Specific binding members comprising human antibody antigen binding domains specific for human transforming growth factor beta (TGFβ) bind specifically isoforms TGFβ2 and TGFβ1 or both, preferentially compared with TGFβ3. Specific binding members may be isolated and utilized in the treatment of disease, particularly fibrotic disease and also immune/inflammatory diseases. Therapeutic utility is demonstrated using in vitro and in vivo models. Full sequence and binding information is provided, including epitope sequence information for particularly advantageous specific binding member which binds the active form of TGFβ2, neutralizing its activity, but does not bind the latent member.

This invention relates to specific binding members for human transforming growth factor (TGFβ) and materials and methods relating thereto. In particular, it relates to specific binding members comprising antibody binding domains; for example, human antibodies. Human antibodies against human TGFβ may be isolated and utilised in the treatment of disease, particularly fibrotic disease and also immune/inflammatory diseases. The isolation of antiself antibodies from antibody segment repertoires displayed on phage has been described (A. D. Griffiths et al. EMBO J. 12, 725-734, 1993; A. Nissim et al. EMBO J. 13, 692-698, 1994; A. D. Griffiths et al. 13, 3245-3260, 1994; C. Barbas et al. Proc. Natl. Acad. Sci. USA 90, 10003-10007 1993; WO93/11236). However, the present invention provides specific antibodies against human TGFβ and further against particular isoforms of TGFβ, which antibodies have unexpected and advantageous properties.
TGFβ is a cytokine known to be involved in many cellular processes such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis and immune and inflammatory responses (A. B. Roberts & M. Sporn 1990 pp419-472 in Handbook of Experimental Pharmacology eds M. B. Sporn & A. B. Roberts, Springer Heidelberg; J. Massague et al. Annual Rev. Cell Biol. 6, 597-646, 1990).
The accumulation of excessive extra-cellular matrix is associated with various fibrotic diseases. Thus there is a need to control agents such as TGFβ including TGFβ1 and TGFβ2 to prevent their deleterious effects in such diseases and this is one application of human antibodies to human TGFβ.
The modulation of immune and inflammatory responses by TGFbetas includes (i) inhibition of proliferation of all T-cell subsets (ii) inhibitory effects on proliferation and function of B lymphocytes (iii) down-regulation of natural-killer cell activity and the T-cell response (iv) regulation of cytokine production by immune cells (v) regulation of macrophage function and (vi) leucocyte recruitment and activation.
A further application of antibodies to TGFβ may be in the treatment of immune/inflammatory diseases such as rheumatoid arthritis, where these functions need to be controlled.
It is a demanding task to isolate an antibody fragment specific for TGFβ of the same species. Animals do not normally produce antibodies to self antigens, a phenomenon called tolerance (G. J. Nossal Science 245, 147-153, 1989). In general, vaccination with a self antigen does not result in production of circulating antibodies. It is therefore difficult to raise human antibodies to human self antigens. There are also in addition, ethical problems in vaccinating humans. In relation to the raising of non-human antibodies specific for TGFβ, there are a number of problems. TGFβ is an immunosuppressive molecule and further, there is strong conservation of sequence between human and mouse TGFβ molecules. Mouse and human TGFβ1 only differ by one amino acid residue, an alanine (human) to serine (mouse) change at a buried residue (R. Derynck et al. J. Biol. Chem. 261, 4377-4379, 1986). Mouse and human TGFβ2 only differ at three residues; residue 59 (T mouse, S human); residue 60 (K mouse, R human) and residue 94 (N mouse; K human). This makes it difficult to raise antibodies in mice against human TGFβ. Further, any antibodies raised may only be directed against a restricted set of epitopes.
Polyclonal antibodies binding to human TGFβ (human TGFβ1 and human TGFβ2) against both neutralising and non-neutralising epitopes have been raised in rabbit (Danielpour et al. Growth Factors 2 61-71, 1989; A. Roberts et al. Growth Factors 3, 277-286, 1990), chicken (R&D Systems, Minneapolis) and turkey (Danielpour et al. J. Cell Physiol. 138, 79-86, 1989). Peptides representing partial TGFβ sequences have also been used as immunogens to raise neutralising polyclonal antisera in rabbits (W. A Border et al. Nature 346, 371-374, 1990; K. C. Flanders Biochemistry 27, 739-746, 1988; K. C. Flanders et al, Growth Factors 3, 45-52, 1990). In addition there have been limited reports of isolation of mouse monoclonals against TGFβ. Following immunisation with bovine TGFβ2 (identical to human TGFβ2), three non-neutralising monoclonal antibodies were isolated that are specific for TGFβ2 and one neutralising antibody that is specific for TGFβ1 and TGFβ2 (J. R. Dasch et al. J. Immunol. 142, 1536-1541, 1989). In another report, following immunisation with human TGFβ1, neutralising antibodies were isolated which were either specific for TGFβ1 or cross-reacted with TGFβ1, TGFβ2 and TGFβ3 (C. Lucas et al. J. Immunol. 145, 1415-1422, 1990). A neutralising mouse monoclonal antibody which binds both TGFβ2 and TGFβ3 isoforms is available commercially from Genzyme Diagnostics.
The present specification discloses the first isolation of human antibodies directed against human TGFβ, including human TGFβ1 and human TGFβ2. A mouse monoclonal antibody directed against human TGFβ1 is available from R&D Systems. This antibody only weakly neutralises TGFβ1 in a neutralisation assay. Neutralising mouse monoclonal antibodies have also been generated from mice immunised with human TGFβ1 peptides comprising amino acid positions 48 to 60 (antibody reactive with TGFβ1, TGfβ2 and TGFβ3) and amino acid positions 86-101 (antibody specific for TGFβ1; M. Hoefer & F. A. Anderer Cancer Immunol. Immunother. 41, 302-308, 1995).
Phage antibody technology (WO92/01047; PCT/GB92/00883; PCT/GB92/01755; WO93/11236) offers the ability to isolate directly human antibodies against human TGFβ. In application WO93/11236 the isolation of antiself antibodies from phage display libraries was disclosed and it was suggested that antibodies specific for TGFβ could be isolated from phage display libraries.
The present application shows that antibodies of differing specificities for TGFβ molecules may be isolated. TGFβ1, TGFβ2 and TGFβ3 are a closely related group of cytokines. They are dimers consisting of two 112 amino acid monomers joined by an interchain disulphide bridge. TGFβ1 differs from TGFβ2 by 27 mainly conservative changes and from TGFβ3 by 22 mainly conservative changes. These differences have been related to the 3D structure (M. Schlunegger & M. G. Grutter Nature 358, 430-434, 1992). The present applicants have isolated inter alia antibodies which are essentially specific for TGFβ1 (very low cross-reactivity with TGFβ2); antibodies which are essentially specific for TGFβ2 (very low cross-reactivity TGFβ1); and antibodies which bind both TGFβ1 and TGFβ2. Hence, these three different types of antibodies, each type with distinctive binding specificities must recognise different epitopes on the TGFβ molecules. These antibodies have low cross-reactivity with TGFβ3 as assessed by binding studies using biosensor assays (e.g. BIACore™), ELISA and radioreceptor assays. The most extensively studied antibody, 6B1 IgG4, shows 9% cross-reactivity with TGFβ3 as compared with TGFβ2, as determined by their relative dissociation constants, determined using a biosensor.
TGFβ isoforms are initially exported from cells as inactive, latent forms (R. Pircher et al, Biochem. Biophys. Res. Commun. 136, 30-37, 1986; L. M. Wakefield et al., Growth Factors 1, 203-218, 1989). These inactive forms are activated by proteases in plasma to generate the active form of TGFβ. It is this active form of TGFβ2 which binds to receptors promoting the deposition of extracellular matrix and the other biological effects of TGFβ. The active form of TGFβ represents a relatively low proportion of TGFβ that is in the plasma. Therefore, for a neutralising antibody against TGFβ to be most effective at preventing fibrosis the antibody should recognise the active but not the latent form. In Example 6, it is demonstrated that a preferred antibody of this invention ("6B1 IgG4 ") recognises the active but not the latent form of TGFβ2.
The epitope of 6B1 IgG4 has been identified using a combination of peptide display libraries and inhibition studies using peptides from the region of TGFβ2 identified from phage selected from the peptide phage display library. This is described in Examples 11 and 14. The sequence identified from the peptide library is RVLSL (SEQ ID NO: 1) and represents amino acids 60 to 64 of TGFβ2 (Example 11). The antibody 6B1 IgG4 has also been shown to bind to a peptide corresponding to amino acids 56 to 69 of TGFβ2 (TQHSRVLSLYNTIN) (SEQ ID NO: 2) with a three amino acid (CGG) extension at the N-terminus. Although, RVLSL is the minimum epitope, 6B1 IgG4 is likely to bind to further adjacent amino acids. Indeed, if the epitope is three dimensional there may be other non-contiguous sequences to which the antibody will bind. 6B1 IgG4 shows much weaker binding to the peptide corresponding to amino acids 56 to 69 of TGFβ1 (CGGTQYSKVLSLYNQHN) (SEQ ID NO: 3).
The results of Example 14 support the assignment of the epitope of 6B1 IgG4 on TGFβ2 to the aminoacids in the region of residues 60 to 64. The peptide used in this example, residues 56 to 69, corresponds to the amino acids of alpha helix H3 (M. P. Schlunegger & M. G. Grutter Nature 358 430-434, 1992; also known as the α3 helix (S. Daopin et al Proteins: Structure, Function and Genetics 17 176-192, 1993). TGFβ2 forms a head-to-tail dimer with the alpha helix H3 (also referred to as the heel) of one subunit forming an interface with finger regions (including residues 24 to 37 and residues in the region of amino acids 91 to 95; also referred to as fingers 1 and 2) from the other subunit (S. Daopin et al supra). It has been proposed that the primary structural features which interact with the TGFβ2 receptor consist of amino acids at the C-terminal end of the alpha helix H3 from one chain together with residues of fingers 1 and 2 of the other chain (D. L. Griffith et al Proc. Natl. Acad. Sci. USA 93 878-883, 1996). The identification of an epitope for 6B1 IgG4 within the alpha helix H3 of TGFβ2 is consistent with 6B1 IgG4 preventing receptor binding and neutralising the biological activity of TGFβ2.
As noted above if the epitope for 6B1 IgG4 is three dimensional there may be other non-contiguous amino acids to which the antibody may bind.
There is earlier advice that antibodies directed against this region of TGFβ2 may be specific for TGFβ2 and neutralise its activity. Flanders et al (Development 113 183-191, 1991) showed that polyclonal antisera could be raised in rabbits against residues 50 to 75 of mature TGFβ2 and that these antibodies recognised TGFβ2 but the TGFβ1 in Western blots. In an earlier paper, K. C. Flanders et al (Biochemistry 27 739-746, 1988) showed that polyclonal antisera raised in rabbits against amino acids 50 to 75 of TGFβ1 could neutralise the biological activity of TGFβ1. The antibody isolated in this application 6B1 IgG4 is a human antibody directed against the amino acids in this region which neutralises the biological activity of human TGFβ2. It is surprising that such a neutralising antibody against TGFβ2 can be isolated in humans (where immunisation with a peptide cannot be used for ethical reasons) directly from a phage display antibody repertoire.
