EP1646655A2 - Ligands de tgf-beta1 - Google Patents

Ligands de tgf-beta1

Info

Publication number
EP1646655A2
EP1646655A2 EP04776554A EP04776554A EP1646655A2 EP 1646655 A2 EP1646655 A2 EP 1646655A2 EP 04776554 A EP04776554 A EP 04776554A EP 04776554 A EP04776554 A EP 04776554A EP 1646655 A2 EP1646655 A2 EP 1646655A2
Authority
EP
European Patent Office
Prior art keywords
seq
ofthe
binding
antibody
monoclonal antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04776554A
Other languages
German (de)
English (en)
Inventor
Heidi Marie Bradley
Kristine Kay Kikly
Joanne Sloan Lancaster
Scott William Rowlinson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1646655A2 publication Critical patent/EP1646655A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention generally relates to compositions related to TGF Beta proteins.
  • it provides purified binding compositions, and related reagents useful, e.g., in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders, and in the assessment ofthe effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of such proteins.
  • TGF-Beta transforming growth factor-beta
  • TGF Beta family members control many cellular functions, and their activity is critical for regulating numerous developmental and homeostatic processes. Mutations in TGF-Beta family ligands are responsible for a number of human diseases, including, e.g., hereditary chondrodysplasia and persistent mullerian duct syndrome (Massague, et al., 2000 Cell 103: 295). In addition, TGF-Beta itself plays an important role in cancer progression by functioning both as an antiproliferative factor and as a tumor promoter, and numerous components ofthe signal transduction pathway are tumor suppressors that are functionally mutated in cancer (de Caestecker, et al., 2000 J. Natl. Cancer Inst. 92:1388).
  • TGF-Beta The specific member TGF Beta 1 is known to be involved in many cellular processes such as cell proliferation and differentiation, migration, differentiation, apoptosis, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and im ⁇ ammatory responses (see, e.g., Roberts, A. B., and Sporn, M. B. (1990) Peptide Growth Factors and Their Receptors, pp. 419-472, Springer-Nerlag, Heidelberg, Germany; Massague, J. (1990) Annu. Rev. Cell Biol. 6, 597-641).
  • TGF-Betal transforming growth factor Betal
  • HSCs hepatic stellate cells
  • myofibroblasts which are the main source of extracellular matrix proteins and particularly of procollagen I and III (see, e.g., Williams & Knapton (1996) Hepatology 23:1268-1275; Border & Noble (1994) N Engl J Med 1:1286-1292; Friedmann SL. (1999) Semin Liver Dis ;19:129-140; Roberts, et al.
  • TGF Beta 1 binding compositions satisfies a need in the art by providing compositions that are useful in the diagnosis, prevention, and treatment of cell proliferative, iirmime/inflammatory, cardiovascular, neurological, fibrotic, and developmental disorders.
  • the present invention is based in part upon the discovery of binding compositions specific and/or selective to TGF Beta 1.
  • the invention provides an isolated, and/or recombinant binding composition which specifically and/or selectively binds TGF Beta 1 isoform over TGF Beta 2 and/or TGF Beta 3 and which neutralizes TGF Beta 1 comprising at least one binding site comprising: at least four contiguous amino acids from QQWNGNPPA (SEQ ID NO: 24)(VL CDR3); at least four contiguous amino acids from QQWDSNPPA (SEQ ID NO: 27)(VL CDR3); at least five contiguous amino acids from YIYPYNGDTGYNQKFKS (SEQ ID NO: 14), wherein one of said at least five contiguous amino acids is D (VH CDR2); or at least five contiguous amino acids from GYYWFAY (SEQ ID NO: 15) (VH CDR3); or an isolated and/or recombinant binding
  • the binding composition further comprises an additional binding site selected from the following: RASSSVSYMH (SEQ ID NO: 22)(1021 NL CDR1); ATS ⁇ LAS (SEQ ID NO: 23)(1021 NL CDR2); GYTFTDY ⁇ MH (SEQ ID NO: 13)(1021 VH CDR1); RASQEISGYLS (SEQ ID NO: 28)(3821 NL CDR1); ATSSLDS (SEQ ID NO: 29)(3821 NL CDR2); and GYTFTDYTMH (SEQ ID NO: 19)(3821 VH CDR1); at least three said binding sites; at least four said binding sites; at least five said binding sites; at least six said bindmg sites; at least seven said binding sites; at least eight said binding sites; at least nine said binding sites; at least ten said binding sites; at least eleven said binding sites; at least twelve said binding sites; at least thirteen said binding sites; at least fourteen said binding sites; at least fifteen said binding sites; at least
  • YIYPYNGDTGYNQKFKS (SEQ ID NO: 14)(1021 VH CDR2); ATSNLAS (SEQ ID NO: 23) (1021 NL CDR2); GYYWFAY (SEQ ID NO: 15) (1021 VH CDR3); QQWNGNPPA (SEQ ID NO: 24)(1021 NL CDR3); or QQWDSNPPA (SEQ ID NO: 27)(2471 NL CDR3); wherein said CDR2 ofthe light chain variable region (LCNR) is ATS ⁇ LAS (SEQ ID NO: 23) (1021 NL CDR2); wherein said CDR2 ofthe heavy chain variable region (HCNR) is YIYPYNGDTGYNQKFKS (SEQ ID NO: 14)(1021 VH CDR2); wherein said CDRl ofthe light chain variable region (LCNR) is RASSSVSYMH (SEQ ID NO: 22)(1021 VL CDRl); wherein said CDRl ofthe
  • a monoclonal antibody ofthe invention is: is humanized; comprises of human or humanized constant regions; is a Fab fragment; is an Fv fragment; is a scFv fragment; is a F(ab)2 fragment; is detectably labeled; is lyophilized; is encoded in an isolated nucleic acid molecule; is encoded in an isolated nucleic acid molecule that is operably linked in an expression vector; is encoded in an isolated nucleic acid molecule that is operably linked in an expression vector that is inco ⁇ orated into a host cell; is a chimeric antibody; is conjugated to another chemical moiety; is sterile; or is a pharmaceutical composition; wherein the CDRl, CDR2, and CDR3 ofthe heavy
  • a binding composition ofthe invention that is a monoclonal antibody that is: a Fab fragment; is an Fv fragment; is an scFv fragment; is a F(ab)2 fragment; is fused to a human constant region; is conjugated to another chemical moiety; comprises a heavy chain constant region selected from: IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM, and IgD; comprises a human light chain constant region; is detectably labeled; is lyophilized; is a fusion protein; is sterile; or comprises a pharmaceutical composition.
  • a binding composition ofthe invention that is a monoclonal antibody that comprises at least one sequence at a CDRl, CDR2, or CDR3 ofthe light chain variable region (LCVR); or at a CDRl, CDR2, or CDR3 ofthe heavy chain variable region (HCVR) selected from: RASQEISGYLS (SEQ ID NO: 28)(3821 VL CDRl), ATSSLDS (SEQ ID NO: 29)(3821 VL CDR2), LQYASSPYT (SEQ ID NO: 30)(3821 VL CDR3), GYTFTDYTMH (SEQ ID NO: 19)(3821 VH CDRl), LITPFYGDAIYNQKFKG (SEQ ID NO: 20)(3821 VH CDR2), and GGLRRGPPFAY (SEQ ID NO: 21)(3821 VH CDR3); wherein said CDR2 ofthe light chain variable region (LCVR) is ATSSLDS (SEQ ID NO: 29) (3821 VL
  • kit embodiments include a kit comprising a described binding composition, and: a compartment comprising a binding composition ofthe invention; and/or instructions for use or disposal of reagents in the kit.
