HK1201859B - Antibodies to tgfbeta - Google Patents

Description

Antibodies to TGF β

The present application is a divisional application of an invention patent application having an application date of 8/2/2006, an application number of 200680008838.9, and an invention name of "antibody against TGF β".

Technical Field

More specifically, the present invention relates to antibody molecules that bind to and preferably neutralize TGF β 1, TGF β 2 and TGF β 3, so-called "pan-specific" antibody molecules, and the use of such antibody molecules.

Background

TGF β was first identified in 1981 (Roberts et al, 1981) in humans there are 3 isoforms TGF β, TGF β 2 and TGF β (Swiss Prot accession numbers P01137, P08112 and P10600, respectively) which in their biologically active state are 25kDa homodimers, comprising two monomers of 112 amino acids connected by interchain disulfide bridges TGF β differs from TGF β by 27 and from TGF β by 22, mainly conservative amino acid changes.

Human TGF β s is very similar to mouse TGF β s-human TGF β 1 differs from mouse TGF β 1 by only 1 amino acid, human TGF β 2 differs from mouse TGF β 2 by only 3 amino acids, and human TGF β 3 is identical to mouse TGF β 3-therefore, it is difficult to produce antibodies against human TGF β s in mice, including transgenic mice.

TGF β s are multifunctional cytokines involved in cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses (Border et al, 1995 a.) dysregulation of TGF β s leads to pathological processes that have been implicated in humans in numerous disease states, such as birth defects, cancer, chronic inflammation, autoimmune and fibrotic diseases (Border et al, 1994; Border et al, 1995 b).

Neutralizing antibodies have been studied as antagonists in a number of animal models of fibrosis (Border et al, 1995 b; Border et al, 1994), such as glomerulonephritis (Border et al, 1990), neural scarring (Logan et al, 1994), cutaneous scarring (Shah et al, 1994) and pulmonary fibrosis (Giri et al, 1993). all of the diseases represented by these models represent an unmet need for novel therapeutic products (Bonewason, 1999; Jackhan, 1998). however, the antibodies used in these and other animal studies have been raised in animals and their therapeutic benefit in humans may be limited because of their potential to induce immunogenic responses and their rapid pharmacokinetic clearance (Vaughan et al, 1998). human antibodies are more desirable for the treatment of TGF β mediated diseases.

For example, antibody fragments comprising only a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together by a short peptide linker, known as single chain fv (scFv), have been widely used, neutralizing TGF β 1(CAT-192) or TGF β 2(CAT-152 or Trabio)^TM) The human antibodies of (1) have been previously generated (EP 0945464, EP 0853661, Thompson et al 1999.) however, most TGF β antibodies available in the art are non-human moreover, the pan-specific monoclonal antibody directed solely to TGF β prior to the present invention was rodent.

Polyclonal antibodies against neutralizing and non-neutralizing epitopes that bind to human TGF β 1 and human TGF β 2 have been generated in rabbits (Danielpour et al, 1989 b; Roberts et al, 1990), chickens (R & D Systems, Minneapolis) and turkeys (Danielpour et al, 1989 c.) peptides representing part of the TGF β sequence have also been used as immunogens to generate neutralizing polyclonal antisera in rabbits (Border et al, 1990; Flanders et al, 1988.) such non-human polyclonal antibodies are not suitable for human therapeutic use.

1D11.16 is a murine pan-specific anti-TGF β antibody that neutralizes human and mouse TGF β 1, TGF β 2 and TGF β 3 in a wide range of in vitro assays (Dasch et al, 1989; Dasch et al, 1996; product page of the R & D System for MAB 1835) and is effective in proof of principle studies in fibrotic animal models (Ling et al, 2003; Miyajima et al, 2000; Schneider et al, 1999; Khanna et al, 1999; Shenkar et al, 1994). however, because 1D11.16 is a murine monoclonal antibody (Dasch et al, 1989; Dasch et al, 1996), it is not suitable for therapeutic use in humans.

Brief Description of Drawings

FIG. 1 shows the neutralization (% inhibition) of fibronectin production induced by TGF β 1(a), TGF β 2(b) or TGF β 3(c) (10pM) from NHLF cells by PET1073G12 germline IgG4 (filled squares) and 1D11.16 (open circles). closed triangles represent the mean. + -. SE mean of n experiments performed in duplicate at the highest concentration (100 nM). The data are shown for the IC₅₀The values are shown in Table 2.

FIG. 2 shows the neutralization (% inhibition) of fibronectin production induced by TGF β 1(a), TGF β 2(B) or TGF β 3(c) (10pM) from NHLF cells by PET1074B9 germline IgG4 (filled squares) and 1D11.16 (open circles). closed triangles represent the mean. + -. SE mean of n experiments performed in duplicate at the highest concentration (100 nM). The data are shown for the IC₅₀The values are shown in Table 2.

FIG. 3 shows the neutralization (% inhibition) of fibronectin production induced by TGF β 1(a), TGF β 2(b) or TGF β 3(c) (10pM) from NHLF cells by PET1287A10 germline IgG4 (filled squares) and 1D11.16 (open circles). closed triangles represent the mean. + -. SE mean of n experiments performed in duplicate at the highest concentration (100 nM). The data are shown for the IC₅₀The values are shown in Table 2.

Summary of The Invention

The subject matter of the embodiments included below is provided in various aspects of the present invention. Further aspects and embodiments of the invention are disclosed in the description herein.

The present invention provides specific binding members for TGF β, in particular human TGF β specific binding members for TGF β 1, TGF β 2 and TGF β 3 are provided in particular preferred embodiments within the invention are antibody molecules, whether intact antibodies (e.g. IgG, such as IgG1 or IgG4) or antibody fragments (e.g. scFv, Fab, dAb), antibody antigen-binding regions and antigen-binding sites for antibodies, such as antibody VH and VL domains comprising such regions are provided.

In one aspect, the invention provides specific binding members for human TGF β comprising the antigen binding site of an antibody, the set of HCDRs, the set of LCDRs, or both and/or the human antibody VH domain, VL domain, or both.

The group of HCDR1, HCDR2 and HCDR3 may have a sequence selected from:

HCDR1SEQ ID NO: 3. HCDR2SEQ ID NO: 4. HCDR3SEQ ID NO: 5 (referred to herein as the "HCDRs group of PET1073G 12");

HCDR1SEQ ID NO: 13. HCDR2SEQ ID NO: 14. HCDR3SEQ ID NO: 15 (referred to herein as the "HCDRs group of PET1074B 9");

HCDR1SEQ ID NO: 23. HCDR2SEQ ID NO: 24. HCDR3SEQ ID NO: 25 (referred to herein as the "HCDRs panel of PET1287a 10").

The set of LCDR1, LCDR2 and LCDR3 may have a sequence selected from:

LCDR1SEQ ID NO: 8. LCDR2SEQ ID NO: 9. LCDR3SEQ ID NO: 10 (referred to herein as the "LCDRs group of PET1073G 12");

LCDR1SEQ ID NO: 18. LCDR2SEQ ID NO: 19. LCDR3SEQ ID NO: 20 (referred to herein as the "LCDRs panel of PET1074B 9");

LCDR1SEQ ID NO: 28. LCDR2SEQ ID NO: 29. LCDR3SEQ ID NO: 30 (referred to herein as the "LCDRs panel of PET1287a 10").

The HCDRs panel of PET1073G12 together with the LCDRS panel of PET1073G12 is referred to herein as the CDRs panel of PET1073G 12.

The HCDRs panel of PET1074B9 together with the LCDRS panel of PET1074B9 is referred to herein as the CDRs panel of PET1074B 9.

The HCDRs panel of PET1287a10 together with the LCDRS panel of PET1287a10 is referred to herein as the CDRs panel of PET1287a 10.

The invention also provides VH domains comprising a panel of HCDRs as disclosed herein, and likewise separately provides VL domains comprising a panel of LCDRs as disclosed herein. Preferably, such VH domains are paired with such VL domains, and most preferably the pairing of VH and VL domains is as in the clones as set out herein.

The invention further provides VH domains comprising the HCDRs group HCDRs 1, HCDR2 and HCDR3, wherein the HCDRs group corresponds to the HCDRs group of PET1073G12, PET1074B9 or PET1287a10 with 1 or 2 amino acid substitutions.

The invention further provides VL domains comprising the set of LCDRs LCDR1, LCDR2 and LCDR3, wherein the set of CDRs corresponds to the set of LCDRs of PET1073G12, PET1074B9 or PET1287a10 with 1 or 2 amino acid substitutions.

The invention also provides specific binding members comprising the antigen binding site of an antibody within such a VH and/or VL domain.

Following the introduction of Multivariate data Analysis Techniques to the computational Chemistry of structure/property-activity relationships (Wold et al, multivariable data Analysis in Chemistry. chemometrics-Mathesics and statics in Chemistry (Ed.: B. Kowalski), D.Reidel Publishing Company, Dordrecht, Holland, 1984(ISBN 90-277) 1846-6), quantitative activity-property relationships of antibodies can be obtained using well-known mathematical Techniques such as statistical regression, pattern recognition and classification (Norman et al, applied research Analysis. William-Interscience; third edition (April1998) ISBN: 0471170828; Abstract Kandel, Imeric Backer, computer-dimensional reaction in Analysis. theory. Press, 12. 12, 1999) ISBN: 1558605525, respectively; david g.t.denison (editor), christopher c.holmes, Bani k.mallick, adian f.m.smith.bayesian Methods for nonlinerearclassification and Regression (Wiley Series in basic and statics). John Wiley & Sons; (July2002), ISBN: 0471490369, respectively; arup k. ghose, vellarkadn. video adaptation Design and Evaluation Principles, Software, Tools, and Applications in Drug discovery. isbn: 0-8247-0487-8). The properties of an antibody can be derived from empirical and theoretical models of antibody sequences (e.g., analysis of likely residues to be contacted or calculated physicochemical properties), function, and three-dimensional structure, and these properties can be considered individually and in combination.

Analysis of antibodies of known atomic structure has elucidated the relationship between the sequence and three-dimensional structure of the binding site of the antibody (Chothia C. et Al, Journal Molecular Biology (1992)227, 799-. These relationships suggest that, in addition to the third region (loop) in the VH domain, the binding site loop has one of a few backbone conformations: regular structures (canonicals structures). It has been shown that the canonical structures formed in a particular loop are determined by their size and the presence of certain residues at key sites within the loop and framework regions (Chothia et Al and Al-Lazikani et Al, supra).

This sequence-structure relationship study can be used to predict those residues in an antibody of known sequence but unknown three-dimensional structure that are important in maintaining the three-dimensional structure of its CDR loops and thus in maintaining binding specificity. These predictions can be confirmed by comparing the predictions with output results from lead optimization experiments (lead optimization experiments). In the structural approach, any freely available or commercial software package such as WAM (Whitelegg, N.R.u. and Rees, A.R (2000) prot.Eng., 12,815-824) can be used to form a theoretical model of the antibody molecule. Protein visualization and analysis software packages such as Insight II (Accelrys, Inc.) or Deep View (Guex, N. and Peitsch, M.C. electrophosphoresis (1997)18, 2714-2723) can then be used to assess possible substitutions at each position in the CDR and FR. This information can then be used to perform substitutions that may have minimal or beneficial effects on activity.

The techniques required to make substitutions within the amino acid sequences of the CDRs, antibody VH or VL domain and specific binding member are generally available in the art variant sequences may be prepared using substitutions which may or may not be predicted to have a minimal or beneficial effect on activity, and tested for the ability to bind and/or neutralize TGF β and/or any other desired property.

As already noted, the present invention provides specific binding members comprising a designated set of CDRs, in particular the set of CDRs of PET1073G12, PET1074B9 and PET1287a10, and the set of CDRs of PET1073G12, PET1074B9 or PET1287a10 with 1 or 2 substitutions within the set of CDRs.

Sets of relevant CDRs within antibody framework regions or other protein scaffolds such as fibronectin or cytochrome B are provided. Antibody framework regions are preferably employed.