The knowledge that the residues of the alpha helix H3 form a neutralising epitope for TGFβ2 means that phage displaying neutralising antibodies are obtainable by selection from phage antibody repertoires by binding to a peptide from this region coupled to a carrier protein such as bovine serum albumin or keyhole limpet haemocyanin. This approach may be applied to select antibodies which are capable of neutralising the biological activity of TGFβ1 by selecting on the peptide TQYSKVLSLYNQHN (SEQ ID NO: 125) coupled to a carrier protein. It is possible that such an approach may be extended to peptides from receptor binding regions of TGFβ isoforms, other than the H3 alpha helix.
It has further been demonstrated by the present inventors that antibodies specific for TGFβ are obtainable by isolation from libraries derived from different sources of immunoglobulin genes: from repertoires of natural immunoglobulin variable domains, e.g. from immunised or non-immunised hosts; and synthetic repertoires derived from germline V genes combined with synthetic CDR3s. The properties of these antibodies in single chain Fv and whole IgG4 format are described.
As noted above WO93/11236 suggested that human antibodies directed against human TGFβ could be isolated from phage display libraries. Herein it is shown that the phage display libraries from which antiself antibodies were isolated in WO93/11236 may be utilised as a source of human antibodies specific for particular human TGFβ and TGFβ isoforms. For instance, in example 1 of the present application, the antibody 1A-E5 specific for TGFβ1 and the antibodies 2A-H11 and 2A-A9 specific for TGFβ2 were isolated from the "synthetic library" described in examples 5 to 7 of WO93/11236 and in Nissim et al. (1994; supra). Also, the phage display library derived from peripheral blood lymphocytes (PBLs) of an unimmunised human (examples 1 to 3 of WO93/11236) was the source for the antibody 1B2 specific for TGFβ1. Phage display libraries made subsequently utilising antibody genes derived from human tonsils and bone marrow, have also provided sources of antibodies specific for human TGFβ. Thus human TGFβ is an example of a human self antigen to which antibodies may be isolated from "large universal libraries". Human antibodies against human TGFβ with improved properties can be obtained by chain shuffling for instance combining the VH domains of antibodies derived from one library with the VL domains of another library thus expanding the pool of VL partners tested for each VH domain. For instance, the antibodies 6B1, 6AH, 6A5 and 6H1 specific for TGFβ2 utilise the 2A-H11 VH domain isolated from the "synthetic library" combined with a light chain from the PBL library.
Thus the VH and VL domains of antibodies specific for TGFβ can be contributed from phage display libraries derived from rearranged V genes such as those in PBLs, tonsil and bone marrow and from V domains derived from cloned germline V segments combined with synthetic CDRs. There are also shown to be a diverse range of antibodies which are specific for TGFβ1 or TGFβ2. The antibodies which have been isolated both against TGFβ1 and TGFβ2 have mainly utilised V genes derived from VH germlines of the VH3 family. A wider variety of light chain variable regions have been used, of both the lambda and kappa types.
Individual antibodies which have been isolated have unexpectedly advantageous properties. For example, the antibodies directed against TGFβ2 (6H1, 6A5 and 6B1) have been shown to bind to TGFβ2 with slow off-rates (off-rate constants koff of the order of 10−3 s−1 and dissociation constants of less than 10−8M) to neutralise TGFβ2 activity in in vitro assays and to be potent in in vivo applications. The antibody 6B1 IgG4 has been shown to bind specifically to TGFβ2 in immunohistochemistry in mammalian tissues and not to cross-react with other antigens in human tissues. The properties of these antibodies may make them particularly suitable for therapeutic applications. The fact that these antibodies share the same heavy chain, shows that VH domains can be effective with a number of different light chains, although there may be differences in potency or subtle changes of epitope with different light chains. As shown in Examples 3 and 4 and Tables 4 and 5, 6B1 IgG4 is the most potent antibody in neutralising TGFβ2 activity in the radioreceptor assay and the TF1 proliferation assay. Its properties may however be expected to be qualitatively similar to the antibodies 6A5 and 6H1 with which it shares a common VH domain. Thus the reduction in neural scarring observed on treatment with 6A5 single chain Fv and 6H1 IgG4 shown in Example 5 would be expected to be reproduced with 6B1. The antibodies directed against TGFβ1 (1AE5, 1AH6 particularly 1B2 and their derivatives) also have unexpectedly advantageous properties. Antibody 27C1/10A6 derived from 1B2 by chain shuffling, spiking and conversion into whole antibody IgG4, has been shown to be potent in an in vitro scarring model. The VH domain of this antibody was derived by site directed "spiking" mutagenesis from the parent antibody 7A3. A large number of spiked clones were obtained which show similar properties in in vitro assays. There can be a number of changes in CDR3 of the VH compared to 27C1, for instance, 28A-H11 differs in 7 of the 14 positions, 2 of which are non-conservative changes. Thus there may be up to 50% of the residues in the VH CDR3 changed without affecting binding properties.
Antibodies specific for human TGFβ, including human TGFβ1 and human TGFβ2, have been shown to be effective in animal models for the treatment of fibrotic diseases and other diseases such as rheumatoid arthritis where TGFβ is overexpressed. Antibodies against TGFβ have been shown to be effective in the treatment of glomerulonephritis (W. A Border et al. Nature 346, 371-374, 1990); neural scarring (A. Logan et al. Eur. J. Neurosci. 6, 355-363, 1994); dermal scarring (M. Shah et al. Lancet 339, 213-214 1992; M. Shah et al. J. Cell Science 107, 1137-1157, 1994; M. Shah et al. 108, 985-1002, 1995); lung fibrosis (S. N. Giri et al. Thorax 48, 959-966, 1993); arterial injury (Y. G. Wolf, L. M. Rasmussen & E. Ruoslahti J. Clin. Invest. 93, 1172-1178, 1994) and rheumatoid arthritis (Wahl et al J. Exp. Medicine 177, 225-230, 1993). It has been suggested that TGFβ3 acts antagonistically to TGFβ1 and TGFβ2 in dermal scarring (M. Shah et al. 1995 supra.). Therefore, antibodies to TGFβ1 or TGFβ2 with apparent low cross-reactivity to TGFβ3, as assessed by binding studies using a biosensor assay (e.g. BIACore™), ELISA or a radioreceptor assay, as disclosed in this application, that is to say antibodies which bind preferentially to TGFβ1 or TGFβ2 compared with TGFβ3, should be advantageous in this and other conditions such as fibrotic conditions in which it is desirable to counteract the fibrosis promoting effects of TGFβ1 and TGFβ2. An antibody which cross-reacts strongly with TGFβ3 has however had an effect in an animal model of rheumatoid arthritis (Wahl et al., 1993, supra).
There are likely to be applications further to the above mentioned conditions, as there are several other in vitro models of disease where antibodies against TGFβ have shown promise of therapeutic efficacy. Of particular importance may be the use of antibodies against TGFβ for the treatment of eye diseases involving ocular fibrosis, including proliferative retinopathy (R. A. Pena et al. (ref. below), retinal detachment and post glaucoma (P. T. Khaw et al., Eye 8 188-195, 1994) drainage surgery. Connor et al. (J. Clin. Invest 83 1661-1666, 1989) showed that much higher levels of TGFβ2 were present in vitreous aspirates from patients with intraocular fibrosis associated with proliferative retinopathy compared with patients with uncomplicated retinal detachment without ocular firbrosis and that the biological activity of this TGFβ2 could be neutralised with antibodies directed against TGFβ2. Moreover, Pena et al. (Invest. Ophthalmology. Vis. Sci. 35: 2804-2808, 1994) showed that antibodies against TGFβ2 inhibit collagen contraction stimulated by TGFβ2. Contraction of the vitreous gel by fibroblasts and other cell types plays a critical role in the proliferative retinopathy disease process, a process thought to be mediated by TGFβ2.
There is other evidence pointing to TGFβ2 being the most important TGFβ isoform promoting intraocular fibrosis. TGFβ2 has been shown to be the predominant isoform of TGFβ in the neural retina, retinal pigment epithelium-choroid and vitreous of the human eye (Pfeffer et al. Exp. Eye Res. 59: 323-333, 1994) and found in human aqueous humour in specimens from eyes undergoing cataract extraction with intraocular lens implantation (Jampel et al. Current Eye Research 9: 963-969, 1990). Non-transformed human retinal pigment epithelial cells predominantly secrete TGFβ2 (Kvanta Opthalmic Res. 26: 361-367, 1994).
Other diseases which have potential for treatment with antibodies against TGFβ include adult respiratory distress syndrome, cirrhosis of the liver, post myocardial infarction, post angioplasty restenosis, keloid scars and scleroderma. The increase level of expression of TGFβ2 in osteoporosis (Erlenbacher et al. J. Cell Biol. 132: 195-210, 1996) means that this is a disease potentially treatable by antibodies directed against TGFβ2.
The use of antibodies against TGFβ for the treatment of diseases has been the subject of patent applications for fibrotic disease (WO91/04748); dermal scarring (WO92/17206); macrophage deficiency diseases (PCT/US93/00998); macrophage pathogen infections (PCT/US93/02017); neural scarring (PCT/US93/03068); vascular disorders (PCT/US93/03795); prevention of cataracts (WO95/13827). The human antibodies against human TGFβ disclosed in this application should be valuable in these conditions.
It is shown herein that the human antibodies both against human TGFβ1 and against human TGFβ2 can be effective in the treatment of fibrosis in animal models of neural scarring and glomerulonephritis in either single chain Fv and whole antibody format. This is the first disclosure of the effectiveness of antibodies directed only against TGFβ2 as sole treatment in these indications, although some effectiveness of antibodies against TGFβ2 only has been observed in a lung fibrosis model (Giri et al. Thorax 48, 959-966, 1993 supra). The effectiveness of the human antibodies against human TGFβ in treatment of fibrotic disease has been determined by measuring a decrease in the accumulation of components of the extracellular matrix, including fibronectin and laminin in animal models.
The evidence of efficacy of the antibodies against TGFβ2 and TGFβ1 describe herein in prevention of neural scarring in the animal model experiment means that these antibodies are likely to be effective in other disease states mediated by TGFβ. For comparison, antisera isolated from turkeys directed against TGFβ isoforms by Danielpour et al. (Cell Physiol. 138: 79-86, 1989) have been shown to be effective in the prevention of dermal scarring (Shah et al. J. Cell Science 108: 985-1002, 1995), neural scarring (Logan et al., supra) and in in vitro experiments relating to proliferative retinopathy (Connor et al., supra).
Terminology
Specific Binding Member
This describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.
Antibody
This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These proteins can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd and diabodies.
It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).
Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), eg prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al, Embo Journal, 10, 3655-3659, (1991).
Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
Antigen Binding Domain
This describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
Specific
This may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
Neutralisation
This refers to the situation in which the binding of a molecule to another molecule results in the abrogation or inhibition of the biological effector function of another molecule.
Functionally Equivalent Variant Form
This refers to a molecule (the variant) which although having structural differences to another molecule (the parent) retains some significant homology and also at least some of the biological function of the parent molecule, e.g. the ability to bind a particular antigen or epitope. Variants may be in the form of fragments, derivatives or mutants. A variant, derivative or mutant may be obtained by modification of the parent molecule by the addition, deletion, substitution or insertion of one or more amino acids, or by the linkage of another molecule. These changes may be made at the nucleotide or protein level. For example, the encoded polypeptide may be a Fab fragment which is then linked to an Fc tail from another source. Alternatively, a marker such as an enzyme, flourescein, etc, may be linked.
Substantial Part
A molecule may comprise only a part of the sequence referred to. The part sequence will be of sufficient length to substantially retain the function of interest of the full-length sequence.
Comprise
This is generally used in the sense of inclusiveness, that is to say permitting the presence of one or more features or components.
The present invention generally provides a specific binding member comprising an antibody antigen binding domain. More particularly it provides a specific binding member for TGFβ, and even more particularly the isoforms TGFβ1 and TGFβ2.
The present invention provides a specific binding member which comprises a human antibody antigen binding domain specific for TGFβ and more particularly for TGFβ1 and/or TGFβ2 and which has low cross reactivity with TGFβ3. The cross-reactivity may be as assessed using any or all of the following assays: biosensor (e.g. BIACore™), ELISA and radioreceptor. The present invention provides specific binding member which comprises a human antibody antigen binding domain specific for TGFβ1 and/or TGFβ2 which binds preferentially to these isoforms compared with TGFβ3.
The TGFβ may be human TGFβ.
The specific binding member may be in the form of an antibody fragment such as single chain Fv (scFv). Other types of antibody fragments may also be utilised such as Fab, Fab′, F(ab′)2, Fabc, Facb or a diabody (G. Winter & C. Milstein Nature 349, 293-299, 1991; WO94/13804). The specific binding member may be in the form of a whole antibody. The whole antibody may be in any of the forms of the antibody isotypes e.g. IgG, IgA, IgE, and IgM and any of the forms of the isotype subclasses eg IgG1 or IgG4.
The specific binding member may also be in the form of an engineered antibody eg bispecific antibody molecules (or fragments such as F(ab′)2) which have one antigen binding arm (i.e. specific binding domain) against TGFβ and another arm against a different specificity. Indeed the specific binding members directed against TGFβ1 and/or TGFβ2 described herein may be combined in a bispecific diabody format. For example the antibodies 31G9 directed against TGFβ1 and 6H1 directed against TGFβ2 may be combined to give a single dimeric molecule with both specificities.
The binding domain may comprise part or all of a VH domain encoded by a germ line gene segment or a rearranged gene segment. The binding domain may comprise part or all of either a VL kappa domain or a VL lambda domain.
The binding domain may be encoded by an altered or variant form of a germ line gene with one or more nucleotide alterations (addition, deletion, substitution and/or insertion), e.g. about or less than about 25, 20, 15, 10 or 5 alterations, 4, 3, 2 or 1, which may be in one or more frameworks and/or CDR'S.
The binding domain may comprise a VH3 gene sequence of one of the following germ lines; the DP49 germ line; the DP53 germ line; the DP50 germ line; the DP46 germ line; or a re-arranged form thereof.
A preferred VH domain for anti-TGFβ2 specific binding members according to the present invention is that of 6H1 VH, whose sequence is shown in FIG. 2(a)(i) (SEQ ID NO: 6). 6H1 may be paired with a variety of VL domains, as exemplified herein. Amino acid sequence variants of 6H1 VH may be employed.
The specific binding member may neutralise the in vitro and/or in vivo effect of TGFβ that is one or more of the isoforms, particularly TGFβ1 and/or TGFβ2.
The specific binding member may be a high affinity antibody. Preferred affinities are discussed elsewhere herein.
The binding domain may comprise part or all of a VH domain having either an amino acid sequence as shown in FIG. 1(a)(i) (SEQ ID NO: 8), (ii) (SEQ ID NO: 111), (iii) (SEQ ID NO: 112) or (iv) (SEQ ID NO: 10) or FIG. 1(c)(i) (SEQ ID NO: 12) or a functionally equivalent variant form of a said amino acid sequence.
The binding domain may comprise part or all of a VH domain encoded by either a nucleotide sequence as shown in FIG. 1(a)(i) (SEQ ID NO: 7), (ii) (SEQ ID NO: 113), (iii) (SEQ ID NO: 114) or (iv) (SEQ ID NO: 9) or FIG. 1(c)(i) (SEQ ID NO: 11) or a functionally equivalent variant form of a said nucleotide sequence.
The binding domain may comprise part or all of a VL domain having either an amino acid sequence as shown in FIG. 1(a)(v) (SEQ ID NO: 14) or FIG. 1(b) (SEQ ID NOS: 16, 18) or a functionally equivalent variant form of a said amino acid sequence.
The binding domain may comprise part or all of a VL domain encoded by either a nucleotide sequence as shown in FIG. 1(a)(v) (SEQ ID NO: 13) or FIG. 1(b) (SEQ ID NOs: 15, 17) or a functionally equivalent variant form of a said nucleotide sequence.
The binding domain may comprise part or all of a VH domain having a variant form of the FIG. 1(a)(i) amino acid (SEQ ID NO: 8), the variant form being one of those as provided by FIG. 3 (SEQ ID NOS: 19 to 35).
The binding domain may comprise part or all of a VH domain having either an amino acid sequence as shown in FIG. 2(a)(i) (SEQ ID NO: 6) or (ii) (SEQ ID NO: 37), (iii) (SEQ ID NO: 116), (v) (SEQ ID NO: 120), (vi) (SEQ ID NO: 122) or a functionally equivalent variant form of a said amino acid sequence.
The binding domain may comprise part or all of a VH domain encoded by either a nucleotide sequence as shown in FIG. 2(a)(i) (SEQ ID NO:5) or (ii) (SEQ ID NO: 36), (iii) (SEQ ID NO: 115), (v) (SEQ ID NO: 119), (vi) (SEQ ID NO: 121) or a functionally equivalent variant form of a said nucleotide sequence.
The binding domain may comprise part or all of a VL domain having either an amino acid sequence as shown in any of FIG. 2(a)(iv) (SEQ ID NO:118) or 2(b)(i) to (vi) (SEQ ID NOS: 39, 41, 43, 45, 47, 124) or functionally equivalent variant form of a said amino acid sequence.
The binding domain may comprise part or all of a VL domain encoded by either a nucleotide sequence as shown in any of FIGS. 2(a)(iv) (SEQ ID NO:117) 2(b)(i) to (vi), (SEQ ID NOS: 38, 40, 42, 44, 46, 123) or a functionally equivalent variant form of a said nucleotide sequence.
The binding domain may be specific for both TGFβ1 and TGFβ2. The binding domain may be specific for both human TGFβ1 and human TGFβ2. The specific binding member may be in the form of scFv.
The binding domain may comprise part or all of a VL domain having either an amino acid sequence as shown in FIG. 4 (SEQ ID NO: 49) or a functionally equivalent variant form of said amino acid sequence. The binding domain may comprise part or all of a VL domain encoded by either the nucleotide sequence as shown in FIG. 4 (SEQ ID NO: 48) or a functionally equivalent variant form of said nucleotide sequence.
In particular, the binding domain may comprise one or more CDR (complementarity determining region) with an amino acid sequence shown in any of the figures. In a preferred embodiment, the binding domain comprises one or more of the CDRs, CDR1, CDR2 and/or CDR3 shown in the Figures, especially any of those shown in italics in FIG. 19 (SEQ ID NOS: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137).
In a preferred embodiment, the binding domain comprises a VH CDR3 sequence as shown, especially as shown in italics in FIG. 19 (SEQ ID NOS: 128, 131, 134, 137). Functionally equivalent variant forms of the CDRs are encompassed by the present invention, in particular variants which differ from the CDR sequences shown by addition, deletion, substitution or insertion of one or more amino acids and which retain ability to bind the antigen and optionally one or more of the preferred characteristics for specific binding members of the present invention as disclosed herein. The specific binding member may comprise all or part of the framework regions shown flanking and between the CDRs in the Figures, especially FIG. 19 (SEQ ID NOS: 61, 63, 65, 67), or different framework regions including modified versions of those shown.
So-called "CDR-grafting" in which one or more CDR sequences of a first antibody is placed within a framework of sequences not of that antibody, e.g. of another antibody is disclosed in EP-B-0239400.
The present invention also provides a polypeptide with a binding domain specific for TGFβ which polypeptide comprises a substantial part or all of either an amino acid sequence as shown in any of FIG. 1(a) (SEQ ID NOS:8, 10, 14, 111, 112), FIG. 1(b) (SEQ ID NOS: 16, 18), FIG. 1(c) (SEQ ID NO: 12), FIG. 2(a) (SEQ ID NOS: 6, 37, 116, 118, 120, 122), FIG. 2(b) (SEQ ID NOS: 39, 41, 43, 45, 47, 124), FIG. 4 (SEQ ID NO:49) or a functionally equivalent variant form of a said amino acid sequence. The polypeptide may comprise a substantial part or all of an amino acid sequence which is a functionally equivalent variant form of the FIG. 1(a)(i) (SEQ ID NO: 8) amino acid sequence, the variant being one of those variants as shown in FIG. 3 (SEQ ID NOS: 19 to 35).
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, as discussed. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion), maybe 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. Alterations may be made in one or more framework regions and/or one or more CDR's.
A specific binding member according to the invention may be one which competes for binding to TGFβ1 and/or TGFβ2 with any specific binding member which both binds TGFβ1 and/or TGFβ2 and comprises part of all of any of the sequences shown in the Figures. Competition between binding members may be assayed easily in vitro, for example by tagging a specific reporter molecule to one binding member which can be detected in the presence of other untagged binding member(s), to enable identification of specific binding members which bind the same epitope or an overlapping epitope.
Preferred specific binding members for TGFβ1 compete for binding to TGFβ1 with the antibody CS37, discussed in more details elsewhere herein.
Preferred specific binding members for TGFβ2 compete for binding to TGFβ2 with the antibody 6B1 discussed in more detail else where herein. They may bind the epitope RVLSL (SEQ ID NO: 1) or a peptide comprising the amino acid sequence RVLSL (SEQ ID NO: 1), particularly such a peptide which adopts an α-helical conformation. They may bind the peptide TQHSRVLSLYNTIN (SEQ ID NO: 2) In testing for this, a peptide with this sequence plus CGG at the N-terminus may be used. Specific binding members according to the present invention may be such that their binding for TGFβ2 is inhibited by a peptide comprising RVLSL (SEQ ID NO: 1), such as a peptide with the sequence TQHSRVLSLYNTIN (SEQ ID NO: 2). In testing for this, a peptide with this sequence plus CGG at the N-terminus may be used.