  • the kit is capable of making a qualitative or quantitative analysis.
  • Other embodiments include: a method of using the binding composition ofthe invention, comprising contacting said binding composition with a biological sample comprising an antigen, thereby forming a TGF Beta 1 bindmg compositiomantigen complex; wherein said biological sample is from a human, and wherein said binding composition is an antibody.
  • the invention provides a method of modulating the physiology or development of a mammal comprising administering a binding composition ofthe invention.
  • references to “an organism” includes, e.g., one or more different organisms
  • reference to “a cell” includes, e.g., one or more of such cells
  • reference to “a method” include, e.g., reference to equivalent steps and methods known to a person of ordinary skill in the art, and so forth.
  • all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references discussed herein are provided solely for their disclosure before the filing date ofthe present application.
  • binding composition is a molecular entity with selective and/or specific binding affinity for at least one other molecular entity or binding partner. Typically, the association will be in a naturally physiologically relevant interaction, either covalently or non-covalent, and may include members of a multi-protein complex, including, without limitation, carrier compounds, dimerization or multimerization partners.
  • a binding composition can be naturally derived (e.g., isolated and/or purified) or synthetically produced, either in whole or in part.
  • a binding composition has at least one region such as, by way of non-limiting example, a surface area, a shape (such as, e.g., a cavity, cleft, crevice, or protrusion), a molecular arrangement, or a spatial configuration, that specifically and/or selectively “fits with,” “binds to,” or is “complementary with” a particular spatial and/or polar organization of an area or region on a binding partner.
  • a binding composition when a binding composition is sufficiently proximate to a potential binding partner, the binding composition and partner will specifically and/or selectively bind each other.
  • Non-limiting examples of a binding composition paired with a binding partner include: antigen-antibody, hapten-binding site, biotin-avidin, hormone-hormone receptor, receptor-ligand, and enzyme-substrate.
  • Non-limiting examples of antibody binding compositions include: antibodies, diabodies, triabodies, tetrabodies, minibodies, Fab fragments (including, such as, e.g., dimeric or trimeric Fabs), Fv fragments, scFv fragments, F(ab)2 fragments, etc. (see, e.g., Hudson & Souriau 2003 Nature Medicine 9:129-34 for non-limiting examples of antibody binding compositions encompassed by the invention).
  • a "binding site” is a specific region, area, or configuration of a molecular entity that takes part in the specific and/or selective binding with another molecular entity.
  • a non-limiting example of a binding site is the contiguous amino acid sequence comprising a complementary determining region (CDR) of an antibody.
  • CDR complementary determining region
  • Another non-limiting example is a binding site formed from the three-dimensional configuration and spatial organization ofthe amino acid sequences comprising the six CDR loops ofthe light and heavy variable chains at the rim ofthe eight-stranded beta barrel of a Fab fragment (see, e.g., Chothia & Lesk, 1987 J. Mol. Biol. 196:901-17).
  • binding composition TGF Beta 1 complex
  • TGF Beta 1 complex refers to a complex of a binding composition and a TGF Beta 1 protein that is formed by specific and/or selective binding ofthe binding composition to the TGF Beta 1 protein.
  • the TGF Beta 1 referred to throughout is a primate TGF Beta 1 protein.
  • the TGF Beta 1 referred to throughout is a human TGF Beta 1 protem.
  • Specific binding ofthe binding composition means that the binding composition has a binding site that recognizes a region ofthe TGF Beta 1 protein, typically in its native active conformation, but possibly in a denatured conformation, e.g., a Western blot.
  • antibodies raised to a TGF Beta 1 protein and recognizing an epitope ofthe TGF Beta 1 protein are capable of fomiing a binding composition:TGF Beta 1 complex by specific binding.
  • TGF Beta 1 complex by specific binding.
  • the formation of a binding compositiomTGF Beta 1 protein complex allows the measurement of TGF Beta 1 protein in a biological sample, e.g., a mixture with other proteins and biologies.
  • specific binding refers 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. Accordingly, a binding composition specific for TGF Beta 1 "will not show any significant binding to molecules other than its specific binding partner(s)" that is, TGF Beta 1.
  • the specified binding composition binds a specific protein and does not significantly bind other proteins present in the sample.
  • Specific bindmg to a binding composition under such conditions may require a binding composition that is selected for its specificity for a particular protein.
  • a binding composition such as an antibody, raised to the human TGF Beta 1 amino acid sequence depicted in SEQ ID NO:X can be selected to obtain antibodies specifically immunoreactive with that TGF Beta 1 protem and not with other proteins.
  • a binding composition could differentiate proteins highly homologous to the human TGF Beta 1 protein, e.g., such as, for example, the other human TGF Beta isoforms (TGF Beta 2, or TGF Beta 3).
  • a binding composition ofthe invention preferably neutralizes TGF Beta 1 and has a dissociation constant (Kd) for TGF Beta 1 preferably of less than about: lOOnM, 95nM, 90nM, 85nM, 80nM, 75nM, 70nM, 65nM, 60nM, 55nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM, 14nM, 13nM, 12nM, 1 InM, or lOnM; more preferably less than about: lOnM, 9.9nM, 9.8nM, 9.7nM, 9.6nM, 9.5nM, 9.4nM, 9.3nM, 9.2nM, 9.1nM 9.0nM, 8.9nM, 8.8nM, 8.7nM, 8.6nM, 8.5nM, 8.4nM, 8.3nM, 8.2nM, 8.
  • Kd dissociation constant
  • InM or 5.0nM even more preferably of less than about 5.0nM, 4.9nM, 4.8nM, 4.7nM, 4.6nM, 4.5nM, 4.4nM, 4.3nM, 4.2nM, 4.1nM 4.0nM, 3.9nM, 3.8nM, 3.7nM, 3.6nM, 3.5nM, 3.4nM, 3.3nM, 3.2nM, 3.
  • the affinity constant (Kd) of a binding composition ofthe invention can be determined using any art method, for example, by BIACoreTM, adapting the method of Karlsson et al., 1991 J. hrrmunol. Methods 145, 299- 240.