In a preferred embodiment, the heavy chain employs human V_H1 family gene. In various embodiments, with human V_HThe heavy chain framework amino acid sequence comprises 1-12, preferably 3-12, and more preferably 3-8 amino acid differences compared to the germline amino acid sequence of the family 1 gene. In certain embodiments, the heavy chain framework sequence is a germline sequence. In a particularly preferred embodiment, the antibody framework region of the heavy chain may be from V_HFamily 1 human DP-10 (V)_H1-69) or human DP-88 (V)_H1-e). Preferably, makeEmbodiments of the human DP-10 gene have non-germline amino acids at residues 27, 78 and 94. In certain embodiments, residue 27 is tyrosine, residue 78 is threonine, and residue 94 is serine or leucine. In certain embodiments, the light chain employs a human vk 3 family gene having 1-5, preferably 1-4, more preferably 1-3 amino acid differences compared to the germline amino acid sequence. In certain embodiments, the light chain framework sequence is a germline human vk 3 family gene sequence. In a particularly preferred embodiment, the light chain framework region may be human DPK-22 (A27). In certain such embodiments, residue 2 is a non-germline amino acid. In certain embodiments, residue 2 is threonine.

In a highly preferred embodiment, there is provided a polypeptide having the sequence of SEQ ID NO: 2, which is referred to as the "PET 1073G12VH domain", or a VH domain having the amino acid sequence of SEQ ID NO: 12, which is referred to as the "PET 1074B9VH domain", or a VH domain having the amino acid sequence of SEQ ID NO: 22, which is referred to as the "PET 1287a10VH domain".

In a further highly preferred embodiment, there is provided a polypeptide having the sequence of SEQ ID NO: 7, which is referred to as the "PET 1073G12VL domain", or a VL domain having the amino acid sequence of SEQ ID NO: 17, which is referred to as the "PET 1074B9VL domain", or a VL domain having the amino acid sequence of SEQ ID NO: 27, which is referred to as the "PET 1287a10VL domain". A highly preferred embodiment provided according to the present invention consists of the PET1073G12VH domain (SEQ ID NO: 2) and the PET1073G12VL domain (SEQ ID NO: 7). Another highly preferred embodiment provided according to the present invention consists of the PET1074B9VH domain (SEQ ID NO: 12) and the PET1074B9VL domain (SEQ ID NO: 17). Another highly preferred embodiment provided according to the present invention consists of the PET1287A10VH domain (SEQ ID NO: 22) and the PET1287A10VL domain (SEQ ID NO: 27). These or any other antibody antigen-binding sites provided according to the invention may be provided in the form of any desired antibody molecule, e.g. scFv, Fab, IgG1, IgG4, dAb, etc., as discussed further elsewhere herein.

In a further highly preferred embodiment, the present invention provides an IgG4 antibody molecule comprising a PET1073G12, PET1074B9 or PET1287a10VH domain, preferably further comprising the corresponding PET1073G12, PET1074B9 or PET1287a10VL domain.

The invention provides other IgG4 or other antibody molecules comprising a PET1073G12, PET1074B9 or PET1287a10VH domain and/or a PET1073G12, PET1074B9 or PET1287a10VL domain, as provided by the HCDRs panel of PET1073G12, PET1074B9 or PET1287a10 included in the antibody VH domain, and/or the LCDRs panel of PET1073G12, PET1074B9 or PET1287a10 included in the antibody VL domain.

For convenience, it is noted herein that "and/or" as used herein is considered a specific disclosure of each of the two specified features or components, with or without the other. For example, "a and/or B" is considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed herein.

As noted, specific binding members are provided in certain embodiments of the invention which bind all 3 isoforms of human TGF β and include PET1073G12, PET1074B9 or PET1287a10VH and/or VL domains, or antigen-binding portions of those domains.

In certain embodiments, the VH domain is paired with a VL domain to provide an antigen binding site. In a preferred embodiment, the PET1073G12VH domain (SEQ ID NO: 2) is paired with the PET1073G12VL domain (SEQ ID NO: 7) such that an antigen binding site is formed comprising the PET1073G12VH and VL domains. In a preferred embodiment, the PET1074B9VH domain (SEQ ID NO: 12) is paired with the PET1074B9VL domain (SEQ ID NO: 17) such that an antigen binding site is formed comprising the PET1074B9VH and VL domains. In a preferred embodiment, the PET1287A10VH domain (SEQ ID NO: 22) is paired with the PET1287A10VL domain (SEQ ID NO: 27) such that an antigen binding site is formed that includes the PET1287A10VH and VL domains. In other embodiments, PET1073G12, PET1074B9 or PET1287a10VH is paired with a VL domain other than the corresponding PET1073G12, PET1074B9 or PET1287a10 VL. Light chain promiscuity is well established in the art.

Similarly, any of the sets of HCDRs disclosed herein may be provided in a VH domain which acts as a specific binding member, alone or in combination with a VL domain. VH domains having the set of HCDRs disclosed herein can be provided, and if such VH domains are paired with VL domains, VL domains having the set of LCDRs disclosed herein can be provided. The pairing of the HCDRs panel and the LCDRs panel may be as disclosed herein for the PET1073G12, PET1074B9 and PET1287a10 antibodies. The framework regions of the VH and/or VL domains may be germline frameworks. The framework regions of the heavy chain domain may be selected from V_HFamily 1, and preferably V_HThe-1 framework is the DP-10 or DP-88 framework the framework regions of the light chain may be selected from the V к 3 family, and a preferred such framework is DPK-22.

One or more CDRs may be taken from a VH or VL domain, the sequences of which are disclosed herein and incorporated into a suitable framework. This is discussed further herein. Other CDRs and sets of CDRs of an antibody as obtained using the methods described herein can also be employed.

Antibody VH domains, antibody VL domains, sets of HCDRs, sets of LCDRs, sets of CDRs, one or more HCDRs, e.g., HCDR3, and/or one or more LCR's, e.g., LCDR3, can be employed in any of the aspects and embodiments of the invention disclosed herein with respect to other molecules, e.g., methods of mutation and selection of antigen binding sites with improved potency.

Variants of the VH and VL domains and CDRs of the invention, including those whose amino acid sequences are listed herein and which may be employed in specific binding members for TGF β, may be obtained by sequence alteration or mutation and screening methods.

As discussed, variable domain amino acid sequence variants of any of the VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention. Particular variants may include one or more amino acid sequence alterations (additions, deletions, substitutions, and/or insertions of amino acid residues), which may be less than about 20 alterations, less than about 15 alterations, less than about 10 alterations, or less than about 5 alterations, 4, 3, 2, or 1 alteration. The alteration may be made in one or more framework regions and/or one or more CDRs.

According to a further aspect of the invention there is provided a human, humanized, chimeric or synthetic specific binding member that competes or cross-competes for binding to an antigen with any specific binding member that binds to the antigen and comprises a specific antibody antigen-binding region, a VH and/or VL domain as disclosed herein, a set of CDRs or HCDR3 as disclosed herein, or a variant of any of these. Competition between binding members can be readily determined in vitro, for example using ELISA and/or by labelling a specific reporter molecule to one binding member, which can be detected in the presence of the other unlabelled binding member, thereby enabling the identification of specific binding members that bind the same epitope or overlapping epitopes. Cross-competition between binding members can be readily determined by performing a reverse assay, for example by inverting labelled and unlabelled binding members to identify pairs that block binding in both directions.

Thus, a further aspect of the invention provides a specific binding member comprising an antigen-binding site of an antibody which competes or cross-competes for binding to TGF- β with a PET1073G12, PET1074B9 or PET1287A10 antibody molecule, in particular PET1073G12, PET1074B9 or PET1287A10scFv and/or IgG4 in various embodiments the antibody is a human, humanized, chimeric or synthetic antibody in a further aspect the invention provides a specific binding member comprising an antigen-binding site of a human, humanized, chimeric or synthetic antibody which competes or cross-competes for binding to TGF- β with an antigen-binding site of the invention, wherein the antigen-binding site of the human, humanized, chimeric or synthetic antibody consists of a VH domain and a VL domain, and wherein the VH and VL domains comprise a set of CDRs as disclosed herein.

Given the information disclosed herein, various methods are available in the art for obtaining human, humanized, chimeric or synthetic antibodies against TGF β, and which may compete or cross-compete for binding to TGF β with PET1073G12, PET1074B9 or PET1287a10 antibody molecules, antibody molecules having the CDRs panel of PET1073G12, PET1074B9 or PET1287a10, antibody molecules having the PET1073G12, PET1074B9 or PET1287a10HCDRs panel, or antibody molecules having the PET1073G12, PET1074B9 or PET1287a10LCDRs panel.

In a further aspect, the present invention provides a method for obtaining one or more specific binding members capable of binding TGF β 1, TGF β 2 and TGF β 3, the method comprising contacting a library of specific binding members according to the invention with said TGF β s, and selecting a specific binding member or members in said library that is capable of binding all of said TGF β s.

The library may be displayed on the surface of phage particles, each particle comprising nucleic acid encoding an antibody VH variable domain (and optionally, if present, also the displayed VL domain) displayed on its surface.

After selecting a specific binding member capable of binding an antigen and being displayed on a phage particle, nucleic acid may be obtained from the phage particle displaying the selected specific binding member. Such nucleic acids may be used for the subsequent production of a specific binding member or antibody VH variable domain (and optionally antibody VL variable domain) by expression of nucleic acids having the sequence of nucleic acids taken from phage particles displaying the selected specific binding member.

The ability to bind to all 3 TGF β isoforms may be further tested, as well as the ability to compete or cross-compete with PET1073G12, PET1074B9, or PET1287a10 (e.g. in scFv format and/or IgG format, such as IgG4) for binding to all 3 human TGF β isoforms.

A specific binding member according to the invention may bind TGF β 1, TGF β 2 and/or TGF β 3 with an affinity of a PET1073G12, PET1074B9 or PET1287a10 antibody molecule (e.g. scFv, or preferably IgG4), or with an affinity greater than one of the aforementioned molecules a specific binding member according to the invention may neutralise TGF β 1, TGF β 2 and/or TGF β 3 with a potency greater than one of the aforementioned molecules, or with a potency of a PET1073G12, PET1074B9 or PET1287a10 antibody molecule (e.g. scFv, or preferably PET1073G12, PET1074B9 or PET1287a10IgG 4).

Specific binding members according to the invention may neutralise naturally occurring TGF β with the potency of a PET1073G12, PET1074B9 or PET1287a10 antibody molecule (e.g. scFv, or preferably IgG4), or with greater potency than one of the above.

Preferred embodiments of the invention preferably include human, humanized, chimeric or synthetic antibodies that neutralize naturally occurring TGF β, wherein the neutralizing potency is equal to or greater than the potency of the TGF β antigen binding site formed by the PET1073G12, PET1074B9 or PET1287a10VH domain and the corresponding PET1073G12, PET1074B9 or PET1287a10VL domain.

In addition to antibody sequences, a specific binding member according to the invention may also comprise other amino acids, for example forming a peptide or polypeptide, such as a folded domain, or conferring another functional characteristic to the molecule other than the ability to bind an antigen. Specific binding members of the invention may carry a detectable label or may be conjugated to a toxin or targeting moiety or enzyme (e.g., via a peptidyl bond or linker).

In a further aspect, the invention provides an isolated nucleic acid comprising a sequence encoding a specific binding member, VH domain and/or VL domain or CDR according to the invention, and a method of making a specific binding member, VH domain and/or VL domain or CDR of the invention, the method comprising expressing the nucleic acid under conditions such that the specific binding member, VH domain and/or VL domain, or CDR is produced and recovered.

Specific binding members according to the invention may be used in methods of treatment or diagnosis of the human or animal body, for example methods of treatment (which may include prophylactic treatment) of a disease or disorder in a human patient which comprises administering to the patient an effective amount of a specific binding member of the invention.

More specifically, specific binding members of the invention may be used to inhibit the activity of any or all of the 3 human TGF β isoforms in vitro or in vivo, such activities include, but are not limited to, TGF β mediated signaling, extracellular matrix (ECM) deposition, inhibition of epithelial and endothelial cell proliferation, promotion of smooth muscle proliferation, induction of type III collagen expression, induction of TGF- β, fibronectin, VEGF, and IL-11 expression, binding of potentially related peptides (Latency Associated Peptide), tumor-induced immunosuppression, promotion of angiogenesis, activation of myofibroblasts, promotion of metastasis, and inhibition of NK cell activity.