TQHSRVLSLYNTIN (SEQ ID NO: 2) corresponds to the alpha helix H3 (residues 56-69) of TGFβ2, as discussed elsewhere herein. The equivalent region in TGFβ1 has the sequence TQYSKVLSLYNQHN (SEQ ID NO: 125). Anti-TGFβ1 antibodies which bind this region are of particular interest in the present invention, and are obtainable for example by panning a peptide with this sequence (or with CGG at the N-terminus) against a phage display library. Specific binding members which bind the peptide may be selected by means of their binding, and may be neutralising for TGFβ1 activity. Binding of such specific binding members to TGFβ1 may be inhibited by the peptide TQYSKVLSLYNQHN (SEQ ID NO: 125) (optionally with CGG at the N-terminus).
A specific binding member according to the present invention which is specific for TGFβ2 may show no or substantially no binding for the latent form of TGFβ2, e.g. be specific for the active form of TGFβ2. 6B1 is shown in Example 6 to have this property.
6B1 is particularly suitable for therapeutic use in the treatment of fibrotic disorders because it has the following advantageous properties. 6B1 binds to TGFβ2 with a dissociation constant of 2.3 nM in the single chain form and 0.89 nM for the whole antibody form, 6B1 IgG4 (Example 13). The antibody 6B1 IgG4 neutralises the biological activity of TGFβ2 in an antiproliferation assay (IC50 2n M; examples 7 and 10) and in a radioreceptor assay (IC50 less than 1 nM; Table 6). The antibody binds to the peptide TQHSRVLSLYNTIN (SEQ ID NO: 2) (TGFβ256-69) from the alpha helix H3 of TGFβ2 and recognises the corresponding peptide from TGFβ1 more weakly. 6B1 recognises the active but not the latent form of TGFβ2 (Example 6), recognises TGFβ2 in mammalian tissues by ICC and does not bind non-specifically to other human tissues (Example 12). The antibody preferentially binds to TGFβ2 as compared to TGFβ3, the cross-reactivity with TGFβ3 being 9% as determined by the ratio of the dissociation constants.
The other antibodies described in this application which contain the 6H1 VH domain, 6H1 and 6A5 have similar properties. The dissociation constants of were determined to be 2 nM for 6B1 IgG4 (Example 2) and 0.7 nM for 6A5 single chain Fv (Table 1). 6H1 IgG4 neutralises the biological activity of TGFβ2 with IC50 values of 12 to 15 nM (Examples 7 and 10). 6A5 and 6H1 inhibit receptor binding of TGFβ2 in a radioreceptor assay with IC50 values of about 1 nM in the single chain Fv format and 10 nM or below in the whole antibody, IgG4 format. Both 6H1 IgG4 and 6A5 scFv were shown to be effective in the prevention of neural scarring (Example 5).
Therefore for the first human antibodies directed against TGFβ2 are provided which have suitable properties for treatment of diseases characterised by the deleterious presence of TGFβ2. Such antibodies preferably neutralise TGFβ2 and preferably have a dissociation constant for TGFβ2 of less than about 100 nM, more preferably about 10 nM, more preferably below about 5 nM. The antibodies preferentially bind to TGFβ2 as compared to TGFβ3, preferably have less than 20% cross-reactivity with TGFβ3 (as measured by the ratio of the dissociation constants) and preferably have less than about 10% cross-reactivity.
The antibody preferably recognises the active but not the latent form of TGFβ2.
For antibodies against TGFβ1, the properties desired for an antibody to be effective in treatment of fibrotic disease are similar. Such antibodies preferably neutralise TGFβ1 and have a dissociation constant for TGFβ1 of less than about 100 nM, more preferably below about 10 nM, more preferably below about 5 nM. The antibodies preferentially bind to TGFβ1 as compared to TGFβ3, preferably have less than about 20% cross-reactivity with TGFβ3 (as measured by the ratio of the dissociation constants) and more preferably have less than about 10% cross-reactivity.
The antibody preferably recognises the active but not the latent form of TGFβ1. The antibody 31G9 has a dissociation constant of 12 nM (Table 5). The antibodies CS37 scFv and 27C1/10A6 IgG4 show IC50 values in a radioreceptor assay of 8 nM and 9 nM respetively, indicating a dissociation contstant in the low nanomolar range. 27C1/10A6 IgG4 was shown to be effective in a neural scarring model. Cross-reactivity of antibodies of the 1B2 lineage with TGFβ3 is very low (Example 9).
In addition to an antibody sequence, the specific binding member may comprise other amino acids, e.g. forming a peptide or polypeptide, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. For example, the specific binding member may comprise a label, an enzyme or a fragment thereof and so on.
The present invention also provides a polynucleotide which codes for a polypeptide with a binding domain specific for TGFβ which polynucleotide comprises a substantial part or all of a nucleotide sequence which codes for either an amino acid sequence as shown in any one of FIG. 1(a) (SEQ ID NOS: 8, 10, 14, 111, 112), FIG. 1(b) (SEQ ID NOS: 16, 18), FIG. 1(c) (SEQ ID NO: 12), FIG. 2(a) (SEQ ID NOS: 6, 37, 116, 118, 120, 122), FIG. 2(b) (SEQ ID NOS: 39, 41, 43, 45, 47, 124), FIG. 4 (SEQ ID NO: 49) or a functionally equivalent variant form of a said amino acid sequence. The polynucleotide may code for a polypeptide with a binding domain specific for TGFβ which polynucleotide comprises a substantial part or all of a nucleotide sequence which codes for an amino acid sequence which is a functionally equivalent variant form of the FIG. 1(a)(i) amino acid sequence (SEQ ID NO:8), the variant being one of those as shown in FIG. 3 (SEQ ID NOS: 19 to 35). The polynucleotide may code for a polypeptide with a binding domain specific for TGFβ which polynucleotide comprises a substantial part or all of a either a nucleotide sequence as shown in any of FIG. 1(a) (SEQ ID NOS: 7, 9, 13, 113, 114), FIG. 1(b) (SEQ ID NOS: 15, 17), FIG. 1(c) (SEQ ID NO: 11), FIG. 2(a) (SEQ ID NOS: 5, 36, 115, 117, 119, 121), FIG. 2(b) (SEQ ID NOS: 38, 40, 42, 44, 46, 123), FIG. 4 (SEQ ID NO: 48) or a functionally equivalent variant form of said nucleotide sequence. The polynucleotide may code for a polypeptide with a binding domain specific for TGFβ which polynucleotide comprises a substantial part or all a nucleotide sequence which codes for a variant form of the FIG. 1(a)(i) amino acid sequence (SEQ ID NO: 8), the variant being one of those as shown in FIG. 3 (SEQ ID NOS: 19 to 35).
The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise least one polynucleotide as above.
The present invention also provides a recombinant host cell which comprises one or more constructs as above.
A specific binding member according to the present invention may be made by expression from encoding nucleic acid. Nucleic acid encoding any specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid thereof. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.
Specific binding members and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic. The term "isolate" encompasses all these possibilities.
The nucleic acid may encode any of the amino acid sequences shown in any of the Figures, or any functionally equivalent form. The nucleotide sequences employed may be any of those shown in any of the Figures, or may be a variant, allele or derivative thereof. Changes may be made at the nucleotide level by addition, substitution, deletion or insertion of one or more nucleotides, which changes may or may not be reflected at the amino acid level, dependent on the degeneracy of the genetic code.
Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others. A common, preferred bacterial host is E. coli.
The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding member, see for recent reviews, for example Reff, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.
Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above.
Following production of a specific binding member it may be used for example in any of the manners disclosed herein, such as in the formulation of a composition, pharmaceutical or a diagnostic product, such as a kit comprising in addition to the specific binding member one or more reagents for determining binding of the member to cells, as discussed. A composition may comprise at least one component in addition to the specific binding member.
The present invention also provides pharmaceuticals which comprise a specific binding member as above, optionally with one or more excipients.
The present invention also provides the use of a specific binding member as above in the preparation of a medicament to treat a condition in which it is advantageous to counteract the fibrosis promoting effects of TGFβ. The condition may be a fibrotic condition characterized by an accumulation in a tissue of components of the extracellular matrix. The components of the extracellular matrix may be fibronectin or laminin.
The condition may be selected from the group consisting of:                glomerulonephritis        neural scarring        dermal scarring        ocular scarring        lung fibrosis        arterial injury        proliferative retinopathy        retinal detachment        adult respiratory distress syndrome        liver cirrhosis        post myocardial infarction        post angioplasty restenosis        keloid scarring        scleroderma        vascular disorders        cataract        glaucoma        proliferative retinopathy.
The condition may be neural scarring or glomerulonephritis.
The present invention also provides the use of a specific binding member as above, in the preparation of a medicament to treat an immune/inflammatory disease condition in which it is advantageous to counteract the effects of TGFβ. Illustrative conditions are rheumatoid arthritis, macrophage deficiency disease and macrophage pathogen infection.
The present invention also provides a method which comprises administering to a patient a therapeutically effective amount of a specific binding member as above in order to treat a condition in which it is advantageous to counteract the fibrosis promoting effects of TGFβ. Fibrotic conditions are listed above.
The present invention also provides a method which comprises administering to a patient a prophylactically effective amount of a specific binding member as above in order to prevent a condition in which it is advantageous to prevent the fibrosis promoting effects of TGFβ. Fibrotic conditions are listed above.
The present invention also provides methods which comprise administering to patients prophylactically and/or therapeutically effective amounts of a specific binding member as above in order to prevent or treat an immune/inflammatory disease condition in which it is advantageous to counteract the effects of TGFβ. Illustrative conditions are stated above.
Thus, various aspects of the invention provide methods of treatment comprising administration of a specific binding member as provided, pharmaceutical compositions comprising such a specific binding member, and use of such a specific binding member in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the specific binding member with a pharmaceutically acceptable excipient.
In accordance with the present invention, compositions provided may be administered to individuals, which may be any mammal, particularly rodent, e.g. mouse, horse, pig, sheep, goat, cattle, dog, cat or human. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibody are well known in the art; see Ledermann J. A. et al. (1991) Int J. Cancer 47: 659-664; Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Further aspects of the invention and embodiments will be apparent to those skilled in the art. In order that the present invention is fully understood, the following examples are provided by way of exemplification only and not by way of limitation.
All documents mentioned herein are incorporated by reference.