  • An antibody binding composition preferably neutralizes TGF Beta 1 and has a dissociation constant (Kd) for TGF Beta 1 of preferably less than about: lOOnM, 95nM, 90nM, 85nM, 80nM, 75nM, 70nM, 65nM, 60nM, 55nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM, 14nM, 13nM, 12nM, l lnM, or lOnM; more preferably less than about: lOnM, 9.9nM, 9.8nM, 9.7nM, 9.6nM, 9.5nM, 9.4nM, 9.3nM, 9.2nM, 9.1nM 9.0nM, 8.9nM, 8.8nM, 8.7nM, 8.6nM, 8.5nM, 8.4nM, 8.3nM, 8.2nM, 8.1nM 8.0nM, 7.9nM, 7.8n
  • InM or 5.0nM even more preferably of less than about 5.0nM, 4.9nM, 4.8nM, 4.7nM, 4.6nM, 4.5nM, 4.4nM, 4.3nM, 4.2nM, 4.1nM 4.0nM, 3.9nM, 3.8nM, 3.7nM, 3.6nM, 3.5nM, 3.4nM, 3.3nM, 3.2nM, 3.1rjM 3.0nM 2.9nM, 2.8nM, 2.7nM, 2.6nM, 2.5nM, 2.4nM, 2.3nM, 2.2nM, 2.1nM 2.0nM, 1.9nM, 1.8nM, 1.7nM, 1.6nM, 1.5nM, 1.4nM, 1.3nM, 1.2nM, l.lnM, LOnM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.55nM, 0.5nM, 0.45nM, 0.4n
  • an antibody binding composition specifically and/or selectively binds TGF Beta 1 as compared to TGF Beta 2 and/or TGF Beta 3, more preferably, an antibody binding composition specifically and/or selectively binds human TGF Beta 1 as compared to human TGF Beta 2 and/or human TGF Beta 3.
  • such an antibody has less than about 20% cross-reactivity with TGF Beta 2 and/or TGF Beta 3 (as measured by the ratio ofthe dissociation constants), more preferably less than about 15% cross-reactivity, and even more preferably has less than about 10% cross-reactivity.
  • an antibody binding composition preferably recognizes the active but not the latent form of TGF Beta 1 , more preferably the active but not the latent form of human TGF Beta 1.
  • a preferred property desired for such an antibody to be effective in the amelioration of fibrotic diseases are similar to those of a binding composition ofthe invention against TGF Beta 1 as described herein.
  • Neutralize or “antagonize” with respect to a binding composition of the invention refers to the situation in which the specific and/or selective binding of a binding composition ofthe invention to another molecular entity results in the abrogation or inhibition ofthe biological effector function ofthe other molecular entity bound with the binding composition.
  • TGF Beta 1 the term “neutralize” or “antagonize” in reference to a binding composition ofthe invention is intended to refer to a binding composition whose binding to or interaction with TGF Beta 1 results in inhibition of a biological activity induced by TGF Beta 1.
  • Inhibition of TGF Beta 1 biological activity can be assessed by measuring one or more in vitro or in vivo indicators of TGF Beta 1 biological activity including, but not limited to, inhibition of receptor binding, inhibition of fibrosis, inhibition of chemotaxis, or inhibition of signal transduction in a TGF Beta 1 binding assay (see, e.g., EP 0 945 464 for non-limiting examples of encompassed neutralization assays).
  • Indicators of TGF Beta 1 biological activity can be assessed by any known in vitro or in vivo assay known in the art.
  • the ability of a binding composition ofthe invention to neutralize or antagonize TGF Beta 1 activity is assessed by use ofthe assay as described in Example 3 herein.
  • the neutralizing activity of a binding composition such as, for example, an antibody described herein can be tested by any method known in the art.
  • testing can be carried out in a modification of an assay for TGF as described by Randall et al (1993) J. Immunol Methods 164, 61-67.
  • This assay is based on the ability of TGF Beta 1 and TGF Beta 2 to inhibit the interleukin-5 (IL-5) induced proliferation ofthe erythroleukaemia cell line TF1 and on the ability to reverse the TGF Beta inhibition with TGF Beta specific binding compositions.
  • IL-5 interleukin-5
  • the assay is reported to be rapid, reproducible and sensitive to less than 500 fg/ml of TGF-beta 1, and 5-10 pg/ml TGF-beta 2.
  • the assay is also reported to be 100- 1000-fold less sensitive to other inhibitory molecules such as interferon-beta, interferon-gamma and TNF-alpha.
  • the assay is also reported to be capable of being made specific for TGF-beta 1 or TGF-beta 2 by including specific neutralizing antibodies for TGF-beta 1 or TGF-beta 2 and to recognize all the readily available recombinant molecular species of these molecules as well as the natural proteins produced from human and bovine platelets and to detect TGF- beta in serum samples.
  • TGF-Beta 1 transforming growth factor betal
  • Increasing TGF-Beta 1 expression in the liver of such animals leads to fibrotic disease states that are reversible by switching off TGF Beta 1 expression — even after liver mass has been reduced 59%.
  • Use of this model permits one to evaluate the effects of binding compositions ofthe invention on inhibiting TGF Beta 1 biological functions by comparing a binding composition's effects with the effects of switching off TGF Beta 1 expression.
  • Antibodies Antibody binding compositions ofthe invention include, e.g., without limitation, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies ofthe invention), and an epitope-binding fragment of any ofthe above.
  • the term "antibody,” as used herein refers to immunoglobulin compositions and immunologically active portions of immunoglobulin compositions, e.g., a binding composition molecule that contains a binding site that specifically and/or selectively binds an antigen.
  • An immunoglobulin composition ofthe invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or a subclass of an immunoglobulin molecule.
  • an antibody is a human antigen-binding antibody fragment ofthe present invention such as, e.g., without limitation, Fab, Fab' and F(ab')2, Fd, 6 single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments may comprise the variable region(s) alone or in combination with the entirety or a portion ofthe following: a hinge region, a CHI, a CH2, or a CH3 domain or combinations thereof. Also included in the invention is, e.g., without limitation, an antigen-binding fragment that also can comprise any combination of variable region(s) with a hinge region, e.g., such as a CHI, CH2, or a CH3 domain or combinations thereof.
  • An antibody ofthe invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, rabbit, goat, guinea pig, camel, horse, or chicken.
  • human antibodies includes, e.g., without limitation, antibodies having an amino acid sequence of a human immunoglobulin including, e.g., without limitation, an antibody isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described herein or, as taught, e.g., in U.S. Patent No. 5,939,598.
  • a binding composition may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of a target protein, polypeptide (or fragment thereof) or may be specific for both a TGF Beta 1 as well as for a heterologous epitope, such as a heterologous TGF Beta isoform or solid support material (see, e.g., WO 2093/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al. (1991) J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893;
  • a binding composition may be described or specified i terms of an epitope(s) or portion(s) of a TGF Beta 1 protein (or fragment thereof) that it recognizes or selectively and/or specifically binds.
  • An epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or as listed in an accompanying Table and/or Figure as described herein.
  • an antibody that specifically binds an epitope, polypeptide, protein, or fragment of a polypeptide or protein may also be specifically excluded from this invention.
  • the present invention can encompass a first (or other) antibody that specifically binds a polypeptide or protein, or fragment thereof, and, at the same time, it can exclude a second (or other) antibody that may also selectively bind the same protein or polypeptide, or fragment thereof, e.g., by binding a different epitope.
  • Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity.
  • Antibodies that do not bind any other analog, ortholog, paralog, or homolog of a target protein, polypeptide (or fragment thereof) are included. Further encompassed by the present invention is an antibody that selectively binds a polypeptide, which is encoded by a polynucleotide that stably hybridizes, under stringent hybridization conditions (as described herein), to a TGF Beta polynucleotide sequence.
  • a binding composition may also be characterized or specified in terms of its binding affinity to a protein or polypeptide (fragment thereof), or epitope.