Because the specific binding members of the invention are pan-specific, i.e., they bind and inhibit the activity of all 3 TGF β isoforms, they are particularly advantageous for treating conditions and diseases (e.g., infections and tumors) involving two or more TGF β isoforms, as well as severe conditions where inhibition of multiple targets is desired.

Specific binding members may be used to treat diseases and conditions including, but not limited to, fibrotic diseases (e.g., glomerulonephritis, neural scarring, skin scarring, pulmonary fibrosis, radiation-induced fibrosis, liver fibrosis, bone marrow fibrosis), burns, immune-mediated diseases, inflammatory diseases (including rheumatoid arthritis), transplant rejection, cancer, dupuytren's contracture, and gastric ulcers. They may also be used to treat, prevent and reduce the risk of the occurrence of renal insufficiency, including, but not limited to: diabetic (type I and type II) nephropathy, radiation nephropathy, obstructive nephropathy, systemic scleroderma, pulmonary fibrosis, allograft rejection, hereditary nephropathy (e.g. polycystic kidney disease, medullary sponge kidney, horseshoe kidney), glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic lupus erythematosus, sjogren's syndrome, begerl's disease, systemic or glomerular hypertension, tubulointerstitial nephropathy, tubulointerstitial acidosis, tuberculosis, and renal infarction. In particular, they are useful when combined with antagonists of the renin-angiotensin-aldosterone system, including, but not limited to: renin inhibitors, Angiotensin Converting Enzyme (ACE) inhibitors, Ang II receptor antagonists (also known as "Ang II receptor blockers"), and aldosterone antagonists. Methods of using specific binding members of the invention in combination with such antagonists are described in PCT/US04/13677, the contents of which are incorporated herein by reference.

Specific binding members of the invention may also be used to treat diseases and conditions associated with ECM deposition, the diseases and conditions include systemic scleroderma, post-operative adhesions, keloids and hypertrophic scarring, proliferative vitreoretinopathy, glaucoma drainage surgery, corneal injury, cataracts, pelonemia, adult respiratory distress syndrome, cirrhosis, scarring after myocardial infarction, restenosis after angioplasty, scarring after subarachnoid hemorrhage, multiple sclerosis, fibrosis after laminectomy, fibrosis after tendon and other repairs, scarring due to tattoo removal, biliary cirrhosis (including sclerosing cholangitis), pericarditis, pleuritis, tracheostomy, CNS eosinophilic injury, hypereosinophilic myalgia syndrome, vascular restenosis, venous occlusive disease, pancreatitis and psoriatic arthropathy.

Specific binding members of the invention are further useful in conditions in which promoting re-epithelialization is beneficial. Such conditions include, but are not limited to, skin diseases such as varicose ulcers, ischemic ulcers (bed 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 therapy, esophageal ulcers (reflux disease), gastric ulcers, small and large intestinal injuries (inflammatory bowel disease).

Still further uses of specific binding members of the invention are in conditions where endothelial cell proliferation is desired, for example in the stabilization of atherosclerotic plaques, the promotion of vascular anastomotic healing, or in conditions where inhibition of smooth muscle cell proliferation is desired, for example in arterial disease, restenosis and asthma.

Specific binding members of the invention may also be used to enhance the immune response to macrophage-mediated infections, such as those caused by Leishmania species (Leishmania spp.), trypanosoma cruzi (trypanosoma cruzi), Mycobacterium tuberculosis (Mycobacterium tuberculosis) and Mycobacterium leprosum (Mycobacterium leprae), as well as the protozoan arcus murinus slurry (Toxoplasma gondii), fungal Histoplasma capsulatum (Histoplasma capsulatum), Candida albicans (Candida albicans), Candida parapsilosis (Candida paropsis) and Cryptococcus neoformans (Cryptococcus neoformans), and Rickettsia such as Rickettsia pusilla (r. prowawakii), Rickettsia crinii (r. coronaviii) and tsutsutsukushi (r. tsukushi). They can also be used to reduce immunosuppression, for example caused by tumour, AIDS or granulomatous diseases.

Specific binding members of the invention are further useful in the treatment of hyperproliferative diseases, such as cancers, including but not limited to breast, prostate, ovarian, gastric, renal, pancreatic, colorectal, skin, lung, cervical and bladder cancers, gliomas, mesotheliomas, and various leukemias and sarcomas, such as kaposi's sarcoma, and in particular in the treatment or prevention of such tumor recurrence or metastasis. In particular, antagonist-specific binding members of the invention are useful for inhibiting cyclosporin-mediated metastasis.

It will, of course, be appreciated that in the context of cancer treatment, "treatment" includes any medical intervention that results in the slowing of tumor growth or the reduction of tumor metastasis and partial remission of the cancer, in order to extend the life expectancy of the patient.

A further aspect of the invention provides nucleic acids, which are generally isolated and encode the antibody VH variable domain and/or VL variable domain disclosed herein.

Another aspect of the invention provides a nucleic acid, generally isolated, encoding an HCDR or LCDR sequence disclosed herein, in particular a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3.4, 5, 13, 14, 15, 23, 24 and 25, or an HCDR selected from SEQ ID NOs: 8.9, 10, 18, 19, 20, 28, 29, 30, most preferably PET1073G12, PET1074B9 or PET1287a10HCDR3 (SEQ ID NOs 5, 15 or 25, respectively). Also provided herein are nucleic acids encoding the CDRs panel of PET1073G12, PET1074B9 or PET1287a10, HCDRs panel of PET1073G12, PET1074B9 or PET1287a10, and LCDRs panel of PET1073G12, PET1074B9 or PET1287a10, as are nucleic acids encoding individual CDRs, HCDRs, LCDRs, and PET1073G12, PET1074B9 or PET1287a10CDRs, HCDRs, LCDRs.

A further aspect provides a host cell transformed with a nucleic acid of the invention.

A still further aspect provides a method of producing an antibody VH variable domain, the method comprising causing expression from an encoding nucleic acid. Such methods may comprise culturing the host cell under conditions for production of the antibody VH variable domain or to cause expression of the antibody VH domain in vivo.

Similar methods for producing VL variable domains and specific binding members comprising VH and/or VL domains are provided as further aspects of the invention.

The production process may comprise steps of isolation and/or purification of the product.

The method of manufacture may comprise formulating the product into a composition comprising at least one additional component, such as a pharmaceutically acceptable excipient.

These and other aspects of the invention are described in further detail below.

Detailed Description

Term(s) for

Specific binding members

This describes members of a molecular pair having binding specificity for each other. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of a molecular pair has a region or cavity on its surface that specifically binds to a region on the surface of the other member of the molecular pair or a cavity therein. Thus, the members of the pair have the property of binding specifically to each other. The present invention relates to specific binding members that bind to a target antigen.

Specific for

For example, a specific binding member specific for human TGF- β will not have significant binding to other non-TGF- β human molecules, however it may cross-react with TGF- β from other species.

The antigen-binding specific binding member includes an antigen binding site. For example, the specific binding member may be an antibody molecule. Antigen binding sites may also be provided by aligning the CDRs on a non-antibody protein scaffold such as fibronectin or cytochrome B and the like (Koide et al, (1998) Journal of Molecular Biology, 284: 1141-1151; Nygren et al (1997) Current Opinion in Structural Biology, Vol.7: 463-469). Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren et al (supra). Protein scaffolds for antibody mimetics are disclosed in WO00/34784, which describes proteins (antibody mimetics) comprising a fibronectin type III domain with at least one randomized loop. Suitable scaffolds in which one or more CDRs, for example a set of HCDRs, are grafted, may be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be a human or non-human protein.

An advantage of a non-antibody protein scaffold is that it can provide antigen binding sites in conserved framework regions that are smaller and/or easier to prepare than at least some antibody molecules. The small size of the specific binding member may confer useful physiological properties, such as the ability to enter cells, penetrate deep into tissues or reach targets in other structures, or the ability to bind within the protein cavity of a target antigen.

The use of antigen binding sites in non-antibody protein scaffolds is reviewed in Wess, 2004. Typically are proteins with a stable backbone and one or more variable loops, wherein the amino acid sequence of the loop is specifically or randomly mutated to generate an antigen binding site with specificity for binding to a target antigen. Such proteins include the IgG binding domains of protein a from staphylococcus aureus (s. aureus), transferrin, tetranectin, fibronectin (e.g. the 10 th fibronectin type III domain) and lipocalin. Other methods include synthetic "Microbodies" (Selecore GmbH) based on macrocyclic oligopeptides (cyclides) which are small proteins with intramolecular disulfide bonds.

In addition to antibody sequences and/or antigen binding sites, specific binding members according to the invention may comprise other amino acids, for example forming peptides or polypeptides, such as folded domains, or conferring another functional characteristic to the molecule other than the ability to bind antigen. Specific binding members of the invention may carry a detectable label or may be conjugated to a toxin or targeting moiety or enzyme (e.g., via a peptidyl bond or linker). For example, a specific binding member may include a catalytic site (e.g., in an enzyme domain) and an antigen binding site, wherein the antigen binding site binds to an antigen and thus targets the catalytic site to the antigen. The catalytic site may inhibit a biological function of the antigen, for example by cleavage.

Although the CDRs may be carried by a scaffold such as fibronectin or cytochrome B as noted (Haan & Maggos, 2004BioCentury, 12 (5): A1-A6; Koide et al, supra; Nygren et al, supra), the structures used to carry the CDRs or sets of CDRs of the present invention will generally have an antibody heavy or light chain sequence or substantial portion thereof in which the CDRs or sets of CDRs are located at positions corresponding to the CDRs or sets of CDRs of the naturally occurring VH and VL antibody variable domains encoded by the rearranged immunoglobulin genes. The structure and position of immunoglobulin variable domains can be determined by reference to Kabat, et al, 1987 and its recent data, which is now available on the Internet (http:// immune. bme. nwu. edu. or using any search engine for "Kabat").

Antibody molecules

This describes immunoglobulins, whether natural or partially or wholly synthetically produced. The term also encompasses any polypeptide or protein that includes the antigen binding domain of an antibody. Antibody fragments comprising an antigen binding domain are molecules such as Fab, scFv, Fv, dAb, Fd and diabodies.

In the genome of human germline cells, the genetic information for the antibody polypeptide chains is contained in multiple gene segments within loci that are spread along different chromosomes. Human heavy chains (VH) are encoded on chromosome 14, kappa light chains (vk) on chromosome 2, and lambda light chains (V λ) on chromosome 22. During the development of B-lymphocytes (antibody-producing cells), gene segments at these loci assemble by recombination, resulting in the formation of the complete antibody heavy or light chain gene (Tonegawa S.Nature, 302, 575-81, 1983). Antibody constant regions (VH, vk and V λ) are largely identical throughout the population, but there is considerable diversity in the variable domains. Such diversity enables the formation of billions of different antibodies, each having specificity for a different target antigen.

Diversity within the variable region of an antibody is generated in several ways. First, at the gene level, there is considerable diversity in antibody variable germline gene sequences. Approximately 50 different VH germline lines (Tomlinson I.M. et al, J.mol.biol., 227, 776-798, 1992), 35 different V.kappa germline lines (Tomlinson I.M. et al, EMBOJ,14,4628-38, 1995) and 30 different V.lambda germline lines (Williams S.C. & Winter G.Eur.J.Immunol, 23, 1456-61, 1993; Kawasaki K. et al, Genome Res, 7, 250-61, 1997) have been described. Antibodies are produced from different combinations of these germline gene sequences. Further diversity is then introduced into antibody variable domains by processes such as somatic recombination and hypermutation (Tonegawa s. nature, 302, 575-81, 1983).

Although there is considerable diversity in antibody variable germline gene sequences, these sequences can be grouped into families based on sequence homology. The 50 different VH gene sequences can be grouped into 7 families, the 35 vk sequences into 6 families, and the 30V λ sequences into 10 families. These groups vary in size from 1 member (VH6 and V κ 4) to at most 21 members (VH3), and the members of each group share a high degree of sequence homology.