A power tool is disclosed, which includes a tip tool driven by an electric motor, a main body for supporting the tip tool and a dust nozzle which is provided to the main body and to which a dust bag or a dust hose is connectable. When the dust nozzle is aligned in a first direction, the dust nozzle is rotatable with respect to the main body around the first direction as a rotation center. When the dust nozzle is inclined with respect to the first direction from a first position to a second position, the dust nozzle is not rotatable around the first direction.

By way of example of the power tool in the related art, a portable circular saw will be explained with reference to FIG. 5, FIG. 6, and FIG. 7 hereunder.
As shown in FIG. 7, the portable circular saw has a main body 1 and a base 3. The main body 1 is provided to the base 3 such that it can turn on a turn pivot 23. A turn adjusting knob 22 engages with a hole portion 21a provided to a depth adjusting plate 21 that is secured to the base 3. This turn adjusting knob 22 can fix the main body 1 to the base 3 in any position of the hole portion 21a. The portable circular saw is constructed such that, when a worker operates a trigger switch after an electric power is fed, an electric motor (not shown) starts to rotate, and then transmits its rotation to a saw blade 2 to cut a worked material.
As shown in FIG. 5, when the base 3 is slid forward (front) in FIG. 5 on the worked material (not shown), the saw blade 2 placed to project from the base 3 cuts the worked material (not shown). In cutting the worked material, the chips as a part of the worked material being cut by the saw blade 2 are produced.
As the mechanism for collecting the chips, there is such a mechanism that, as shown in FIG. 5, a dust nozzle 66 is coupled to a top portion of a saw cover 55 and then a chip recovering means as a dust hose 9 of the dust collector (not shown) is fitted to the dust nozzle 66. The chips when sucked by the dust collector (not shown) moves from the saw cover 55 to the dust nozzle 66, and are collected by the dust collector via the dust hose 9.
In FIG. 6, a dust bag 10 is fitted to the dust nozzle 66. In this case, a force of scattering the chips is applied to the chips by the saw blade 2 in the direction of a fitting axis X of the saw cover 55. Therefore, the chips enter into the dust nozzle 66 from the saw cover 55 by its own scattering force, and then enter into the dust bag 10. As a result, the chips are collected by the dust collector.
The dust nozzle 66 shown in FIG. 5 and FIG. 6 is provided to turn on the fitting axis X of the saw cover 55 along a groove provided in the circumferential direction of the saw cover 55. This fitting axis X of the saw cover 55 also acts as a fitting axis of the dust nozzle 66. In the cutting work by the portable circular saw, the worker can turn the dust hose 9 and the dust bag 10 together with the dust nozzle 66 in any direction.
In this case, as shown in FIG. 5 or FIG. 6, an angle α between the fitting axis X of the saw cover 55 and a fitting axis Y of the dust hose 9 or the dust bag 10 is always kept constant, and is set to about 45 degree, for example. Also, although not shown, the fitting axis Y of the dust bag 10 or the dust hose 9 is identical to the fitting axis X of the saw cover 55, and is set to about 0 degree, for example.

