-82, fused with the C-terminal region of TGF&bgr;1 from residues 83-112. Finally, TGF&bgr;1-&bgr;2 (40-112) corresponds to the N-terminal region of TGF&bgr;1 from residues 1-39, and the central and C-terminal region of TGF&bgr;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&bgr;1 and TGF&bgr;2 were profiled for inhibition of binding of SL15S scFv and CS37 scFv to immobilized TGF&bgr;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 5×1010 in 50 &mgr;l) was added for 30 minutes to a preblocked ELISA plate coated with TGF&bgr;1 at 0.5 &mgr;g/ml.
[0217] For inhibition analysis, the same concentration of phage was used and a dilution series of TGF&bgr; concentrations set up for each of the TGF&bgr; chimeric molecules described above (Gift from J. Burmester, National Institutes of Health) and the TGF&bgr;1 and 2 isoforms.
[0218] These were incubated overnight at 4° 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&bgr;1. Furthermore, the mutant molecule TGF&bgr;1-&bgr;2(92-98) which comprises TGF&bgr;1 with the TGF&bgr;2 sequence from residues 92-98 also inhibits CS37 and SL15S scFv binding to TGF&bgr;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&bgr;1 and TGF&bgr;2).
EXAMPLE 6 Acceleration of Corneal Epithelial Wound Healing in the Presence of the Anti-TGF&bgr;1 Monoclonal Antibody SL15 A IgG4 (CAT-192)[0221] TGF&bgr;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&mgr;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 &mgr;l of serum-free T8 medium was added dropwise to the epithelial surface and limbus, containing (i) 10 ng/ml TGF&bgr;1; (ii) 1001 &mgr;g/ml anti-TGF&bgr;1 mAb; (iii) a combination of (i) and (ii); (iv) 100 &mgr;g/ml null antibody (2G6 IgG4), (v) 100 &mgr;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&bgr;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&bgr;1 demonstrated delayed re-epithelialisation by 24 hours and this inhibitory effect was abolished by anti-TGF&bgr;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&bgr;1 mAb did not adversely affect corneal architecture 3 TABLE 3 The effect of CAT192 or TGF&bgr;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−1) n Medium 199 (100 &mgr;l) 1.56 ± 0.07 8 Vehicle (100 &mgr;l) 1.57 ± 0.08 8 Null antibodyIgG4 (10 &mgr;l) 1.48 ± 0.09 8 TGF&bgr;1 (1 ng) 1.01 ± 0.09&tgr; 8 CAT192 (10 &mgr;l) 2.11 ± 0.09* 8 Table 3 legend: Bovine cornea were treated with 100 &mgr;l of either serum free Medium 199 (control) or medium containing vehicle (for antibody and TGF&bgr;1) null IgG4 isotope-matched antibody, TGF&bgr;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&bgr;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; &tgr;P0.01 compared to the vehicle treated group
[0224] These results confirm the important role of endogenous TGF&bgr;1 in corneal epithelial repair and provide indication that the topical application of the anti-TGF&bgr;1 mAb SL15A IgG4 (CAT-192) may be used to promote improved wound healing in vivo
Claims
1. An isolated specific binding member capable of binding TGF&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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&bgr;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
Type: Application
Filed: Sep 13, 2002
Publication Date: May 15, 2003
Inventors: Julia Elizabeth Thompson (Cambridgeshire), Simon Nicholas Lennard (Linton), Alison Jane Wilton (Cambridge), Peta Sally Helena Braddock (Huntingdon), Sarah Leila Du Fou (Herfordshire), John Gerald McCafferty (Cambridgeshire), Louise Anne Conroy (Cambridge), Philip Ronald Tempest (Cambridgeshire)
Application Number: 10243265
International Classification: A61K039/395; C07K016/22;