Antibodies can be aligned to VH and VL germline sequence databases to determine their closest germline match and to identify any amino acid changes introduced by somatic hypermutation. Studies have shown that the human immune system employs certain germline (e.g. VH3DP47) in preference to other germline (e.g. VH2) during the course of the immune response (Knappik a. et al, j.mol.biol, 296, 57-86, 2000). However, populations of antibodies isolated by phage display typically employ a wide range of germline genes, even when isolated against a single antigen (Edwards B. et al, J.mol Biol, 334, 103-118, 2003).

Monoclonal and other antibodies can be taken and recombinant DNA techniques used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve linking DNA encoding the variable region of an immunoglobulin to a constant region, or introducing the Complementarity Determining Regions (CDRs) of an antibody into the constant region plus framework regions of a different immunoglobulin. See, for example, EP-A-184187, GB2188638A or EP-A-239400, and ｃA number of subsequent documents. The hybridoma or other cell producing the antibody may be subjected to genetic mutations or other alterations that may or may not alter the binding specificity of the antibody produced.

Because antibodies can be modified in a variety of ways, the term "antibody molecule" should be construed to encompass any specific binding member or substance that has the antigen-binding site of an antibody with the desired specificity. Thus, this term encompasses antibody fragments and derivatives, including any polypeptide comprising an antigen-binding domain, whether natural or wholly or partially synthetic. Thus chimeric molecules comprising the antigen binding domain of an antibody fused to another polypeptide or an equivalent are included. The cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023, as well as in ｃA number of subsequent documents.

Further techniques available in the field of antibody engineering have enabled the isolation of human and humanized antibodies. For example, human hybridomas can be prepared as described by Kontermann et al (Kontermann R and Dubel Stefan; Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN: 3540413545). Phage display this alternative established technique for generating specific binding members has been described in detail in a number of publications such as Kontermann et al (supra) and W092/01047 (discussed further below). Transgenic mice, in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes, while leaving other components of the mouse immune system intact, can be used to isolate human antibodies against human antigens (Mendez et al, 1997). Monoclonal or polyclonal human antibodies can also be made in other transgenic animals, such as goats, cattle, sheep, rabbits, and the like.

Synthetic antibody molecules can be prepared by expression from genes generated by means of oligonucleotides synthesized and assembled in suitable expression vectors, for example as described by Knappik et al (supra) or Krebs et al, journal Immunological Methods 254200167-84.

It has been shown that fragments of intact antibodies can perform the function of binding antigen. Examples of binding fragments are (i) a Fab fragment consisting of VL, CL, VH and CHI domains; (ii) (ii) a fragment of Fd consisting of VH and CHI domains; (iii) (ii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH domains (Ward, E.S. et al, Nature341, 544-546(1989), McCafferty et al (1990) Nature, 348, 552-554); (v) an isolated CDR region; (vi) a F (ab')2 fragment which is a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv) in which the VH domain and the 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-; (viii) bispecific single chain Fv dimers (PCT/US 92/09665); and (ix) "diabodies", which are multivalent or multispecific fragments constructed by gene fusion (WO/13804; F. Holliger et al, Proc. Natl. Acad. Sci. USA906444-6448, 1993). Fv, scFv or diabody molecules can be stabilized by introducing a disulfide bridge connecting the VH and VL domains (Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996). Small antibodies (minibodies) comprising scFv linked to the CH3 domain may also be prepared (S.Hu et al, Cancer Res., 56, 3055-3061, 1996).

dAbs (domain antibodies) are small monomeric antigen-binding fragments of antibodies, i.e., the variable regions of the heavy or light chains of antibodies (Holt et al, 2003). VH dAbs occur naturally in camelids (e.g. camels, llamas) and can be produced by immunising a camelid with a target antigen, isolating antigen-specific B cells and cloning the dAb gene directly from individual B cells. dAbs can also be produced in cell culture. Their small size, good solubility and temperature stability make them particularly physiologically useful and suitable for selection and affinity maturation. A specific binding member of the invention may be a dAb which comprises a VH or VL domain substantially as set out herein, or a VH or VL domain comprising a set of CDRs substantially as set out herein.

When bispecific antibodies are used, these may be conventional bispecific antibodies, which may be prepared in various ways (Holliger, P. and Winter G. Current Opinion Biotechnol.4, 446-449(1993)), e.g.by chemical methods or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Examples of bispecific antibodies include BiTE^TMThose of the art, where the binding domains of two antibodies with different specificities can be used and linked directly via a flexible short peptide. This combines the two antibodies on a short single polypeptide chain. Diabodies and scFvs can be constructed without the Fc region using only variable domains, which effectively reduces the effect of anti-idiotypic reactions.

Bispecific diabodies, unlike bispecific whole antibodies, can also be particularly useful because they can be readily constructed and expressed in e.coli (e.coli.) diabodies (and many other polypeptides such as antibody fragments) with appropriate binding specificity can be readily selected from libraries using phage display (W094/13804.) if one arm of a diabody is held constant, e.g., with specificity for TGF β, a library can be prepared in which the other arm is variable and an antibody with appropriate specificity can be selected.

Antigen binding sites

This describes a portion of a specific binding member, such as an antibody molecule, which contacts and is partially or fully complementary to the other member of the binding pair, i.e. the antigen. In an antibody molecule, an antigen binding site may be referred to as an antibody antigen-binding site, and includes a portion of an antibody that specifically binds to and is fully or partially complementary to a target antigen. When the antigen is large, the antibody may bind only a specific portion of the antigen, which portion is referred to as an epitope.

Antigen binding domains

The antigen binding domain is a portion of a specific binding member that includes an antigen binding site and binds a target antigen. In certain embodiments, the antigen binding domain may be provided by one or more antibody variable domains (e.g., so-called Fd antibody fragments consisting of VH domains) or antigen binding portions thereof. In certain embodiments, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

Specific binding members may be glycosylated, either naturally or by various eukaryotic cell systems (e.g., CHO or NS0(ECACC85110503) cells), or they may be unglycosylated (e.g., if produced by expression in prokaryotic cells). Glycosylation can also be intentionally altered, for example by inhibiting fucosylation, in order to increase ADCC activity of the resulting antibody. Thus, any of the specific binding members of the invention may be expressed so as to minimise or eliminate fucosylation.

In certain embodiments, a CDR or VH or VL domain of the invention will be identical or highly similar to the designated region whose sequence is listed herein. It is contemplated that 1-5, preferably 1-4 or 1 or 2, or 3 or 4 amino acid substitutions may be made in the CDR and/or VH or VL domain. VH or VL domains and CDRs and sets of CDRs that are highly similar to those whose sequences are given herein are encompassed by aspects of the invention as are sequences substantially as those listed herein.

The structures used to carry the CDRs or sets of CDRs of the present invention will generally have an antibody heavy or light chain sequence or a substantial portion thereof in which the CDRs or sets of CDRs are located at positions corresponding to the CDRs or sets of CDRs of the naturally occurring VH and VL antibody variable domains encoded by the rearranged immunoglobulin genes. The structure and position of immunoglobulin variable domains can be determined by reference to Kabat, E.A. et al (Sequences of Proteins of immunological interest, fourth edition, US Department of Health and Human services, 1987) and its latest literature, which is now available on the Internet (http:// immunological. by. nwu. edu. or using any search engine for "Kabat"). CDRs are defined according to Kabat et al.

The CDRs may also be carried by other scaffolds such as fibronectin or cytochrome B.

Preferably, the CDR amino acid sequences substantially as set out herein are carried as CDRs in a human variable domain or a substantial part thereof. The HCDR3 sequence as substantially set out herein represents a preferred embodiment of the invention and preferably carries each of these as HCDR3 in a human heavy chain variable domain or substantial part thereof.

The variable domains employed in the present invention may be obtained or derived from any germline or rearranged human variable domain or may be synthetic variable domains based on consensus or actual sequences of known human variable domains. The CDR sequences of the invention (e.g., CDR3) can be introduced into a CDR-deficient (e.g., CDR3) variable domain repertoire using recombinant DNA techniques. Preferred germline frameworks have been identified herein.

For example, Marks et al (Bio/Technology, 1992,10: 779-783) describes a method for generating antibody variable domain repertoires in which consensus primers (consensus primers) directed to or adjacent to the 5' end of the variable domain region are directed against the human VH geneThe consensus primers of framework region 3 of (a) were used in combination to provide a VK variable domain repertoire lacking CDR 2. Marks et al further describe how this repertoire can be combined with the CDR2 of a particular antibody. Using similar techniques, the CDR 3-derived sequences of the invention may be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains combined with cognate (cognate) VL or VH domains to provide specific binding members of the invention. The library can then be displayed in a suitable host system, such as the phage display system of WO92/01047, or any of a number of subsequent references, including Kay, B.K., Winter, J., and McCafferty, J. (1996)Phage Display of Peptides andProteins：A Laboratory ManualSan Diego: academic Press, thereby allowing selection of an appropriate specific binding member. The reservoir may be made up of 10⁴Consisting of more than one single member, e.g. 10⁶To 10⁸Or 10¹⁰And (4) each member. Other suitable host systems include yeast display, bacterial display, T7 display, ribosome display, covalent display, and the like.

A similar shuffling or combining technique is also disclosed by Stemmer (Nature, 1994, 370: 389-391), who describes a technique related to the β -lactamase gene, but reviews that this approach can be used to generate antibodies.

A further alternative is to generate novel VH or VL regions carrying CDR-derived sequences of the invention, wherein random mutagenesis of one or more selected VH and/or VL genes is used to generate mutations within the entire variable domain. Such techniques are described by Gram et al (1992, proc.natl.acad.sci., USA,89: 3576-3580), they use error-prone PCR. In preferred embodiments, 1 or 2 amino acid substitutions are made in the HCDRs and/or LCDRs panels.

Another method that may be used is to direct mutagenesis to the CDR regions of the VH or VL genes. Such techniques are described by Barbas et al (1994, proc.natl.acad.sci., USA,91: 3809-.263: 551-567).

All the above techniques are known per se in the art and do not form part of the present invention per se. Given the disclosure provided herein, the skilled person will be able to use such techniques to provide specific binding members of the invention by using routine methods in the art.

A further aspect of the invention provides a method for obtaining an antigen binding site of an antibody specific for a TGF β antigen, the method comprising providing a VH domain which is an amino acid sequence variant of said VH domain by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of a VH domain as set out herein, optionally combining the VH domain so provided with one or more VL domains, and testing the VH domain or VH/VL combination to identify a specific binding member or antigen binding domain which is specific for TGF β and optionally has one or more preferred properties, preferably the ability to neutralise TGF β activity, said VL domain may have an amino acid sequence substantially as set out herein.

Similar approaches may be employed wherein one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

In a preferred embodiment, the PET1073G12, PET1074B9 or PET1287a10VH domains may be mutated to provide one or more VH domain amino acid sequence variants, which may be combined with one or more VL domains.

A further aspect of the invention provides a method for making a specific binding member specific for all 3 isoforms of human TGF β, said method comprising:

(a) providing a starting repertoire of nucleic acids encoding a VH domain that includes a CDR3 to be replaced or lacks a CDR3 encoding region;

(b) combining the depot with a donor nucleic acid encoding an amino acid sequence substantially as set out herein for HCDR3 such that the donor nucleic acid is inserted within the CDR3 region of the depot so as to provide a product depot of nucleic acid encoding a VH domain;

(c) expressing the nucleic acid of the product reservoir;

(d) selecting a specific binding member specific for at least one TGF- β isoform, and

(e) recovering the specific binding member or the nucleic acid encoding it.

The method may further comprise the step of performing binding and neutralisation assays with each of the 3 TGF β isoforms to identify specific binding members that bind to and neutralise all 3 isoforms.

Furthermore, a similar approach may be taken wherein the LCDR3 of the invention is combined with a repertoire of nucleic acids encoding a VL domain that includes the CDR3 to be replaced or lacks the CDR3 encoding region.