Power tool with chips ejecting mechanism


A composition, test device and method of determining the presence or concentration of proteins, such as albumin, in a liquid test sample, such as urine. The test device includes a test pad with a carrier matrix incorporating a indicator reagent composition capable of interacting with proteins to produce a detectable response. The indicator reagent composition includes an indicator dye that is capable of interacting with albumin and undergoing a detectable color transition; a buffer; a hydrophobic polymeric compound having the general structural formula: ##STR1## wherein A is ##STR2## and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number in the range of 0 to about 20, z is a number in the range of 0 to about 20, the sum y+z is a number in the range of about 2 to about 20, and R.sub.2 and R.sub.3 are selected, independently, from the group consisting of hydrogen, an alkyl group, an aralkyl group, and an aryl group;. R.sub.1 is either methylene or oxygen;. n is a number in the range of one to about 8; and. E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when R.sub.1 is oxygen; and a suitable carrier which is water. The indicator reagent composition provides an improved color resolution in the test samples raving negative protein and trace amount of protein.

Albumin is the most abundant plasma protein, generally constituting slightly over one-half of the total protein in mammalian plasma. In the human body, albumin has the important role of regulating the water balance between blood and tissues, and of functioning as a transport molecule for various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and drugs like sulfonamides and barbiturates, that are only sparsely soluble in water. An albumin deficiency can restrict the transport of sparsely water soluble materials throughout the body and a deficiency is signaled in an individual by an abnormal accumulation of serous fluid, or edema. Therefore, it is clinically important to determine whether an individual has a deficiency of serum albumin.
Likewise, it is clinically important to determine if an individual is excreting an excess amount of protein. A normal functioning kidney forms urine in an essentially two step process. Blood flows through the glomerulus, or glomerular region of the kidney. The capillary walls of the glomerulus are highly permeable to water and low molecular weight components of the blood plasma. Albumin and other high molecular weight proteins cannot pass through these capillary walls and essentially are filtered out of the urine so that the protein is available for use by the body. The liquid containing the low molecular weight components passes into the tubules, or tubular region, of the kidney where reabsorption of some urine components, such as low molecular weight proteins; secretion of other urine components; and concentration of the urine occurs. AM a result, through the combined processes of the glomerulus and tubules, the concentration of proteins in urine should be minimal. Therefore, abnormally high amounts of albumin in urine must be detected and related to a physiological dysfunction.
A relatively high concentration of albumin in the urine of an individual usually is indicative of a diseased condition. For example, the average normal concentration of protein in urine varies from about 10 mg/dL (milligrams per deciliter) to about 20 mg/dL, with approximately one-fifth of the total urinary protein being serum albumin. However, in a majority of diseased states, urinary protein levels increase appreciably, such that albumin accounts for from about 60 percent to about 90 percent of the excreted protein. The presence of an abnormal increased amount of protein in the urine, known as proteinuria, is one of the most significant indicators of renal disease, and may be indicative of various other non-renal related diseases.
Therefore, in order to determine if an individual has an albumin deficiency or to determine if an individual excretes an excess amount of protein, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive protein detection assays have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of protein in urine and serum, the methods based on dye binding techniques have proven especially useful because dye binding methods are readily automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are capable of interacting with a protein, such as albumin, and that are capable of changing color upon interaction with a protein absent any change in pH. When a pH indicator dye interacts with, or binds to, a protein, the apparent pK.sub.a (acid dissociation constant) of the indicator dye is altered and the dye undergoes a color transition, producing the so-called "protein-error" phenomenon. In methods utilizing the dye binding technique, an appropriate buffer maintains the pH indicator dye at a constant pH to prevent a color transition of the pH indicator dye due to a substantial shift in pH. Due to the "protein-error" phenomena, the pH indicator dye undergoes a color transition upon interaction with protein that is identical to the color change arising because of a change in the pH. Examples of pH indicator dyes used in the dry phase assay of proteins that are capable of interacting with or binding to proteins and exhibiting "protein-error" color transitions include tetrabromophenol blue (TBPB) and tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.
Although pH indicator dyes have been used extensively in protein assays, several disadvantages still exist in protein assay methods utilizing indicator dyes. For example, methods based upon pH indicator dyes cannot sufficiently differentiate, quantitatively, between a trace protein concentration of about 15 to about 30 mg/dL and a negative protein concentration below about 15 mg/dL. A negative protein concentration is the normal background amount of protein present in urine, and is clinically insignificant. A trace protein concentration shows a slightly elevated amount of protein in urine and is clinically significant. An assay showing a trace amount of protein requires a confirmatory assay to conclusively show that an elevated amount of protein is present in the urine. Although several simple quantitative assays are available for the determination of the total protein content in a test sample, the majority of these assay methods, with the notable exception of the simple colorimetric reagent test strip, require the precipitation of protein to make quantitative protein determinations. Accordingly, the confirmatory assays are more time consuming and expensive than the test strip assays used to screen the urine samples for protein content. Therefore, a need exists for a test strip assay that substantially reduces the number of false positive assays for a trace amount of protein.
The colorimetric reagent test strip utilizes the previously discussed ability of proteins to interact with certain acid-base indicators and to alter the color of the indicator without any change in the pH. For example, when the indicator tetrabromophenol blue (TBPB) is buffered to maintain a constant pH of approximately 3, the test pad remains a yellow color upon contact with a test sample that does not contain protein. However, for test samples containing protein, the presence of protein causes the buffered dye to impart either a greenish-yellow color, a green color or a blue color to the test pad, depending upon the concentration of protein in the test sample. Consequently, the development of a greenish-yellow color in the test pad of a dry phase test strip can be interpreted as a trace amount of protein or as a negative amount of protein.
Some colorimetric test strips used in protein assays have a single test area consisting of a small square pad of a carrier matrix impregnated with a buffered pH indicator dye, such as tetrabromophenol blue. Other colorimetric test strips are multideterminant reagent strips that include one test area, or test pad, for the protein assay as described above, and further include several additional test pads on the same strip to permit the simultaneous assay of other urinary constituents, like pH. For both types of colorimetric test strips, the assay for protein in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test pad of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the indicator dye, quantitative assays for protein can be performed and are reported as negative, trace, or one "plus" to four "plus". A negative reading, or yellow color, indicates that the urine contains less than about 15 mg/dL protein, as demonstrated by the lack of a color transition of the indicator dye. A trace reading, or greenish-yellow color, indicates that the urine contains from about 15 to about 30 mg/dL of protein. The one "plus" to four "plus" readings, signified by color transitions of green through increasingly dark shades of blue, are approximately equivalent to urine protein concentrations of 30, 100, 300, and over 2000 mg/dL protein, respectively, and serve as reliable indicators of increasingly severe proteinuria. Therefore, differentiating between a negative assay (yellow color) and a trace assay (greenish-yellow color) is important for an accurate protein analysis.
In accordance with the above-described method, an individual can readily determine, visually, that the protein content of a urine sample is in the range of 0 mg/dL to about 30 mg/dL. However, the color differentiation afforded by the presently available commercial test strips is insufficient to allow an accurate determination of urinary protein content between a sample having less than about 15 mg/dL protein (negative) and a sample including from about 15 to about 30 mg/dL protein (trace). The inability to differentiate between low urinary protein concentrations is important clinically because a healthy person usually has a urine protein level in the range of about 2 mg/dL to about 20 mg/dL. Therefore, it is clinically important to determine precisely the urine protein content of an individual, rather than merely estimating the protein content at some value less than about 30 mg/dL.
A trace reading for urinary protein is considered a positive assay, and confirmation of a positive test strip reading is required. The prevalent method of confirming a test strip positive protein assay is the turbidimetric sulfosalicylic acid method, abbreviated as SSA. A high frequency of false positive assays, requires confirmatory testing for each false positive assay, and the attendant added cost. Therefore, it is important that a screening test for protein, like a test strip assay, provide a low frequency of false positive readings.
Trace proteinuria is defined as protein excretion slightly above normal proteinuria. Normal excretion of protein is 50-150 mg/24 hours and 200-300 mg/24 hours in pregnancy. Using an average urine volume of 1250 mL/24 hrs, concentration units of 4-12 mg/dL (16-20 mg/dL in pregnancy) are calculated. Since 24 hour urine volumes vary from about 700 mL to more than 2000 mL, the range of normal protein is considerably wider, and, as expected, the more concentrated urine samples of higher specific gravity (SG) contain more protein. Trace proteinuria, then, is the concentration of protein that falls between negative and one "plus" (30 mg/dL). However, since normal proteinuria covers a range of protein concentrations, trace proteinuria also covers a range of protein concentrations. The trace protein concentration also is dependent on the specific gravity, or SG, of the sample.