  • a preferred binding affinity of a binding composition includes, e.g., a binding affinity that demonstrates a dissociation constant or Kd of less than about: 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 "3 M, 5 X 10 "4 M, 10 "4 M, 5 X 10 "5 M, 10 "5 M, 5 X 10 "6 M, 10- 6 M, 5 X 10 "7 M, 10 _7 M, 5 X 10 '8 M, 10 _8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10- 10 M, 10 0 M, 5 X 10 'n M, 10- ⁇ M, 5 X 10- 12 M, 10 "12 M, 5 X 10 _ 13 M, 10 "13 M, 5 X 10 "14 M, 10 "14 M, 5 X 10 "15 M, or 10 _15 M.
  • the invention also encompasses antibodies that competitively inhibit binding of a binding composition to an epitope of a TGF Beta 1 as determined by any known art method for determining competitive binding, e.g., the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
  • Antibodies ofthe present invention may act as antagonists of TGF Beta 1 (or fragment thereof).
  • an antibody or binding composition of present invention can disrupt, e.g., an interaction, either partially or completely, of TGF Beta 1 with its cognate receptor/ligand.
  • antibodies ofthe present invention bind an antigenic epitope of TGF Beta 1, or a portion thereof.
  • antibodies that bind a ligand and prevent it binding to a receptor are also encompassed.
  • ligand-binding antibodies that inhibit receptor activation without inhibiting receptor binding are also included.
  • Antibodies ofthe present invention may be used, e.g., without limitation, to purify, detect, or target a TGF Beta 1 (or fragment thereof) for, e.g., in vitro and/or in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and/or quantitatively measuring levels of TGF Beta 1 (or fragment thereof) ofthe present invention in a biological sample (see, e.g., Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, cur. ed.).
  • a binding composition ofthe present invention may be used either alone or in combination with other compositions.
  • a binding composition such as an antibody may be recombinantly fused to a heterologous polypeptide at the N- or C-terminus, or chemically conjugated (including covalently and non-covalently conjugations) to a polypeptide or other compositions.
  • antibodies ofthe present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins (see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387).
  • a binding composition ofthe invention includes, e.g., derivatives that are modified, e.g., by the covalent attachment of any type of molecule to, e.g., an antibody such that the covalent attachment does not prevent the antibody from generating an anti- idiotypic response.
  • an antibody derivative includes, e.g., antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, e.g., but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, a derivative may contain one or more non-classical amino acids.
  • a binding composition may be generated by any suitable known art method.
  • adjuvants may be used to increase an irnmunological response depending on the host species, these include, e.g., without limitation, Freund's (complete and incomplete), mineral gels such as e.g., aluminum hydroxide, surface active substances such as e.g., lysolecithin, plutonic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as, e.g., BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • corynebacterium parvum Such adjuvants are known in the art.
  • Monoclonal antibodies can be prepared using a variety of art known techniques including, e.g., the use of hybridoma, recombinant, and phage display technologies, or combinations thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, e.g., in Harlow, et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, current edition); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-68 1 (Elsevier, N.Y., 1981).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • mice are immunized with a polypeptide ofthe invention or a cell expressing such a polypeptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested, and splenocytes isolated.
  • the splenocytes are then fused by known techniques to any suitable myeloma cells; e.g., SP20 cells (available from the ATCC).
  • Hybridomas are then selected and cloned by limited dilution.
  • the hybridoma clones are then assayed by art known methods to discover cells that secrete antibodies that bind a binding composition polypeptide (or fragment thereof).
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody ofthe invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a binding composition polypeptide.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain ofthe heavy chain.
  • a binding composition can also be generated using various phage display methods known in the art in which functional antibody domains are displayed on the surface of phage particles, which carry a polynucleotide sequence encoding them.
  • a phage display method is used to display antigen- binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage that express an antigen binding domain that binds an antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • phage used in these methods are filamentous phage including, e.g., fd and M 13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods contemplated for use include, e.g., those of Brinkman, et al., J. Immunol. Methods
  • Fab, Fab' and F(ab')2 fragments can also be employed using art known methods such as, e.g., WO 92122324; Mullinax, et al., BioTechniques 12(6):864-869 (1992); and Sawai, et al., AJRI 34:26-34 (1995); and Better, et al., Science 240:1041-1043 (1988).
  • Examples of producing single-chain Fvs and antibodies include, e.g., U.S. Patents 4,946,778 and 5,258,498; Huston, et al., Methods in Enzymology 203:46-88 (1991); Shu, et al., Proc. Natl. Acad. Sci. USA
  • a chimeric antibody is a molecule in which different portions ofthe antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Humanized antibodies are antibody molecules from non-human species that bind a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues ofthe human framework regions are substituted with a corresponding residue from a CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by any known art method, e.g., by (1) modeling the interactions of a CDR and framework residues to identify framework residues important for antigen binding and/or (2) by sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Patent No. 5,585,089, Riechmann, et al., Nature 332:323 (1988).
  • Antibodies can be humanized using a variety of known techniques icluding, e.g., CDR-grafting (see, e.g., EP 239,400; WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (see, e.g., EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka, et al., Protein Engineering 7(6):805-814 (1994); Roguska, et al, Proc. Natl. Acad. Sci.
  • CDR-grafting see, e.g., EP 239,400; WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089)
  • veneering or resurfacing see, e.g., EP 592,106; EP 519,596
  • Human antibodies can be made using a variety of known methods including, e.g., phage display methods described herein using antibody libraries derived from human immunoglobulin sequences (see, e.g., U.S. Patent Nos. 4,444,887 and 4,716,111; and WO 98/46645, WO 98/150433, WO 00/58513104 WO 98/124893, WO 98/116654, WO 96/134096, WO 96/133735, and WO 91/10741).
  • Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • human variable regions, constant regions, and diversity regions may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional (separately or simultaneously) with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion ofthe JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a (or fragment thereof) polypeptide ofthe invention.
  • Monoclonal antibodies directed against the antigen can be obtained from an immunized, transgenic mouse using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies.
  • antibodies ofthe invention can, in turn, be used to generate anti-idiotype antibodies that "mimic" a polypeptide (or fragment thereof) ofthe invention using known techniques (see, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. (1991) Immunol. 147(8):2429-2438).
  • antibodies that bind and competitively inhibit polypeptide multimerization and/or competitively inhibit binding of a polypeptide ofthe invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize a polypeptide and/or its ligand.
  • anti-idiotypes, or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize a polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide ofthe invention (or fragment thereof) and/or to bind its ligand/receptor, and thereby block its biological activity.
  • the invention further provides a nucleic acid molecule comprising a polynucleotide sequence encoding a binding composition ofthe invention and/or a fragment thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to a polynucleotide that encodes a binding composition, preferably, that specifically and/or selectively binds a mature polypeptide or protein ofthe invention, preferably, a binding composition that binds to TGF Beta 1.
  • the polynucleotides may be obtained, and the nucleotide sequence ofthe polynucleotides determined, by any known art method.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier, et al., (1994) BioTechniques 17:242), which, briefly described, involves synthesizing overlapping oligonucleotides containing portions ofthe sequence encoding the antibody, annealing, and ligating those oligonucleotides, then, amplifying the ligated oligonucleotides using a polymerase chain reaction.