Similarly, one or more, or all 3 CDRs may be grafted into a repertoire of VH or VL domains, which are then screened for specific binding members specific for all human TGF β isoforms.

The VH domain may have a germline sequence and in one preferred embodiment is DP-10 or DP-88. The VL domain sequence may have a germline sequence and in a preferred embodiment is DPK-22.

In a preferred embodiment, one or more of PET1073G12, PET1074B9 or PET1287a10HCDR1, HCDR2 and HCDR3, or the HCDRs group of PET1073G12, PET1074B9 or PET1287a10, and/or one or more of PET1073G12, PET1074B9 or PET1287a10LCDR1, LCDR2 and LCDR3, or the LCDRs group of PET1073G12, PET1074B9 or PET1287a10 may be employed.

A substantial portion of an immunoglobulin variable domain will comprise at least the 3 CDR regions, along with their intervening framework regions. Preferably, the portion will further comprise at least about 50% of either or both of the 1 st and 4 th framework regions, the 50% being the C-terminal 50% of the 1 st framework region and the N-terminal 50% of the 4 th framework region. Additional residues at the N-terminus or C-terminus of the essential part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of a specific binding member of the invention 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 link the variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein tags, as discussed in more detail elsewhere herein.

Although in a preferred aspect of the invention, specific binding members comprising a VH and VL domain pair are preferred, a single binding domain based on VH or VL domain sequences constitutes a further aspect of the invention. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner.

In the case of either single specific binding member, these domains may be used to screen for complementary domains capable of forming a two-domain specific binding member capable of binding to 3 isoforms of human TGF β.

This can be done by phage display screening methods using the so-called stepwise dual combinatorial approach as disclosed in WO92/01047, in which a single colony comprising an H or L chain clone is used to infect a complete clone library encoding the other chain (L or H), and the resulting two-chain specific binding members are selected according to phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al (supra).

Specific binding members of the invention may further comprise an antibody constant region or portion thereof. For example, the VL domain may be attached at its C-terminus to an antibody light chain constant domain, including human C_κOr C_λChains, preferably C_κAnd (3) a chain. Similarly, a specific binding member based on a VH domain may be attached at its C-terminus to all or part of an immunoglobulin heavy chain (e.g. a CH1 domain) derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM, and any of the subclasses of isotypes, preferably IgG1 and IgG 4. IgG4 is preferred. IgG4 is preferred for some applications because it does not bind complement and does not produce effector functions. IgG1 is preferred when effector function is desired. Effector function can also be increased by manipulating the glycosylation state of the antibody, for example, by reducing fucose content, by employing methods known in the art. The heavy chain may or may not have a C-terminal lysine residue. Any synthetic or other constant region variant that possesses these properties and stabilizes the variable region is also preferred for use in embodiments of the invention.

Also within the present invention are heterogeneous preparations of the specific binding members, or antigen-binding fragments thereof, disclosed herein. For example, such preparations may be mixtures of antibodies having full-length heavy chains and heavy chains lacking C-terminal lysine, having various degrees of glycosylation, having derivatized amino acids (e.g., N-terminal glutamic acid cyclized to form pyroglutamic acid residues), and/or having deamidated forms of the heavy and/or light chains.

Specific binding members of the invention may be labelled with a detectable or functional label. Detectable labels include radioactive labels, e.g.¹³¹I or⁹⁹TCs, which may be attached to the 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 can be detected via binding to a specific cognate detectable moiety, such as labeled avidin.

The specific binding members of the invention are designed for use in a method of diagnosis or treatment of a human or animal subject, preferably a human.

In some embodiments, the specific binding members of the invention inhibit binding of TGF β, 2 and/or 3 to a cell surface TGF β receptor or receptor complex (including, but not limited to, complexes comprising receptor serine/threonine kinase type I or type II and proteoglycan β -glycan (TGF β type III receptor)), thus, the invention includes methods for inhibiting binding of TGF β to a cell surface TGF β receptor or receptor complex, including the steps of contacting TGF β with a specific binding member of the invention and detecting inhibition of binding to the receptor or receptor complex.

Thus, further aspects of the invention provide methods of treatment comprising administration of a specific binding member provided, pharmaceutical compositions comprising such specific binding members, and the use of such specific binding members in the manufacture of a medicament for administration, for example in a method of manufacturing a medicament or pharmaceutical composition, the method comprising formulating the specific binding member with a pharmaceutically acceptable excipient.

Specific binding members of the invention may be administered by injection (e.g. subcutaneously, intravenously, intraluminally (e.g. after tumour resection), intralesionally, intraperitoneally or intramuscularly), by inhalation, or topically (e.g. intraocularly, intranasally, rectally, intralesionally, dermally) or orally. The route of administration may be determined by the physicochemical characteristics of the product, by special consideration of the disease, by the dosage or dosing interval, or by the requirements for optimal efficacy or minimization of side effects.

It is envisaged that anti-TGF β treatment will not be limited to administration by a health care professional therefore, subcutaneous injection, especially using a needle-free device, may be appropriate.

According to the present invention, the provided compositions can be administered to an individual in need thereof. Administration is preferably in a "therapeutically effective amount", which is sufficient to show benefit to the patient. Such benefit may be at least amelioration of at least one symptom of a particular disease or disorder. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. The prescription of treatment, e.g., decisions regarding dosage, etc., can be determined based on preclinical and clinical studies, the design of which is well within the level of skill in the art.

The precise dose will depend on many factors, including whether the antibody is for diagnosis or treatment, the size and location of the area to be treated, 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. Typical antibody doses will be 100 μ g-1 mg for systemic applications and 1 μ g-1 mg for topical applications. Generally, the antibody will be a whole antibody, preferably of the IgG4 isotype. This is a single therapeutic dose for adult patients, which can be scaled up for children and infants, and can also be scaled up for other antibody formats by molecular weight and activity. Treatment may be repeated once a day, twice a week, once a month, or at other intervals, as determined by the physician. In a preferred embodiment of the invention, the treatment is periodic and the period between administrations is about 2 weeks or more, preferably about 3 weeks or more, more preferably about 4 weeks or more, or about once a month.

The specific binding members of the invention will generally be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.

Thus, the pharmaceutical compositions according to the invention and for use according to the invention may contain, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers 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. Such materials can include, for example, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and physiologically compatible substances, and the like. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Further examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering substances which enhance the shelf life or effectiveness of the antibody. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, topical, by inhalation or by injection, e.g. intravenously. In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids, for example containing an inert diluent or an assimilable edible carrier. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions typically comprise a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Physiological saline solution, glucose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. The specific binding member (and other ingredients, if desired) may also be encapsulated in a hard or soft shell gelatin capsule, compressed into a tablet, or incorporated directly into the diet of the subject. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets (ingestribletables), buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In order to administer the compounds of the present invention by means other than parenteral administration, it may be necessary to coat the compound with or co-administer the compound with a material that prevents its inactivation.

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 a suitable pK, isotonicity and stability. Those skilled in the art will be well able to prepare suitable solutions, for example, using an isotonic vehicle such as sodium chloride injection, ringer's injection, lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired.

The compositions may be administered simultaneously or sequentially, either alone or in combination with other treatments, depending on the condition being treated.

Specific binding members of the invention may be formulated in liquid, semi-solid or solid forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the desired form of administration, therapeutic application, physicochemical properties of the molecule and the route of delivery. The formulation may comprise an excipient, or a combination of excipients, such as: sugars, amino acids, and surfactants. Liquid formulations may contain a wide range of antibody concentrations and pH. Solid formulations can be produced, for example, by lyophilization, spray drying, or drying via supercritical fluid techniques.

In general, therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by: the required amount of specific binding member in a suitable solvent is admixed with one or a combination of ingredients as listed above, if desired, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained by: for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the compositions agents delaying absorption, for example, monostearate salts and gelatin.

In certain embodiments, the antibody composition active compound may be prepared with carriers that protect the antibody from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, e.g., Sustainated and Controlled Release Drug Delivery Systems (J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

As noted, such binding may occur in vivo, e.g., after administration of the specific binding member or nucleic acid encoding the specific binding member to a patient, or it may occur in vitro, e.g., in an ELISA, Western blot, immunocytochemistry, immunoprecipitation, affinity chromatography, or cell-based assay, or in an ex vivo-based therapeutic approach (e.g., one in which a cell or bodily fluid is contacted with a specific binding member according to the invention ex vivo and then administered to a patient).

The amount of specific binding member that binds to TGF β may be determined the quantitative result may be related to the amount of antigen in the test sample, which may be of diagnostic interest.

Also provided is a kit comprising a specific binding member or antibody molecule according to any aspect or embodiment of the invention, as an aspect of the invention. In the kits of the invention, the specific binding member or antibody molecule may be labelled to allow its reactivity in the sample to be determined, for example, as described further below. The components of the kit are generally sterile and in sealed vials or other containers. The kit may be employed in diagnostic assays or other methods in which antibody molecules are useful. The kit may comprise instructions for using the components in a method, such as a method according to the invention. Auxiliary materials that assist or enable the performance of such methods may be included in the kits of the invention.

The reactivity of the antibody in the sample may be determined by any suitable method. Radioimmunoassay (RIA) is one possibility. The radiolabeled antigen is mixed with unlabeled antigen (test sample) and allowed to bind to the antibody. The bound antigen is physically separated from the unbound antigen and the amount of radioactive antigen bound to the antibody is determined. The more antigen in the test sample, the less radioactive antigen is bound to the antibody. Competitive binding assays can also be used with non-radioactive antigens, using an antigen or analog linked to a reporter molecule. The reporter molecule may be a fluorescent dye, phosphor or laser dye having spectrally separated absorption or emission characteristics. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin or texas red. Suitable chromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal or particulate materials such as latex beads, which are colored, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause a detectable signal that can be observed by the naked eye, detected or recorded electronically. These molecules may be, for example, enzymes that catalyze reactions that produce or change color or that cause a change in electrical properties. They may be molecularly excitable such that electronic transitions between energy states result in characteristic spectral absorption or emission. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed. The signal generated by a single antibody-reporter conjugate can be used to derive quantifiable absolute or relative data of the relevant antibody binding in the sample (normal and test).

The invention also provides the use of a specific binding member as above for measuring the level of an antigen in a competition assay, that is to say a method of measuring the level of an antigen in a sample by employing a specific binding member as provided by the invention in a competition assay. This may be the case where physical separation of bound and unbound antigen is not necessary. It is a possibility to link a reporter molecule to a specific binding member such that upon binding a physical or optical change occurs. The reporter molecule may directly or indirectly generate a detectable and preferably measurable signal. The linkage of the reporter molecule may be direct or indirect, covalent (e.g., via a peptide bond) or non-covalent. Linkage via a peptide bond may be the result of recombinant expression of a gene fusion encoding the antibody and reporter molecule.

The invention also provides for direct measurement of antigen levels, for example in a biosensor system, by employing a specific binding member according to the invention.

The manner of determining binding is not a feature of the present invention and a person skilled in the art will be able to select a suitable manner according to his preference and general knowledge.

As noted, in various aspects and embodiments, the invention extends to a human, humanized, chimeric or synthetic specific binding member that competes for binding to TGF β (TGF β 1, 2 and/or 3) with any specific binding member defined herein, e.g. PET1037GR, PET1074B9 or ET1287a10IgG 4.

Competition may be determined using, for example, an ELISA in which TGF β is immobilised to a plate and a first labelled binding member (reference binding member) is added to the plate together with one or more other unlabelled binding members the presence of unlabelled binding member that competes with the labelled binding member is observed by a reduction in the signal emitted by the labelled binding member.

In competition assays, peptide fragments of the antigen, in particular peptides comprising the epitope of interest, may be employed. Peptides having an epitope sequence plus one or more amino acids at either end can be used. Such peptides may be said to "consist essentially of" the specified sequence. Specific binding members according to the invention may be such that their binding to an antigen is inhibited by a peptide having or including a given sequence. In the tests for this purpose, peptides with either sequence plus one or more amino acids can be used.