The variation of protein concentration with SG does not effect the confirmatory SSA assay, and therefor protein precipitation in the SSA assay is considered indicative of clinical proteinuria. However, the SSA method also has limitations because it is a qualitative method and because procedures vary between clinical laboratories. Accordingly, the problem with a trace reading for protein provided by a screening test, such as a dry phase test strip, is that the reading must correspond to a protein range that is not well defined for clinical samples; that is dependent on the SG of the sample; and that overlaps with the normal, or negative, protein range. Further, trace readings are usually confirmed as positive by a qualitative method that has not been standardized, but is the method of choice in the art because the method is easy, requires no instrumentation, and detects clinical proteinuria in the presence of normal protein.
Of course, the protein content of a urine sample can be determined more precisely by quantitative 24 hour protein precipitation techniques. However, these tests are time consuming and relatively expensive. Furthermore, the precipitation tests must be run in a laboratory by trained personnel, and therefore are unavailable for the patient to perform at home to quickly determine urine protein content and to monitor the success or failure of a particular medical treatment.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying urine for protein content that allows visual differentiation of protein levels in the ranges of 0 mg/dL to about 15 mg/dL and about 15 mg/dL to about 30 mg/dL, and upwards to between about 100 mg/dL to about 300 mg/dL. By providing such an accurate method of determining urine protein concentration in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results, such that a diagnosis can be made without having to wait up to one day for assay results and medical treatment can be commenced immediately. In addition, the test strip method can be performed by the patient at home to more precisely monitor low levels of protein in urine and monitor the success of the medical treatment the patient is undergoing, without providing a large number of false positive assays that require unnecessary, time consuming and costly confirmatory testing. Finally, the method and composition used in a protein assay should be independent of the specific gravity of the urine to provide an accurate protein assay.
For example, the current urinary protein reagent test strips contain an octahalosulfophthalein protein indicator, e.g., tetrabromophenol blue (TBPB), as the indicator dye. When these strips are dipped into albumin-free urine samples of low to medium specific gravity, e.g., SG less than 1,020, the strips turn to a yellowish-green color. When the same strips are dipped into an albumin-free, high SG urine sample, e.g., SG equal to or greater than 1,020, the strips turn to a greenish-yellow color. This greenish-yellow color easily can be interpreted incorrectly as a clinically significant trace concentration of albumin (10-15 mg/dL). However, even with low SG urine samples, the negative color is difficult to differentiate from a true trace color.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy protein assay of urine by utilizing a test strip that includes a test pad comprising a carrier matrix incorporating a new and improved indicator reagent composition. Surprisingly and unexpectedly, the indicator reagent composition of the present invention essentially eliminates the interfering effects of specific gravity on the assay of urine samples including negative to trace amounts of protein. The new and improved indicator reagent composition of the present invention enhances visual color resolution by essentially eliminating the development of an interfering greenish-yellow color by high SG test samples including a negative amount of albumin. Therefore the sensitivity of the assay is enhanced, allowing urine protein concentrations to be determined accurately at levels of about 30 mg/dL or less, and precluding costly confirmatory testing arising from a false positive screening assay for albumin. In addition, the method of the present invention also can be used to determine the presence or concentration of higher concentrations of proteins, such as from about 100 mg/dL to about 2000 mg/dL, in a test sample.
Proteinuria resulting from abnormally high albumin levels depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease. Proteinuria can be intermittent or continuous, with transient, intermittent proteinuria usually being caused by physiological or functional conditions rather than by renal disorders. Therefore, accurate assays of urine and other test samples for protein must be available for both laboratory and home use. The assays must permit the detection or measurement of the proteins of interest, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the protein assay method could be utilized in a dip-and-read format for the easy and economical, qualitative or quantitative determination of protein in urine or other test samples.
Furthermore, any method of assaying for protein in urine or other test samples must yield accurate, trustworthy and reproducible results by utilizing a method that provides a detectable or measurable color transition as a result of an interaction between the indicator reagent composition and the protein, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with a test sample component other than protein. Moreover, it would be advantageous if the protein assay method is suitable for use in dry reagent strips for the rapid, economical and accurate determination of protein in urine and other test samples. Additionally, the method and test pad, comprising the carrier matrix and the indicator reagent composition, utilized in the assay for protein, and the new indicator reagent composition, should not adversely affect or interfere with the other test reagent pads that are present on multideterminant test pad strips.
Although a dry phase chemistry test strip utilizing a dye, such as tetrabromophenol blue or tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein, has been used extensively for several years, no dry phase test strip has utilized a test pad comprising a carrier matrix, such as a filter paper, homogeneously impregnated with an indicator reagent composition including a hydrophobic polymeric compound as depicted above in general structural formula (I). The indicator reagent composition responds to urinary proteins and is essentially independent of urine specific gravity, thereby essentially eliminating the development of an interfering greenish-yellow color in the test pad by high SG samples including a negative amount of protein. Therefore, the assay exhibits an improved visual color resolution and an increased assay sensitivity, especially at lower protein concentration levels, to substantially reduce the number of false position assays. Surprisingly and unexpectedly, because of the essential elimination of the interferences related to urine specific gravity, the method of the present invention facilitates the dry phase test strip assay of urine and other test sample for albumin, especially at albumin levels of 0 mg/dL to about 30 mg/dL.
The prior art contains numerous references relating to the wet phase and the dry phase chemistry utilized in the pH indicator dye method of assaying urine for proteins. For example, Keston U.S. Pat. No. 3,485,587 discloses the basic dye binding technique used to assay for proteins at a constant pH. Keston teaches utilizing a single indicator dye, maintained at a constant pH slightly below the pK.sub.a (acid dissociation constant) of the dye and impregnated into a dry test paper, like filter paper, to determine the presence or concentration of albumin by monitoring the color transition of the dye. Free et al., in U.S. Pat. No. 3,095,277, also disclose a method of detecting the albumin content of liquid test samples by incorporating a suitable indicator composition into a bibulous carrier, like untreated filter paper. Similarly, Atkinson et al., in U.S. Pat. No. 3,438,737, disclose a test device comprising a test composition impregnated into an untreated bibulous matrix, such as filter paper, wood strips, synthetic plastic fibrous materials, nonwoven fabrics and woven fabrics for detecting protein in fluids.
Rittersdorf et al., in U.S. Pat. No. 4,013,416, disclose a test strip wherein an absorbent carrier is impregnated with an octahalosulfophthalein pH indicator dye, a buffer and a water-insoluble polypropylene glycol having a molecular weight of from about 500 to about 10,000 daltons. Rittersdorf et al. teach that the water-insoluble polypropylene glycol reduces the reactivity of the indicator dye with nitrogen containing compounds, such as metabolites of pharmaceuticals, thereby reducing the blank reaction in test strips. Rittersdorf et al. also teach only that water-insoluble propylene glycols are useful, e.g. polyethylene glycols are not useful. Rittersdorf et al. do not teach or suggest the usefulness of a polymer including a hydrocarbon, or essentially a hydrocarbon, backbone including pendant polyoxyalkylene units. In contrast, the indicator reagent composition of the present invention includes a hydrophobic polymeric compound having a hydrocarbon, or essentially hydrocarbon, backbone including 1 to about 8 alkylphenol units, like nonylphenol, linked by a methylene group or oxygen group, wherein the phenol moiety of each alkylphenol unit is ethoxylated and/or propoxylated to include about 2 and up to about 20 ethoxy and/or propoxy units in total.
The above-cited references do not teach or suggest, either alone or in combination, that an indicator reagent composition including a hydrophobic polymeric compound, as depicted above in general structural formula (I), can be used in a diagnostic device to achieve a more accurate determination of the amount of an analyte, like protein, and especially low amounts of an analyte, in a test sample. The references also do not teach or suggest, alone or in combination, that such an indicator reagent composition substantially reduces the number of false positive assays for albumin by essentially eliminating the effects of urine specific gravity in the assay for urinary proteins.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased sensitivity in the detection and measurement of proteins in a liquid test sample, such as a biological fluid, like urine. Surprisingly and unexpectedly, by utilizing an indicator reagent composition, comprising an indicator dye, a buffer and a hydrophobic polymeric compound depicted by general structural formula (I), homogeneously impregnated into a carrier matrix, an assay of a test sample including a negative amount of protein (e.g., less than about 15 mg/dL) can be differentiated from an assay of a test sample including a trace amount of protein (e.g., about 15 to about 30 mg/dL) more accurately. Accordingly, the number of false positive assays is reduced substantially, and the number of unnecessary and costly confirmatory assays also is reduced. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase test strip assay of urine and other test samples for proteins by utilizing a test pad, comprising a carrier matrix having homogeneously incorporated therein an indicator reagent composition comprising an indicator dye, a buffer and a hydrophobic polymeric compound of general structural formula (I), that provides an accurate protein assay for samples including a negative to low trace amount of protein, and that is independent of the specific gravity of the test sample.