  • oligonucleotides e.g., as described in Kutmeier, et al., (1994) BioTechniques 17:242
  • a polynucleotide encoding an antibody can be generated from nucleic acid of any suitable source. If a clone containing a nucleic acid molecule encoding a particular antibody is not available, but, however, the sequence ofthe antibody molecule is known, then a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source.
  • the source may be an antibody cDNA library, or a cDNA library generated from poly A+ RNA, isolated from any tissue or cell expressing the antibody of interest, such as, e.g., a hybridoma cells selected to express an antibody ofthe invention by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a polynucleotide sequence of interest or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody, a nucleic acid molecule for the antibody can be generated.
  • a hybridoma cells selected to express an antibody ofthe invention by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a polynucleotide sequence of interest or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a
  • Amplified nucleic acids may be cloned into replicable cloning vectors using any known art method.
  • the nucleotide sequence ofthe antibody may be manipulated using any known art method, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. to generate antibodies having a different amino acid sequence to create amino acid substitutions, deletions, and/or insertions (see, e.g., Sambrook, et al, and Ausubel, et al., eds., cur. ed., Current Protocols in Molecular Biology, John Wiley & Sons, NY).
  • the aniino acid sequence ofthe heavy and/or light chain variable domains may be inspected to identify the sequences of complementarity determining regions (CDRs) by known methods, e.g., by comparing known amino acid sequences of other heavy and chain light variable regions to determine regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody as here for the mAbs 1021 , 2471 , and 3821.
  • the framework regions may be naturally occurring or consensus framework regions, and are preferably human framework regions (for a listing of human framework regions see, e.g., Chothia, et al. (1998) J.
  • the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specifically and/or selectively binds TGF Beta 1 (or fragment thereof).
  • one or more amino acid substitutions may be made within the framework regions to improve binding ofthe antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable-region, cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • chimeric antibody techniques can be used by, e.g., splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity (see, e.g., Morrison, et al, Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger, et al., Nature 312: 604-608 (1984); Takeda, et al., Nature 314:452-454 (1985)).
  • a chimeric antibody e.g., humanized antibodies
  • techniques can be adapted to produce single chain antibodies (see, e.g., U.S. Patent No. 4,946,778; Bird, Science 242:423-42 (1988); Huston, et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward, et al, Nature 334:544-54 (1989)).
  • Single chain antibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an ammo acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra, et al. (1988) Science 242: 1038- 1041).
  • a binding composition polynucleotide "fragment” encompasses a short polynucleotide of a nucleic acid molecule, which is a portion of a sequence described in a Table herein or a portion of a polynucleotide sequence of a S ⁇ Q ID NO: .
  • Polynucleotide fragments ofthe invention encompass a polynucleotide sequence that is preferably at least about 15 nucleotides, more preferably at least about: 20, 21, 22, 24, 26, or 29 nucleotides, favorably at least about: 30, 32, 34, 36, 38, or 39 nucleotides, and even more preferably, at least about: 40, 42, 44, 46, 48, or 49 nucleotides, desirably at least about: 50, 52, 54, 56, 58, or 59 nucleotides, particularly at least about 75 nucleotides, or at least about 150 nucleotides in length.
  • "at least about” includes, e.g., a specifically recited value (e.g.,
  • nucleotide 20nt and a value that is larger or smaller by one or more nucleotides (e.g., 5, 4, 3, 2, or 1), at either terminus or at both termini.
  • these fragments encode a polypeptide possessing biological activity as defined herein, e.g., immunogenicity, or antigenicity.
  • a polynucleotide fragment can be used as a probe or primer as discussed herein.
  • the present invention also encompasses a polynucleotide that stably hybridizes to a polynucleotide fragment described herein under either stringent or lowered stringency hybridization conditions.
  • polypeptides encoded by a polynucleotide fragment or a hybridized polynucleotide stably bound to a polynucleotide fragment ofthe invention encompasses an amino acid sequence that is a portion of a sequence in a Table herein or a portion of a polynucleotide sequence of a S ⁇ Q ID NO:.
  • Protein and/or polypeptide fragments or segments may be "free-standing, 5' ' or they may comprise part of a larger polypeptide or protein, of which the fragment or segment forms a portion or region, e.g., a single continuous region of a SEQ JD NO: herein connected in a fusion protein.
  • a polypeptide segment can have a length of contiguous amino acids that is at least about: 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 120, 130, 140, or 150 contiguous amino acids in length.
  • a plurality will be at least two, more usually at least three, and preferably at least: four, five, six, seven, eight, nine, or more. While minimum lengths of a segment are provided, maximum lengths of various sizes are also encompassed for any specific plurality of segments, e.g., a plurality of three segments in toto could have one segment of length 7 contiguous amino acids, and two additional non- overlapping segments, each of which has a length of 12.
  • Features of one ofthe different genes should not be taken to limit those of another ofthe genes.
  • polypeptide fragments or segments that characterize structural or functional domains, such as, fragments, or combinations thereof, that comprise e.g., alpha-helix, and alpha-helix forming regions, beta-sheet, and beta-sheet-forming regions, turn, and turn-forming regions, coil, and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, loop regions, hairpin domains, beta-alpa-beta motifs, helix bundles, alpha/beta barrels, up and down beta barrels, jelly roll or swiss roll motifs, transmembrane domains, surface-forming regions, substrate binding regions, transmembrane regions, linkers, immunogenic regions, epitopic regions, and high antigenic index regions.
  • Polypeptide fragments of a sequence herein falling within conserved domains are specifically encompassed by the present invention. Moreover, polynucleotides encoding these domains are also encompassed. Other preferred polypeptide segments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a binding composition polypeptide (or fragment thereof). The biological activity ofthe fragments may include, e.g., an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention. Preferably, the polynucleotide fragments encode a polypeptide that demonstrates a functional activity.
  • the phrase "functional activity" encompasses a polypeptide segment that can accomplish one or more known functional activities.
  • Such functional activities include, e.g., without limitation, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an antibody binding composition to a polypeptide ofthe invention], immunogenicity (ability to generate antibody that binds to a polypeptide ofthe invention), ability to form multimers with a polypeptide ofthe invention, and the ability to bind to a receptor or ligand of a polypeptide ofthe invention.
  • the functional activity of a polypeptide can be assayed by various methods.
  • immunoassays known in the art can be used, including, e.g., without limitation, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.)
  • agglutination assays e.g., gel agglutination assays, hemagglutination assays, complement fixation assays, immunofluorescence as
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope ofthe present invention.
  • binding can be assayed, e.g., by using reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting (see generally, Phizicky, et al. (1995) Microbial. Rev. 59:94-123).
  • physiological correlates of binding of a polypeptide to its substrates can be assayed with common techniques.
  • assays described herein can routinely be applied to measure the ability of a binding composition ofthe invention (its fragments, variants derivatives and analogs thereof) to modulate a related biological activity (either in vitro or in vivo) of TGF Beta 1.
  • An antibody binding composition of the invention can be produced by any known art method, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • Recombinant expression of a binding composition ofthe invention, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody ofthe invention or a single chain antibody ofthe invention, requires construction of an expression vector containing a polynucleotide sequence that encodes the antibody.