Specific binding members that bind to a particular peptide can be isolated from a phage display library, for example, by panning with the peptide.

The invention further provides an isolated nucleic acid encoding a specific binding member of the invention. The nucleic acid may comprise DNA and/or RNA. In a preferred aspect, the invention provides a nucleic acid encoding a CDR or set of CDRs of the invention as defined above, or an antibody antigen-binding site, or a VH domain or a VL domain, or an antibody molecule, for example scFv or IgG 4.

The invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one polynucleotide as above.

The invention also provides a recombinant host cell comprising one or more of the constructs as above. Nucleic acids encoding any of the CDRs or sets of CDRs or VH domains or VL domains or antigen binding sites provided or antibody molecules such as scFv or IgG4 themselves form an aspect of the invention, as do methods of producing the encoded products, including expression from nucleic acids encoding them. Expression may conveniently be effected by culturing a recombinant host cell comprising the nucleic acid under appropriate conditions. After production by expression, the VH or VL domain, or specific binding member, may be isolated and/or purified using any suitable technique, and then used as appropriate.

Specific binding members, VH and/or VL domains and encoding nucleic acid molecules and vectors according to the invention may be provided in isolated and/or purified form (e.g. from their natural environment) in substantially pure or homogeneous form, or in the case of nucleic acids, free or substantially free of other nucleic acids or gene sources than the sequence encoding a polypeptide having the desired function. The nucleic acids according to the invention may comprise DNA and RNA, or may be wholly or partially synthetic. When referring to the nucleotide sequences listed herein, DNA molecules having the specified sequences are encompassed, and RNA molecules having the specified sequences with U instead of T are encompassed, unless the context requires otherwise.

Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, insect cells, fungi, yeast and transgenic plants and animals. Mammalian cell lines useful in the art for the expression of heterologous polypeptides include Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retinal cells, and many others. A common, preferred bacterial host is E.coli.

Expression of antibodies and antibody fragments in prokaryotic cells such as E.coli is well established in the art for a review see, e.g., Pl ü ckthun, A.Bio/Technology9: 545-551(1991). Expression in cultured eukaryotic cells is known in the artThe skilled person is also available as an option for the production of specific binding members, e.g.Chadd HE and Chamow SM (2001)110Current Opinion in Biotechnology12: 188-; andersen DC and Krummen L (2002) Current Opinion in Biotechnology13: 117; larrick JW and Thomas DW (2001) Current Opinion in Biotechnology12：411-418。

Suitable vectors can be selected or constructed that contain suitable regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other suitable sequences. Suitably, the vector may be a plasmid, a virus such as a bacteriophage or phagemid, or an adenovirus, AAV, lentivirus, or the like. For further details, see, e.g.Molecular Cloning：A Laboratory ManualThird edition, Sambrook and Russell, 2001, Cold spring Harbor Laboratory Press. Many known techniques and procedures for manipulating nucleic acids, such as preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis are described inCurrent Protocols in Molecular BiologySecond edition, Ausubel et al (editor), John Wiley&Sons，1986；Short Protocols in Molecular Biology：A Compendium of Methods from Current Protocols in Molecular BiologyAusubel et al (editor), John Wiley&Sons, fourth edition, 1999. The disclosures of Sambrook et al and Ausubel et al (both) are incorporated herein by reference.

Accordingly, a further aspect of the invention provides a host cell comprising a nucleic acid as disclosed herein. Such host cells may be in vitro or may be cultured. Such host cells may be in vivo. The presence of the host cell in vivo may allow intracellular expression of a specific binding member of the invention as an "intrabody" or intrabody. Intrabodies can be used in Gene Therapy (Marasco WA (1997) Gene Therapy,4(1)：11)。

a still further aspect provides a method comprising the steps ofA method for introducing a 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 retroviruses or other viruses (e.g., vaccinia, or baculovirus for insect cells). Introduction of nucleic acids into host cells, particularly eukaryotic cells, viral systems or plasmid-based systems may be used. The plasmid system may be maintained episomally or may be introduced into host cells or artificial chromosomes (Csonka E et al (2000) Journal of Cell Science, 113: 3207-3216; Vanderbyl S et al (2002) Molecular Therapy,5(5): 10. the introduction may be a random or targeted integration of one or more copies at a single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using phage.

Introduction may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing the host cell under conditions for expression of the gene.

In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by inclusion of sequences that facilitate recombination with the genome according to standard techniques.

The invention also provides a method comprising using a construct as described above in an expression system to express a specific binding member or polypeptide as described above.

In a preferred embodiment, the precursor B cells are transfected or infected ex vivo and then transplanted into a patient in need thereof, in another embodiment, the precursor B cells or other cells are infected in vivo using a virus known to infect the cell type of interest.

In a more preferred method, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain of an anti-TGF β antibody or antigen-binding portion thereof and expressing said nucleic acid molecule.

Dose correction from species to species generally requires only adjustment of body weight if the active agent is an antibody that acts in or near the vascular system. In acute cases, the effective dose of the antibody of the invention is 0.5-5 mg/kg in rats and mice. Thus, for long-term dosing, administration of 0.3-10 mg/kg with a half-life (expected to be 21 days in humans) may be considered. The preferred dosage is sufficient for efficacy, but low enough to facilitate optimal administration. For example, a dose of less than 50mg is advantageous for subcutaneous administration. Intravenous administration is preferred in early clinical trials and can be used as a delivery route for severe disease if the dose is high and the dosing interval is long. Subcutaneous injection is generally more convenient than intravenous delivery because it allows self-administration. However, subcutaneous injection has the potential to increase any immune response against the product. For localized diseases, topical administration can minimize the amount of product required and maximize the concentration of the site of action. Significant safety (therapeutic window) advantages can be conferred by topical administration, avoiding any potential side effects that may develop from long-term systemic administration.

Further aspects and embodiments of the invention will be apparent to those skilled in the art in light of this disclosure, including the following experimental illustrations. All documents mentioned anywhere in this specification are incorporated by reference.

Example 1

Production of anti-TGF β ScFvs

ScFv naive (nave) antibody library

A large single chain fv (scFv) human antibody library obtained from spleen lymphocytes of 20 donors and cloned into a phagemid vector (Hutchings et al, 2001) was used for selection.

ScFv-directed selection libraries

A1 D11.16VH-human VL library was constructed and used to select mouse-human chimeric antibodies with the desired binding properties. The human light chains from these chimeric antibodies were then cloned into human VH-VL and human VH (1D11CDR3) -VL receptor libraries. These libraries are screened for human antibodies with the desired binding properties.

Selection of ScFv phage libraries

Recombinant human TGF β 1 and TGF β 2 were supplied by Genzyme Corp. (Framingham, MA), and TGF β 3 was purchased from R & DSystems.

Briefly, after incubation with the library, the immobilized antigen that has been pre-coupled to paramagnetic beads and bound to phage is recovered by magnetic separation while unbound phage is washed away.

The selection was made according to the manufacturer's recommendations using TGF β 1, TGF β 2, or TGF β 3 coupled to Dynabeads M-270 amine (Dynal.) alternatively, biotinylated TGF β 1 or TGF β 2 was selected for use, which was prepared according to the manufacturer's instructions (EZ linkNHS LC Biotin, Pierce) using the primary amine specific reagent succinimidyl-6- (biotinannido) hexanoate.

The output from the selection process was tested as a periplasmic preparation in a high throughput screening based on a competition assay that measures the ability of scFvs present in the periplasmic preparation to compete for binding to TGF β with 1D11.16 or recombinant human TGF β soluble receptor II-Fc chimeras (sRII, R & D Systems).

Samples that compete with 1D11.16 or sRII in high throughput screening were subjected to DNA sequencing as described in Vaughan et al (1996) and Osbourn et al (1996). clones were expressed and purified as scFvs or IgGs and their ability to neutralize TGF β s in MLEC and/or NHLF assays was evaluated as described in examples 4 and 5, respectively.A purified scFv preparation was prepared as described in example 3 of WO 01/66754. the protein concentration of the purified scFv preparation was determined using the BCA method (Pierce). A purified IgG preparation was prepared as described in example 3 below.

Example 2

Optimization of anti-TGF β scFvs

ScFvs generated to bind and neutralize TGF β as described in example 1 DNA mutagenesis and/or combinatorial techniques were used to increase the neutralizing potency of these antibodies to TGF β 1 and/or TGF β 2 and/or TGF β 3 antibodies significantly improved in potency to TGF β 1 and/or TGF β 2 and/or TGF β 3 were generated by selecting and screening phage antibody libraries as essentially described in example 1.

These are DP-10/1-69 and DP-88/1-e for the heavy chain (both members of the VH1 germline family), and DPK22/A27 for the light chain (V)_κFamily 3) these lines appear to provide a structural framework particularly suited for highly potent TGF β neutralizing pan antibodies, which is unpredictable because the 1D11.16VH gene segment is closest to human germline DP-7 and the 1D11.16VL gene segment is closest to human germline L16.

In the MLEC proliferation assay, PET1073G12, PET1074B9 and PET1287a10scFvs showed potencies that approached or exceeded the potencies of 1D11.16 for all 3 TGF β isoforms.

The derived amino acid sequences of the PET1073G12, PET1074B9 and PET1287a10VH and VL gene segments were aligned with the known human germline sequences in the VBASE database (Tomlinson et al, 1997) and the closest human germline identified by sequence similarity. The closest human germline gene for the VH gene segment of PET1073G12 and PET1074B9 was identified as DP-10/1-69(VH1 germline family) and the closest human germline gene for the VH gene segment of PET1287A10 was identified as DP-88/1-e (VH1 germline family). The closest human germline gene for the VL gene segments of PET1073G12, PET1074B9 and PET1287A10 was identified as DPK22/A27 (V)_κ3 germline families) framework residues that differ from the germline are changed to germline residues using site-directed mutagenesis, provided that such changes do not cause the resulting antibody to lose more than 3-fold the potency of any TGF β isoform in the MLEC proliferation assay.

In germlined PET1073G12 and germlined PET1074B9, all framework residues are germlined except for two residues in VH and one residue in VL. For the amino acid sequence of germlined PET1073G12, the amino acid sequence for VH is set forth in seq id NO: 2, and as described in SEQ ID NO: 7. For the amino acid sequence of germlined PET1074B9, the amino acid sequence in SEQ ID NO: 12, and as described in SEQ ID NO: 17.

In germlined PET1287a10, all VH and VL framework residues are germlined. For the amino acid sequence of germlined PET1287a10, the amino acid sequence in SEQ ID NO: 22, and the amino acid sequence depicted in SEQ ID NO: 27 is described.

Example 3

Production of IgG4s

Germlined scFvs PET1073G12, PET1074B9 and PET1287a10 were converted from the scFv format to the IgG4 format by subcloning their VH and VL domains into vectors expressing the complete antibody heavy and light chains, respectively. VH gene segments were amplified from pCantab6, a vector expressing scFv, and cloned into the peu8.1(+) vector containing the human γ 4 heavy chain constant domain and regulatory elements to express the entire heavy chain in mammalian cells. Similarly, VL gene segments were amplified from pCantab6, a scFv expressing vector, and cloned into the peu3.1(-) vector containing the human kappa light chain constant domain and regulatory elements to express the entire light chain in mammalian cells. pEU3.l (-) and pEU8.1(+) vectors are based on the vectors described by Persic et al (1987) and modified to introduce an oriP sequence to increase the yield of antibody produced (Shen. et al, 1995; Langle-Rouuult et al, 1998). After cloning, the VH and VL domains of all 3 antibodies were sequenced to confirm that no mutations were introduced during cloning.

Vectors for expressing PET1073G12, PET1074B9 and PET1287a10 heavy and light chains were transfected into EBNA-293 cells (Invitrogen). After gene expression and secretion in cell supernatants, PET1073G12, PET1074B9 and PET1287a10IgG4 were purified by protein a affinity chromatography (Amersham). Purified antibody preparations were sterile filtered and stored in Phosphate Buffered Saline (PBS) at 4 ℃ prior to evaluation. IgG concentrations were determined spectrophotometrically using extinction coefficients based on IgG amino acid sequences as described in Mach et al (1992). Purified IgG was analyzed by SEC-HPLC using a Biosep-SEC-S2000 column (Phenomenex) to check for aggregation or degradation of the protein. Reformatted human IgG4 whole antibody was compared to the 1D11.16 antibody in MLEC and NHLF cell-based assays as described in examples 4 and 5, respectively.