Composition and device for urinary protein assay and method of using the same


A motor includes a rotor portion including a shaft and a rotor magnet rotatable about a center axis, an armature arranged opposite to the rotor magnet to generate a torque therebetween, a circuit board arranged below the armature in an axial direction substantially parallel to the center axis and including a control circuit controlling rotation of the rotor portion, and a base portion including a bearing holder which supports the armature on its outer side surface and the shaft on its inner side surface via a bearing member. The armature is pressed against the bearing holder in the axial direction with an elastic member.

1. Field of the Invention
The present invention relates to an axial fan motor.
2. Description of the Related Art
An axial fan motor for cooling heat generating electronic parts is generally mounted on household electric appliances and various OA/IT equipment. Recently, demand for a large air flow capacity of the axial fan has increased because of the increase in the heat generated by, and the reduction in product costs, of household electric appliance and various OA/IT equipment. When the air flow capacity of an axial fan motor is increased, however, there is a tendency that noise due to an electromagnetic exciting force and rotation of vanes in the axial fan is increased. Further, a demand for noise reduction has increased for the pursuit of a more comfortable environment, and improvements corresponding thereto have been developed.
In JP-A-10-159792, vibration reduction is intended by providing vibration isolating rubber between a motor shaft and a fan boss.
In JP-A-2003-269381, vibration reduction is intended by providing vibration isolating rubber between an inner ring member fitted onto a motor shaft and an outer ring member fitted into a fan boss.
In JP-A-2003-286997, vibration reduction is intended by providing vibration isolating rubber between a fan shaft and a fan body.
In JP-A-07-213005, vibration reduction of a fan motor is intended by the use of an attenuation material.
In all of the above-described patent documents, a vibration attenuation material such as a vibration isolating rubber, etc., is provided in order to achieve vibration reduction, but cannot be considered to be a fundamental measure for reducing vibration since a vibration source and the vibration isolating rubber are in contact with each other.
Meanwhile, it is desirable that a stator core and a sleeve, which supports the stator core, be separated from each other as far as possible for the purpose of vibration reduction and noise reduction, and it is preferred that a contact area between the stator core and the sleeve be made small. Recently, recycling of resources is advocated, and products which may be disassembled and recycled are desired for axial fan motors.
A stator core and a sleeve, which supports the stator core, in conventional fan motors and blowers are joined together by measures such as bonding, caulking, welding, etc., but when a stator core is fixed to the sleeve by such measures, disassembly cannot be performed unless the sleeve is broken, so that recycling of the resource cannot be achieved.


A zoom lens includes, in order from object side to image side, a first lens unit having a negative refractive power and a second lens unit having a positive refractive power. During zooming, the first lens unit and the second lens unit move so that the distance between the first lens unit and the second lens unit changes. The first lens unit includes at least one positive lens and at least one negative lens. The total length of the zoom lens at the wide angle end, the back focal length at the wide angle end, the focal length of the zoom lens at the telephoto end, the focal length of the first lens unit, the focal length of the second lens unit, and the refractive index of the material of the at least one positive lens included in the first lens unit are each appropriately set according to mathematical conditions.

Field of the Invention
The present invention relates to a zoom lens and an image pickup apparatus including the zoom lens, and in particular, to a zoom lens that can be used as an image pickup optical system of an image pickup apparatus, such as a digital still camera, a video camera, a monitoring camera, a broadcast camera, or a film camera.
Description of the Related Art
It is required that a zoom lens used as image pickup optical system of an image pickup apparatus have a high optical performance. For example, for a zoom lens of a monitoring camera, it is required that the zoom lens have a super-wide view angle so that a wide area can be monitored with a single monitoring camera. Moreover, it is required that the zoom lens have a small F-number so that a clear image can be captured even at night. Furthermore, it is required that the entire zoom lens be small so that the monitoring camera can be easily set inconspicuously in monitoring locations.
Two-unit zoom lenses are known that include, from the object side to the image side, a first lens unit having a negative refractive power and a second lens unit having a positive refractive power. Two-unit zoom lenses perform zooming by moving the first and second lens units. Japanese Patent Application Laid-Open No. 8-320435 describes a small zoom lens having an angle of view of about 80 degrees at the wide angle end, an F-number of 2 at the wide angle end, and a zoom ratio in the range of about 3 to 6. Japanese Patent Application Laid-Open No. 2009-204699 describes a small zoom lens having an angle of view of about 140 degrees at the wide angle end, an F-number of 1.2 at the wide angle end, and a zoom ratio of about 3.5.

Zoom lens and image pickup apparatus including zoom lens


The present invention relates to an ink cartridge, an ink cartridge assembly and a method for detecting the installation of the ink cartridge. A plurality of ink cartridges are detachably mounted on an inkjet printer including a light receiver and a plurality of apparatus electrical contacts which are commonly connected with a line, the ink cartridge comprises a container electrical contact electrically connected with corresponding apparatus electrical contact an information storage device, a light-emitting portion configured to emit light towards the light receiver, and a control portion configured to control the light-emitting portion to emit light, wherein, the control portion of the ink cartridge controls the light-emitting portion to emit light when receiving an emission instruction sent by the inkjet printer. Therefore, the stability of the ink cartridge installation detection can be fully guaranteed and the phenomenon of installation detection error can be avoided.

A color inkjet printer is provided with an ink cartridge assembly as required. Ink of different colors is refilled into different ink cartridges, and all the ink cartridges are mounted on the printer. In order to ensure the normal printing of the inkjet printer and avoid the print error caused by the fact that the ink cartridge is mounted at a wrong position, generally, whether the ink cartridge is correctly mounted at an appropriate position in the inkjet printer must be detected in the process of ink cartridge installation. FIG. 1 is a schematic diagram illustrating the ink cartridge installation detection in the prior art. As illustrated in FIG. 1, supposing the inkjet printer is provided with four ink cartridges, each ink cartridge is respectively mounted at corresponding ink cartridge mounting position; a correct position corresponding to the ink cartridge a is a position A; a correct position corresponding to the ink cartridge b is a position B; a correct position corresponding to the ink cartridge c is a position C; and a correct position corresponding to the ink cartridge d is a position D.
An ink cartridge chip is provided with a light-emitting portion, e.g., a light-emitting diode (LED) lamp; and a light receiving portion capable of receiving light emitted by the light-emitting portion is disposed on a housing of the inkjet printer. The light-emitting portion of the detected ink cartridge will be turned on; the light receiving portion corresponds to a correct position at which the ink cartridge must be disposed; if the light receiving portion can receive light with light quantity reaching a default threshold at the position, it indicates that the detected ink cartridge has been correctly mounted; and if the light quantity does not reach the default threshold, it indicates that the ink cartridge is not mounted at the correct position and an installation error will be prompted. For instance, as illustrated in FIG. 1, when the ink cartridge b is detected, the light-emitting portion on the ink cartridge b is turned on and emits light, and the light receiving portion corresponds to the correct position B at which the ink cartridge b must be disposed; and if the light receiving portion can receive light with light quantity reaching the default threshold at the position, it indicates that the ink cartridge b has been mounted at the correct position. Subsequently, the ink cartridges a to d are integrally driven by a carriage in the inkjet printer to move along an arrowhead direction as shown in FIG. 1; the light receiving portion corresponds to the position C and the ink cartridge c is turned on; and whether the ink cartridge c is correctly mounted is detected by the above method. The process is continued until all the ink cartridges are detected.
However, in the actual detection process, the case that the ink cartridge is mounted at the correct position but is determined to be not mounted at the correct or appropriate position as the light receiving portion does not receive the light with enough light quantity will usually occur. In the final analysis, the root cause of the above problem is that: in the actual production process, due to unavoidable manufacturing error, the light quantity of light emitted by each LED lamp has difference. Therefore, when the light quantity of the light emitted by the LED lamp of the detected ink cartridge is small, as the emitted light is scattered light, the phenomenon that the light quantity of the light arriving at the light receiving portion does not reach the threshold and hence the light receiving portion determines that the ink cartridge is not mounted even if the ink cartridge has been correctly mounted in place tends to occur.
Moreover, from the viewpoint of the inkjet printer, the inkjet printer can only be adapted to specific ink cartridges and cannot be adapted to non-specific ink cartridges which are not provided with LED lamps and cannot respond to on instructions and the like, and hence the ink cartridge selection range of users can be limited and the use cost of the users can be increased. But from the viewpoint of the specific ink cartridges, the production cost of the specific ink cartridges is relatively high. Moreover, due to the limitation of the factors such as the settings of the LED lamps and the self functions of the chips, the specific ink cartridges are likely to become disposable products, namely the specific ink cartridges will be discarded after used up. Therefore, not only the use cost of the users can be increased but also recyclable resources such as the LED lamps can be wasted due to the discarding of the specific ink cartridges; and the environmental pollution can be easily caused.

Ink cartridge, ink cartridge assembly and method for detecting the installation of ink cartridge