  • the vector for the production ofthe antibody molecule may be produced by known recombinant DNA technology techniques. Methods known in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, e.g., without limitation, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the invention thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule ofthe invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably-linked to a promoter.
  • Such vectors may include, e.g., the nucleotide sequence encoding the constant region ofthe antibody molecule (see, e.g., WO 86/05807; WO 89/01036; or U.S. Patent No. 5,122,464) and the variable domain ofthe antibody may be cloned into such a vector for expression ofthe entire heavy or light chain.
  • an expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured to produce an antibody.
  • the invention includes, e.g., host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chain antibody ofthe invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in a host cell for expression ofthe entire immunoglobulin molecule, as detailed herein or known in the art.
  • a variety of host-expression vector systems may be utilized to express antibody molecules ofthe invention. Such host-expression systems represent vehicles by which any coding sequence of interest may be produced and subsequently purified.
  • host- expression system cells may also represent an antibody molecule ofthe invention in situ.
  • These cells include, e.g., without limitation, microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences
  • yeast e.g., Saccharomyces, Pichia
  • insect cell systems infected with recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 cells harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, - .O- are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking, et al. (1986) Gene 45:101 ; Cockett, et al. (1990) Bio/Technology 8:2).
  • a number of expression vectors may be advantageously selected depending upon the intended use ofthe expressed antibody molecule.
  • vectors that direct the expression of high levels of fusion protein products, which are readily purified may be desirable.
  • Such vectors include, e.g., without limitation, the E. coli expression vector pUR278 (Ruther, et al., EMBO J. 2: 1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pLN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3 101- 3 109 (1985); Van Heeke and Schuster, J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include, e.g., thrombin, or factor Xa protease cleavage sites so that the cloned target gene product can be released from a GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the AcNPV virus grows in Spodopteru frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • an AcNPV promoter e.g., the polyhedrin promoter
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome using in vitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region El or E3) results in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (see, e.g., Logan and Shenk, (1984) Proc. Natl. Acad. Sci. USA 8 1 :355-359).
  • Specific initiation signals may be required for efficient translation of inserted antibody coding sequences. These signals include, e.g., the ATG initiation codon, and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame ofthe desired coding sequence to ensure proper translation ofthe entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner, et al., Methods in Enzymol. 153:5 1-544 (1987)).
  • a host cell strain may be chosen that modulates the expression ofthe inserted sequence, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of a protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of a foreign protein that is expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper: processing ofthe primary transcript, glycosylation, and phosphorylation may be used.
  • Such mammalian host cells include, e.g., without limitation, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and, in particular, breast cancer cell lines such as, e.g., BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, e.g., CRL7030 and Hs578Bst.
  • breast cancer cell lines such as, e.g., BT483, Hs578T, HTB2, BT20 and T47D
  • normal mammary gland cell line such as, e.g., CRL7030 and Hs578Bst.
  • stable expression is preferred.
  • cell lines that stably express an antibody molecule may be engineered.
  • host cells can be transformed with controlled by appropriate expression control elements (e.g., promoter, a polynucleotide sequence enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, a polynucleotide sequence enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • selectable marker in a recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and to form foci that can be subsequently cloned and expanded into cell lines. This method can be used to engineer cell lines that express an antibody molecule.
  • Such engineered cell lines are particularly useful in screening and evaluating compounds that interact either directly or indirectly with an antibody molecule ofthe invention.
  • a number of selection systems can be used, including, e.g., without limitation, herpes simplex virus ti ymidine kinase (Wigler, et al., Cell 11 :223 (1977)) in the cells, hypoxanthine-guanine phosphoribosyltransferase in hgprt- cells (Szybalska and Szybalski, Proc. Natl. Acad. Sci.
  • anti- metabolite resistance can be used as the basis of selection for the following genes: dhfr — which confers resistance to methotrexate (Wigler, et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare, et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt— which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci.
  • neo hich confers resistance to the aminoglycoside G-20418 (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991)); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 19 1-2 17 (1993); May, 1993, TJB TECH 1 1(5): 155-215); and hygro — which confers resistance to hygromycin (Santerre, et al., Gene 30: 147 (1984)).
  • Expression levels of an antibody molecule can be increased by vector amplification (see, e.g., Bebbington and Hentschel, Vol. 3 (Academic Press, New York, 1987)).
  • a marker in the vector-system expressing antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies ofthe marker gene. Since the amplified region is associated with the antibody gene, production ofthe antibody also increases (Grouse, et al., Mol. Cell. Biol. 3:257 (1983)).
  • the host cell may be co-transfected with two expression vectors ofthe invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers that enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used that encodes, and is capable of expressing, both heavy and light chain polypeptides.
  • the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
  • Coding sequences for heavy and light chains may comprise cDNA or genomic
  • an antibody molecule ofthe invention may be purified by any known method, e.g., by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other technique for protein purification.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or any other technique for protein purification.
  • a binding composition or fragments thereof can be fused to heterologous polypeptide sequences to facilitate purification using any art known method or one described herein.
  • the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a polypeptide (or portion thereof, preferably comprising at least: 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 contiguous amino acids ofthe polypeptide) to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • Antibodies fused or conjugated to a polypeptide may also be used in in vitro immunoassays and in purification methods using known art methods (see e.g., Harbor, et al., supra, and WO 9312 1232; EP 439,095; Naramura et al. (1994) Immunol.
  • the present invention further includes compositions comprising a polypeptide (or fragment thereof) fused or conjugated to an antibody domain other than a variable region.
  • a polypeptide ofthe invention or fragment thereof may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion that is fused to a polypeptide ofthe invention may comprise a constant region, a hinge region, a CHI domain, a CH2 domain, and/or a CH3 domain or any combination of whole domains or portions thereof.
  • a polypeptide (or fragment thereof) may also be fused or conjugated to an antibody portion described herein to form multimers.
  • Fc portions fused to a polypeptide ofthe invention (or fragment thereof) can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
  • a polypeptide, polypeptide fragment may be fused or conjugated to an antibody portion described herein or known in the art to increase the in vivo half-life. Further, a polypeptide, polypeptide fragment, may be fused or conjugated to an antibody portion to facilitate purification.
  • One example uses chimeric proteins comprising the first two domains ofthe human CD4-polypeptide and various domains of the constant regions ofthe heavy or lighl chains of mammalian immunoglobulins. (see, e.g., EP 394,827; Traunecker, et al. (1988) Nature 33 1 :84-86).
  • a polypeptide, polypeptide fragment, fused or conjugated to an antibody having disulfide-linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone (see, e.g., Fountoulakis, et al. (1995) 3. Biochem. 270: 3958-3964).
  • the Fc part of a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties (see, e.g., EP A232, 262).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified can be favored.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (see, e.g., Bennett, et al. (1995) J. Molecular Recognition 8:52-58; Johanson, et al. (1995) J. Biol. Chem. 270:9459-9471).
  • a bmding composition (or fragment thereof) can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available.
  • Hexa-histidine provides for convenient purification of a fusion protein (Gentz, et al. (1989) Proc. Natl. Acad. Sci. USA 86:821-824).