Example 4

Neutralizing potency of anti-TGF β antibodies in a TGF β dependent MLEC proliferation assay

The neutralizing efficacy of purified antibody preparations against the biological activity of human TGF β was evaluated using a Mink Lung Epithelial Cell (MLEC) proliferation assay.

The MLEC proliferation assay is based on the assay described by Danielpour et al (1989 a.) this assay works on the principle that when TGF β, TGF β or TGF β 3 is added to mink lung epithelial cells, this results in the inhibition of serum-induced cell proliferation³H]Thymidine uptake to measure proliferation. The potency of an antibody is defined as 50% (IC)₅₀) In nM, neutralizes the antibody concentration of TGF β 1, TGF β 2 or TGF β 3 at a single concentration.

MLEC proliferation assay protocol

MLEC bed board

The MLEC line was obtained from the American type culture Collection (Cat. # CCL-64). Cells were grown in minimal essential medium (MEM, Gibco) containing 10% fbs (Gibco), 1% penicillin/streptomycin (Gibco), and 1% MEM nonessential amino acid solution (Gibco). Confluent cells from T-175 flasks were detached from the flasks, spun down, washed, and resuspended in MLEC assay medium made from MEM containing 1% FBS, 1% penicillin/streptomycin, and 1% MEM non-essential amino acid solutions. Then, the cells are culturedAliquots were labeled with trypan blue, counted on a hemocytometer, and cell stocks diluted to 1.75 × 10 using assay media⁵Cells/ml. 100 μ l of this suspension was added to each well of a tissue culture flat bottom 96 well plate and incubated for 3-5 hours.

Preparation of TGF β/antibody solutions

Working solutions of 6ng/ml (6 times the final assay concentration) of TGF β 1, TGF β 2, or TGF β 3, and 3 times the final maximum assay concentration of antibody (including controls such as 1D11.16) were prepared in MLEC assay medium the final concentration of TGF β (1ng/ml or 40pM) in the assay corresponded to a concentration that induced approximately 80% inhibition of cell proliferation (i.e., EC) compared to the control without TGF β₈₀Value).

Establishment of dilution plates

Samples of test and control antibodies were titrated in MLEC assay media in 3-fold dilution steps and incubated in the presence or absence of TGF β 1, TGF β 2 or TGF β 3 all relevant controls were included in each experiment, test 1D11.16 and/or the appropriate reference antibody and TGF β 1, TGF β 2 or TGF β 3 titrations were performed, the completed plates were placed in a humidified tissue culture incubator for 1 hour ± 15 minutes.

Addition of TGF β/antibody solution to plated cells

After the appropriate incubation time, 100 μ Ι from each well of the dilution plate was transferred to plated MLEC and the plate was returned to the incubator 44 ± 2 hours.

³Addition of [ H]-thymidine

To each well was added 25. mu.l of 10. mu. Ci/ml [ [ solution ] ]³H]Thymidine (diluted in PBS) (0.25. mu. Ci/cell). The plate was then returned to the incubator for 4 hours ± 30 minutes.

Cell harvesting

To each well 100 μ L trypsin-EDTA (0.25%, Gibco) was added, the plates were incubated in an incubator for 10 minutes, and cells were harvested using Tomtec or Packard96 well cell harvesters.

Data accumulation and analysis

The data from the harvested cells were read using β -plate reader (TopCount, Packard.) the data were analyzed to obtain IC₅₀And a standard deviation value. IC was obtained using prism2.0(GraphPad) software₅₀The value is obtained.

Results

Purified PET1073G12, PET1074B9 and FET1287a10 germlined IgG4s were tested in MLEC proliferation assay together with 1D 11.16. IgG4s was produced as described in example 3. IC (integrated circuit)₅₀Arithmetic mean ± standard deviation (where IC₅₀The concentration of antibody required to neutralize 50% 40pM TGF β 1, TGF β 2, or TGF β 3) is shown in table 1.

IC for PET1073G12 and PET1287A10IgG4s₅₀The mean data shows that these antibodies have a potency similar or close to that of 1D11.16 for TGF β 1, TGF β 2 or TGF β 3.

IC₅₀The mean data suggest that PET1074B9IgG4 is significantly more potent for TGF β 1 (although a complete dose response curve was not obtained in the MLEC assay.) as a means for comparison, 1D11.16 showed 12% neutralization of TGF β 1 at a concentration of 91pM and PET1074B9 showed 78% neutralization at a similar concentration of 92pM furthermore, PET1074B9 was also tested in a normal human pulmonary fibroblast (NHLF) fibronectin production assay with 1D11.16 (example 5). the results obtained in the NHLF assay confirm those obtained in the MLEC assay that PET1074B9 has potency similar to that of 1D11.16 for TGF β 2 and TGF β 3, and PET1074B9 is more potent for TGF β 1 than 1D 10711.16.

Example 5

anti-TGF β antibodies in a TGF β 3-dependent NHLF cell assayNeutralizing effect of

TGF β s is a potent stimulator of fibronectin production in cultured fibroblasts (Ignotz and Massaoue, 1986), exerting its effect via activation of the c-Jun N-terminal kinase pathway (Hocevar et al, 1999).

NHLF cell assay protocol

NHLF cells from Clonetics^TMObtained and contained 5% C0 at 37 ℃₂Is maintained in complete fibroblast growth medium-2 (FGM-2). At 90-100% confluence, fibroblasts were plated in 1.5ml FGM-2 medium (1.5X 10)⁵Per well, 24 well format) and allowed to adhere for 24 hours at 37 ℃. Cells were washed with serum-free Fibroblast Basal Medium (FBM) and serum starved overnight in 1.5ml FBM supplemented with human insulin (100. mu.g/ml), gentamicin/amphotericin (50. mu.g/ml) and ascorbic acid (50. mu.g/ml) and incubated at 37 ℃ for 24 hours. All experiments were performed on cells between passages 3-6.

Preparation of TGF β and antibody solutions

Working solutions of 25ng/ml (1nM) of TGF β 1, TGF β 2, or TGF β 3, as well as working solutions of antibodies (including controls such as 1D11.16) are prepared in assay media the final concentration of TGF β (250pg/ml or 10pM) in the assay corresponds to a concentration that induces stimulation of approximately 80% fibronectin production (i.e., EC) compared to the control without TGF β₈₀Value).

Establishment of dilution plates

Samples of test and control antibodies were serially diluted in assay media in 10-fold dilution steps and preincubated for 30 minutes in the presence or absence of TGF β 1, TGF β 2, or TGF β 332 NHLF cells were incubated in 2 ml/well assay media at 37 ℃ for 48 hours after which 0.5ml aliquots of the media supernatant were taken for fibronectin analysis by ELISA.

Human fibronectin ELISA

Using Technoclone^TMFresh or frozen (-20 ℃) NHLF supernatant samples were analyzed by a human fibronectin antigen ELISA kit comprising a human anti-fibronectin capture monoclonal antibody (clone 6FN) and a HRP-conjugated monoclonal anti-fibronectin secondary antibody. The method comprises the following steps:

Nunc-Immuno^TMMaxisorp^TM96-well plates were used in coating buffer (12 mM Na in distilled water)₂CO₃、35mM NaHC0₃Anti-fibronectin capture antibody (1. mu.g/well) in 0.01% (w/v) thimerosal, pH9.6) was coated for 16 hours at 4 ℃. The capture antibody was removed and each well blocked with 100. mu.l dilution buffer (1% (w/v) BSA in PBS) for 1 hour at 37 ℃. After 3 washes with wash buffer (250 μ g/well 0.5% (v/v) Tween20 in PBS), human plasma fibronectin standards and samples were added to the plates and incubated for 1 hour at 37 ℃. The plates were then washed 3 times and incubated with an anti-fibronectin HRP secondary antibody (100 μ g/well in dilution buffer) for 30 minutes at 37 ℃. The plate was washed 3 times with wash buffer and 100. mu.g/well of Tetramethylbenzidine (TMB) substrate was added to the plate. After incubation of the plates at room temperature for 20 minutes, the reaction was stopped with 100. mu.g/well of 2M sulfuric acid. The absorbance at 450nm was then measured using a Dynex MRX plate reader.

Data analysis

Data are provided as a percentage of control response to TGF β isoforms under test (100%). pIC50 geometric means and 95% confidence limits were evaluated using four-parameter logistic curve fitting (Prism2, GraphPad Software, San Diego, USA).

Results

Purified PET1073G12, PET1074B9 and PET1287a10 germlined IgG4s were tested in the NHLF fibronectin production assay together with 1D 11.16. IgG4s was produced as described in example 3. IC (integrated circuit)₅₀Arithmetic mean ± standard deviation (where IC₅₀The antibody concentration required to neutralize 50% 10pM TGF β 1, TGF β 2, or TGF β 3) is shown in table 2.

Example 6

Potency in IL-11 Induction assay

We used an a549 cell (human lung epithelial cancer cell) IL-11 induction assay to evaluate the neutralizing potency of purified antibody preparations against the biological activity of human TGF β.

We maintained A549 cells (ATCC, Part #: CCL-185) in growth/assay medium (435mL DMEM, 50mL Fetal Bovine Serum (FBS), 5mL penicillin/streptomycin, 5mL modified Eagle medium non-essential amino acids, 5mL100X L-glutamine; all from Gibco/Invitrogen). At approximately 90% confluence, we plated the cells in 100. mu.L growth/assay medium and let the cells stand at 37 ℃ with 5% CO₂The wall was attached for 24 hours.

Preparation of TGF β and antibody solutions

We prepared working solutions of TGF β 1(1.8ng/ml), TGF β 2(4.2ng/ml) or TGF β 3(4.2ng/ml) and antibodies (including controls) in growth/assay media.

Establishment of dilution plates

We serially diluted test and control antibody samples in growth/assay media in 5-fold (TGF β 2 or TGF β 3) or 10-fold (TGF β 1) dilution steps and preincubated for 75 minutes at 37 ℃ in the presence or absence of TGF β 1, TGF β 2 or TGF β 3, We incubated A549 cells in 200. mu.l/well assay media at 37 ℃ for 18-24 hours, after 18-24 hours, 100. mu.l aliquots of the culture supernatant were taken for IL-11 analysis by ELISA.

Example 7

To determine the biological efficacy of the human pan-neutralizing (pan-neutralizing) TGF- β monoclonal antibody for the treatment of chronic kidney disease characterized by pathogenic fibrosis and other clinical indications, we investigated the effect of the antibody in a rat Unilateral Urethral Obstruction (UUO) model.

Adult Sprague Dawley rats weighing 250-280 g (about 6 weeks) (Tastic Farms, Germantown, NY) were housed in an environment of controlled air, temperature and light. Rats undergoing UUO received a lower abdominal midline laparotomy to expose the left kidney and upper ureters. We ligated the ureters with silk suture at the lower end of the kidney and a second ligation was performed approximately 0.2cm below the first. Sham operated rats received the same surgical protocol but no ureteral ligation.

We administered the antibody intraperitoneally to rats starting on the day of ureteral ligation, with a course of 3 weeks, 13C4 and 1D11 at 5mg/kg (3 times per week), and the human pan-neutralizing antibody was given to rats at 5mg/kg (every 5 days). at the end of 3 weeks, we sacrificed the rats, perfused the kidneys with PBS for 3 minutes, and harvested perfused kidneys for analysis of mRNA, determination of collagen content, and histological examination.

To assess the extent of tissue fibrosis, we determined the total content of tissue collagen by biochemical analysis of hydroxyproline in hydrolyzed extracts as described by Kivirikko et al. The assay is based on the observation that essentially all hydroxyproline in animal tissue is found in collagen.

We also performed the Sircol collagen assay for total collagen content. The Sircol collagen assay measures the amount of total acid/pepsin soluble collagen based on the specific binding of sirius red dye to tissue collagen side chains.