  • Other peptide tags useful for purification include, e.g., the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, et al. (1984) Cell 37:767) and the "flag" tag.
  • the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagriostically to, for-example, monitor the development or progression of a tumor as part of a clinical testing procedure to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include, e.g., various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
  • the detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, e.g., an art known linker) using established techniques (see, e.g., U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention).
  • suitable enzymes include, e.g., without limitation, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include, e.g., without limit, streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include, e.g., without limitation, umbelliferone, fluorescein, fluorescein iso hiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoeryfhrin;
  • an example of a luminescent material includes, e.g., without limitation, luminol;
  • examples of bioluminescent materials include, e.g., without limitation, luciferase, luciferin, and aequorin;
  • examples of a suitable radioactive material includes, e.g., I 125 ,
  • an antibody ofthe invention may be conjugated to a moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., an alpha-emitter such as, e.g., Bi 213 .
  • a cytotoxin e.g., a cytostatic or cytocidal agent
  • a therapeutic agent e.g., a radioactive metal ion
  • a radioactive metal ion e.g., an alpha-emitter such as, e.g., Bi 213 .
  • a cytotoxin or cytotoxic agent can include, e.g., any agent that is detrimental to a cell such as, e.g., without limitation, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • any agent that is detrimental to a cell such as, e.g., without limitation, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mit
  • Therapeutic agents include, e.g., without limitation, anti-metabolites (such as, e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (such as, e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (such as, e.g., daunorubicin (formerly, daunomycin) and doxorubicin), antibiotics (such as, e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and
  • a conjugate ofthe invention can be used to modify a given biological response, a therapeutic agent or drug moiety is not to be construed as being limited to typically classical chemical therapeutic agents.
  • a drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, e.g., a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin; or a protein such as, e.g., tumor necrosis factor, a-interferon, B-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent (such as, e.g.,
  • TNF-alpha TNF-beta
  • AIM I see, e.g., WO 97/33899
  • AIM II see, e.g., WO 97/34911
  • Fas Ligand see, e.g., Takahashi, et al., Int.
  • VEGI see, e.g., WO 99/23105
  • athrombotic agent or an anti-angiogenic agent e.g., angiostatin or endostatin
  • biological response modifiers such as, e.g., lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or purification ofthe target antigen.
  • Such solid supports include, e.g., without limitation, glass, cellulose, poly-acrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • Techniques for conjugating a therapeutic moiety to an antibody are known, see, e.g., Amon, et al., "Monoclonal Antibodies For mimunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld, et al. (eds.), pp. 243-56 (Alan R. Liss, Inc.1985); Hellstrom, et al., “Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson, et al.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described U.S. Patent No. 4,676,980.
  • An antibody, with or without a therapeutic moiety conjugated to it, administered alone, or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
  • An antibody (or fragment thereof) ofthe invention may be utilized for immunophenotyping of cell lines and biological samples.
  • the translation product of a TGF Beta 1 may be useful as a cell specific marker, or more specifically, as a cellular or tissue specific marker (which is differentially expressed at various stages of differentiation and/or maturation of particular cell types).
  • Monoclonal antibodies directed against a specific epitope, or combination of epitopes permit screening of cell populations expressing such a marker.
  • Various techniques can be used using an antibody ofthe invention (or fragment thereof) to screen for cells expressing a marker(s) including, e.g., without limitation, magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (see, e.g., U.S.
  • Patent 5,985,660 or Morrison, et al. (1999) Cell 96737-49).
  • These techniques permit screening of cell populations such as, e.g., might be found with hematological malignancies (e.g., ntinimal residual disease (MRD) in acute leukemic patients) and "non-self cells in transplantations to prevent Graft- ersus-Host Disease (GVHD).
  • hematological malignancies e.g., ntinimal residual disease (MRD) in acute leukemic patients
  • GVHD Graft- ersus-Host Disease
  • these techniques permit screening of hematopoietic stem and progenitor cells, which are capable of undergoing proliferation and/or differentiation, as might be found, e.g., in human umbilical cord blood.
  • MRD ntinimal residual disease
  • GVHD Graft- ersus-Host Disease
  • Immunoassays A particular protein can be measured by a variety of immunoassay methods including, e.g., without limitation, competitive and non-competitive assay systems using techniques such as, e.g., without limitation, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.
  • sandwich assays e.g., without limitation, western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, a
  • Immunoassays for measurement can be performed by a variety of methods known to those skilled in the art.
  • immunoassays to measure the protein can be either competitive or noncompetitive binding assays.
  • competitive binding assays the sample to be analyzed competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
  • the capture agent is an antibody specifically reactive with a TGF Beta 1 protein as described herein.
  • concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample.
  • the target protein present in the sample competes with labeled protein for binding to a specific binding composition, for example, a binding composition, such as an antibody, that is specifically and/or selectively reactive with the target protein.
  • the binding composition may be bound to a solid surface to effect separation of bound-labeled protein from the unbound-labeled protein.
  • the competitive binding assay may be conducted in liquid phase and a variety of techniques known in the art may be used to separate the bound-labeled protein from the unbound-labeled protein. Following separation, the amount of bound labeled protein is determined. The amount of protein present in the sample is inversely proportional to the amount of labeled protein binding.
  • a homogeneous immunoassay may be performed in which a separation step is not needed. In these immunoassays, the label on the protein is altered by the binding ofthe protein to its specific binding composition. This alteration in the labeled protein results in a decrease or increase in the signal emitted by label, so that measurement ofthe label at the end ofthe immunoassay allows for detection or quantitation ofthe protein.
  • Viable cells could also be used to screen for the effects of drugs on a TGF beta protein mediated function (e.g., second messenger levels, such as, e.g., cell proliferation; inositol phosphate pool changes, transcription using a luciferase-type assay; and others).
  • a separation step e.g., a proximity-sensitive detection system.
  • Qualitative or quantitative analysis of proteins may also be determined by a variety of noncompetitive immunoassay methods. For example, a two-site, solid phase sandwich immunoassay may be used. In this type of assay, a binding composition for the protein, for example an antibody, is attached to a solid support.
  • a second protein-binding composition which may also be an antibody, and which binds the protein at a different site, is labeled. After binding at both sites on the protein has occurred, the unbound- labeled binding composition is removed and the amount of labeled binding composition bound to the solid phase is measured. The amount of labeled binding composition bound is directly proportional to the amount of protein in the sample.
  • Western blot analysis can be used to determine the presence of proteins in a sample. Electrophoresis is carried out, for example, on a tissue sample suspected of containing the protein. Following electrophoresis to separate the proteins, and transfer of the proteins to a suitable solid support, e.g., a nitrocellulose filter, the solid support is incubated with an antibody reactive with the protein. This antibody may be labeled, or alternatively may be detected by subsequent incubation with a second labeled antibody that binds the primary antibody.
  • the immunoassay formats described above employ labeled assay components.
  • the label may be coupled directly or indirectly to the desired component ofthe assay according to methods well known in the art.
  • a variety of labels and methods may be used.
  • a radioactive label incorporating ⁇ H, 125 ⁇ 35g 5 14 or 32p was used.