UUO rats treated with human pan-neutralizing monoclonal antibody showed a 43.4% decrease in hydroxyproline content (1.98. + -. 0.26. mu.g/mg dry tissue) when compared to the PBS-treated group (3.5. + -. 0.3. mu.g/mg dry tissue, p < 0.05). The reduction in total solubilized collagen in the affected kidneys was further supportive of the reduction in renal fibrosis as determined by a Sirius red dye-based assay (sham: 18.5 ± 2.6, PBS: 69.3 ± 3.8, and human pan-neutralizing monoclonal antibody: 35.6 ± 5.2 μ g/100mg tissue, p <0.05 vs PBS).

We also evaluated the ability of human pan-neutralizing anti-TGF- β monoclonal antibodies to reduce tissue fibrosis by immunohistochemical examination.

In control animals, urethral blockage for 3 weeks resulted in extensive destruction of the tubular structure of the kidney, with significant swelling, cell atrophy and necrosis/apoptosis, tissue inflammation and tubulointerstitial dilation (with significant fibrosis). There is little evidence for glomerular damage. On the other hand, rats treated with 1D11 or human pan-neutralizing monoclonal antibody showed preservation of renal structures as judged by impaired tubular enlargement and disintegration, reduced inflammatory infiltrate (cell formation) and reduced tubulointerstitial dilation and fibrosis.

We also measured the effect of treatment with a human pan-neutralizing anti-TGF- β monoclonal antibody on gene expression regulated by TGF- β.

Significant reduction in TGF- β 1mRNA was seen in UUO animals treated with human pan-neutralizing monoclonal antibody compared to animals treated with PBS or with 13C4 control antibody a significant reduction in mRNA levels of type III collagen was also seen in occluded kidneys treated with human and murine anti-TGF- β antibodies compared to those treated with PBS or 13C4, indicating reduced collagen synthesis.

We further demonstrated the efficacy of the human pan-neutralizing anti-TGF- β monoclonal antibody in reducing self-induced TGF- β synthesis by measuring total renal TGF- β 1 protein.

The occluded kidney showed a significant increase in total tissue TGF- β 1 compared to sham operated animals whereas occluded rats dosed with human pan-neutralizing monoclonal antibody showed a 75% reduction in tissue TGF- β 1 levels, significantly lower than those recorded for the two control groups by comparison, murine 1D11 antibody reduced tissue TGF- β 1 levels by 45% compared to the control group.

The above results demonstrate that neutralization of TGF- β with a human pan-neutralizing anti-TGF- β monoclonal antibody effectively disrupts TGF- β autocrine-regulatory loop and concomitantly prevents TGF- β 1 production and collagen III mRNA expression.

We further determined the effect of human pan-neutralizing anti-TGF- β monoclonal antibodies on smooth muscle actin (α -SMA) expression as an indirect indicator of TGF- β inhibition.

We detected α -SMA protein by standard Western blot analysis.

Rats with occluded kidneys showed a significant up-regulation of α -SMA protein when compared to sham operated animals (data not shown) as measured by western blotting of tissue homogenates the occluded rats dosed with human pan-neutralizing anti-TGF- β monoclonal antibody showed a significant reduction in measurable α -SMA expression (75%, compared to PBS control).

These results demonstrate the efficacy of human pan-neutralizing anti-TGF- β monoclonal antibodies in reducing collagen deposition in fibrotic kidneys, clearly indicating that the antibodies are effective inhibitors of renal collagen production and deposition in this model of severe renal injury and tubulointerstitial fibrosis because the tissue fibrotic processes in organs such as the lung, liver or kidney share a common mechanism or pathway, the skilled artisan will understand that the antibodies can be used to treat chronic kidney disease characterized by pathogenic fibrosis as well as other clinical indications.

Certain preferred claims of the invention are described below.

SEQ ID NO1

Nucleotide sequence of coding PET1073G12VH

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAG

GTCTCCTGCAAGGCTTCTGGATACACCTTCAGTAGCAATGTTATCAGCTGGGTGCGC

CAGGCCCCTGGACAAGGGCTCGAGTGGATGGGGGGGGTCATCCCTATTGTTGATATT

GCGAACTACGCACAGAGATTCAAGGGCAGAGTCACGATTACCGCGGACGAATCCACT

AGTACAACTTACATGGAGTTGAGCAGCCTGAGGTCTGAGGACACGGCCGTGTATTAC

TGTGCGAGCACACTTGGTCTCGTCCTGGATGCTATGGACTACTGGGGTCAGGGTACG

TTGGTCACCGTCTCCTCA

SEQ ID NO2

Amino acid sequence of PET1073G12VH

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDI

ANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCASTLGLVLDAMDYWGQGT

LVTVSSSEQ ID NO3

Amino acid sequence of PET1073G12HCDR1

SNVIS

SEQ ID NO4

Amino acid sequence of PET1073G12HCDR2

GVIPIVDIANYAQRFKG

SEQ ID NO5

Amino acid sequence of PET1073G12HCDR3

TLGLVLDAMDY

SEQ ID NO6

Nucleotide sequence of coding PET1073G12VL

GAAACGGTACTCACGCAGTCTCCAGGTACCCTGTCTTTGTCTCCAGGGGAAAGAGCC

ACCCTCTCCTGCAGGGCCAGTCAGAGTCTTGGCAGCAGCTACTTAGCCTGGTATCAG

CAGAAACCTGGTCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCACCT

GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGTACCGACTTCACTCTCACCATC

AGCCGACTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATGCTGACTCA

CCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

SEQ ID NO7

Amino acid sequence of PET1073G12VL

ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAP

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIK

SEQ ID NO8

Amino acid sequence of PET1073G12LCDR1

RASQSLGSSYLA

SEQ ID NO9

Amino acid sequence of PET1073G12LCDR2

GASSRAP

SEQ ID NO10

Amino acid sequence of PET1073G12LCDR3

QQYADSPIT

SEQ ID NO11

Nucleotide sequence of coding PET1074B9VH

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAG

GTCTCCTGCAAGGCTTCTGGATACACCTTCAGTAGCAATGTTATCAGCTGGGTGCGC

CAGGCCCCTGGACAAGGGCTCGAGTGGATGGGGGGGGTCATCCCTATTGTTGATATT

GCGAACTACGCACAGAGATTCAAGGGCAGAGTCACGATTACCGCGGACGAATCCACT

AGTACAACTTACATGGAGTTGAGCAGCCTGAGGTCTGAGGACACGGCCGTGTATTAC

TGTGCGCTGCCACGCGCTTTCGTCCTGGATGCTATGGACTACTGGGGTCAGGGTACG

TTGGTGACCGTCTCCTCA

SEQ ID NO12

Amino acid sequence of PET1074B9VH

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDI

ANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGT

LVTVSS

SEQ ID NO13

Amino acid sequence of PET1074B9HCDR1

SNVIS

SEQ ID NO14

Amino acid sequence of PET1074B9HCDR2

GVIPIVDIANYAQRFKG

SEQ ID NO15

Amino acid sequence of PET1074B9HCDR3

PRAFVLDAMDY

SEQ ID NO16

Nucleotide sequence of coding PET1074B9VL

GAAACGGTACTCACGCAGTCTCCAGGTACCCTGTCTTTGTCTCCAGGGGAAAGAGCC

ACCCTCTCCTGCAGGGCCAGTCAGAGTCTTGGCAGCAGCTACTTAGCCTGGTATCAG

CAGAAACCTGGTCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCACCT

GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGTACCGACTTCACTCTCACCATC

AGCCGACTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATGCTGACTCA

CCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

SEQ ID NO17

Amino acid sequence of PET1074B9VL

ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAP

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIK

SEQ ID NO18

Amino acid sequence of PET1074B9LCDR1

RASQSLGSSYLA

SEQ ID NO19

Amino acid sequence of PET1074B9LCDR2

GASSRAP

SEQ ID NO20

Amino acid sequence of PET1074B9LCDR3

QQYADSPIT

SEQ ID NO21

Nucleotide sequence encoding PET1287A10VH

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAA

GTGTCCTGCAAGGCTTCTGGAGGCACCTTCAGCACCTCTTTCATCAATTGGGTGCGA

CAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGATATA

ACAAACTACGCACAGAAATTTCAGAGCAGAGTCACTATTACCCCGGACAAATCCACG

AGCACCGCCTACATGGAGCTGAGCAGCCTGCGCTCTGAGGACACGGCTGTGTATTAC

TGCGCACGCGGAAATGGTAACTACGCCCTGGATGCTATGGACTACTGGGGTCAGGGT

ACGTTGGTCACCGTCTCCTCA

SEQ ID NO22

Amino acid sequence of PET1287A10VH

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTSFINWVRQAPGQGLEWMGGIIPIFDI

TNYAQKFQSRVTITADKSTSTAYMELSSLRSEDTAVYYCARGNGNYALDAMDYWGQG

TLVTVSS

SEQ ID NO23

Amino acid sequence of PET1287A10HCDR1

TSFIN

SEQ ID NO24

Amino acid sequence of PET1287A10HCDR2

GIIPIFDITNYAQKFQS

SEQ ID NO25

Amino acid sequence of PET1287A10HCDR3

GNGNYALDAMDY

SEQ ID NO26

Nucleotide sequence encoding PET1287A10VL

GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC

ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTTGCCTGGTACCAG

CAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACT

GGCATCCCTGACAGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATC

AGCCGCCTGGAGCCTGAAGATTTCGCAGTTTATTACTGTCAGCAATATTATGATAGT

CCCATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

SEQ ID NO27

Amino acid sequence of PET1287A10VL

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYFAWYQQKPGQAPRLLIYGASSRAT

GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYYDSPITFGQGTRLEIK

SEQ ID NO28

Amino acid sequence of PET1287A10LCDR1

RASQSVSSSYFA

SEQ ID NO29

Amino acid sequence of PET1287A10LCDR2

GASSRAT

SEQ ID NO30

Amino acid sequence of PET1287A10LCDR3

QQYYDSPIT

SEQ ID NO31

WGQGTLVTVSS

Note: CDRs are defined according to Kabat et al (1991)

TABLE 1

The anti-proliferative effects of TGF β 1, TGF β 2 and TGF β 3 on MLEC were neutralized using IgG4s or 1D11.16 germlined PET1073G12, PET1074B9 or PET1287A10 the total number of data points for each mean is indicated by the number of n and represents an independent titration of each antibody, # IC₅₀Values cannot be determined because the antibody is too effective in the tested concentration range and a complete dose response curve is not obtained.

TABLE 2

Potency of PET1073G12, PET1074B9 or PET1287a 10-germlined IgG4s or 1D11.16 in NHLF assay. The total number of data points for each mean is indicated by the number of n and represents an independent titration for each antibody.

Claims (7)

1. Use of a monoclonal human antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of systemic sclerosis, wherein the antibody or antigen-binding fragment thereof binds to and neutralizes human TGF β 1, TGF β 2 and TGF β 3, and wherein the V of the antibody_HThe domain is in SEQ ID NO: 2, and V of said antibody_LThe domain is in SEQ ID NO: shown in fig. 7.

2. Use according to claim 1, wherein the systemic sclerosis is systemic scleroderma.

3. Use according to claim 1 or 2, wherein the antibody belongs to human IgG₄Isoform subtype.

4. Use according to claim 3, wherein the light chain of the antibody is of the human kappa type.

5. Use of a monoclonal human antibody or antigen-binding fragment thereof for the manufacture of a medicament for treating breast, prostate, ovarian, gastric, renal, pancreatic, colorectal, skin, lung, cervical, bladder, glioma, mesothelioma, leukemia or sarcoma, wherein the antibody or antigen-binding fragment thereof binds and neutralizes human TGF β 1, TGF β 2 and TGF β 3, and wherein the V of the antibody_HThe domain is in SEQ ID NO: 2, and V of said antibody_LThe domain is in SEQ ID NO: shown in fig. 7.

6. Use according to claim 5, wherein the antibody belongs to human IgG₄Isoform subtype.

7. Use according to claim 6, wherein the light chain of said antibody is of the human kappa type.