  • Non-radioactive labels include proteins, which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies, which can serve as specific-binding pair members for a labeled protein.
  • the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation.
  • Antibody binding compositions reactive with a particular protein can also be measured by a variety of immunoassay methods.
  • immunoassay methods for a review of immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see Stites and Terr (eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, A Laboratory Manual, supra.
  • immunoassays to measure antisera reactive with the targeted protein can be either competitive or noncompetitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
  • the capture agent is a purified recombinant protein.
  • Other sources of proteins, including isolated or partially purified naturally occurring protein, may also be used.
  • Noncompetitive assays include sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents.
  • One of the binding compositions is used as a capture agent and is bound to a solid surface.
  • the second binding composition is labeled and is used to measure or detect the resultant complex by visual or instrument means.
  • a number of combinations of capture agent and labeled binding composition can be used.
  • a variety of different immunoassay formats, separation techniques, and labels can be used similar to those described above for the measurement of a protein. Exemplary immunoassays (without limitation) are described herein.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodiumdeoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, l%Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4°C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4°C, washing the beads in lysis buffer, and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodiumde
  • the ability ofthe antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., Western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters are modifiable to increase binding of an antibody to an antigen and to decrease background (e.g., by pre-clearing the cell lysate with sepharose beads). Further discussion of immunoprecipitation protocols can be found in, e.g.,
  • Western blot analysis generally comprises preparing a protein sample, electrophoresis ofthe sample through polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight ofthe antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF, or nylon, then blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (that recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • An ELISA assay comprises preparing an antigen, coating the well of a 96 well microtiterplate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition ofthe antigen of interest to the coated well.
  • a second antibody conjugated to a detectable compound may be added following the addition ofthe antigen of interest to the coated well.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction can be determined by, e.g., using a competitive binding assay.
  • a competitive binding assay is a radioimmunoassay comprising incubating labeled antigen (e.g., using H or I) with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and then detecting the amount of antibody bound to the labeled antigen.
  • the affinity ofthe antibody of interest for a particular antigen and the binding off-rates can be determined from the data by, e.g., Scatchard plot analysis.
  • Competition with a second antibody can also be determined using, e.g., radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., H or I) in the presence of increasing amounts of an unlabeled second antibody.
  • a labeled compound e.g., H or I
  • the present invention further encompasses binding composition-based therapies that involve administering an antibody binding composition to an animal, preferably a mammal, most preferably a primate (e.g., a human), to modulate, treat, inhibit, effect, or ameliorate one or more ofthe disclosed diseases, disorders, or conditions described herein.
  • a primate e.g., a human
  • TGF Beta 1 antibodies specific for human TGF Beta 1 have been shown to be effective in animal models for the treatment of fibrotic diseases and other diseases such as rheumatoid arthritis where the TGF receptor (TGFR) is overexpressed.
  • Antibodies against TGFR have been shown to be effective in the treatment of glomerulonephritis (W.A Border et al. Nature 346, 371-374, 1990); neural scarring (A.
  • binding compositions ofthe invention would also be effective in the amelioration of conditions, states, or diseases, such as, e.g., fibrotic diseases and immune/inflammatory conditions associated with TGF Beta 1.
  • Therapeutic binding compositions ofthe invention include, e.g., without limitation, antibodies ofthe invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acid molecules encoding antibodies ofthe invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
  • An antibody ofthe invention can be used to modulate, treat, inhibit, ameliorate, or prevent diseases, disorders, or conditions associated with aberrant expression and/or activity of a TGF Beta 1 polypeptide (or fragment thereof), including, e.g., without limitation, any one or more ofthe diseases, disorders, syndromes or conditions described herein.
  • the treatment, amelioration, and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a TGF Beta 1 polypeptide includes, e.g., without limitation, ameliorating symptoms associated with those diseases, disorders, or conditions.
  • Bindmg compositions ofthe invention such as, e.g., antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary ofthe ways in which an antibody binding composition ofthe present invention may be used therapeutically includes, e.g., without limitation, binding a TGF Beta 1 polypeptide (or a fragment thereof) locally or systemically in the body or by direct cytotoxicity ofthe antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
  • CDC complement
  • ADCC effector cells
  • An antibody of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3, and IL-7), e.g., that serve to increase the number or activity of effector cells that interact with the antibody.
  • An antibody ofthe invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy, and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that ofthe patient is preferred.
  • human antibodies, fragments derivatives, analogs, or nucleic acid molecules are administered to a human patient for therapy or prophylaxis. It is preferable to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against TGF Beta 1 polypeptides (fragments or regions thereof), for both immunoassays directed to and treatment, inhibition, amelioration or prevention therapy of syndromes, diseases, conditions, or disorders related to TGF Beta 1. Such antibodies, fragments, regions, or portions will preferably have an affinity for human TGF Beta 1.
  • binding affinities for a binding composition ofthe invention include, e.g., those with a dissociation constant or Kd less than about: 5 X 10 "2 M, 10 “2 M, 5 X 10 "3 M, 10 _3 M, 5 X lO ⁇ M, 10 "4 M, 5 X 10 "5 M, 10 " 5 M, 5 X 10- 6 M, 10- 6 M, 5 X 10 "7 M, 10 "7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 '9 M, 10 "9 M, 5 X 10 " 10 M, 10- 10 M, 5 X 10 "n M, 10 1 M, 5 X 10 "12 M, 10 _12 M, 5 X 10 _13 M, 10 "13 M, 5 X 10 '14 M, 10 "14 M, 5 X 10 "15 M, or 10 '15 M.
  • Pfaysieal Variants The invention also encompasses polypeptides having substantial amino acid sequence similarity with an amino acid sequence described herein. Amino acid sequence similarity, or sequence identity, is determined by optimizing residue matches. This changes when considering conservative substitutions as matches. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Time Warps. String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison Chapter One.
  • the invention encompasses, but is not limited to, polypeptides that are functionally related to a polypeptide encoded by the specific sequence identifiers ofthe present application.
  • Functionally related polypeptides include any polypeptide sharing a functional characteristic with a binding composition ofthe present invention (e.g., the ability to selectively and/or specifically bind TGF Beta 1).
  • Such functionally related polypeptides include, without limitation, additions or substitutions of amino acid residues within the amino acid sequence encoded by the sequences described herein; particularly, those that result in a silent change, thus producing a functionally equivalent polypeptide.
  • Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphiphatic nature ofthe residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine
  • positively charged (basic) amino acids include arginine, lysine, and histidine
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • non-classical amino acids or chemical amino acid analogs may be substituted or added into a polypeptide sequence.
  • Non-classical amino acids include, e.g., without limitation, D-isomers ofthe common amino acids; 2,4-diaminobutyric acid; a- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6- amino hexanoic acid, Aid, 2-amino isobutyric acid, 3 -amino propionic acid, ornifhine, norleucine, norvaline, hydroxyproiine, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids
  • the amino acid can be either dextrorotary (D ) or levorotary (L). While random mutations can be made to a nucleic acid molecule (using well known random mutagenesis techniques) and the resulting polypeptides can be tested for activity, site-directed mutations of coding sequences can be engineered (using well known site-directed mutagenesis techniques) to generate mutant with increased function (e.g., increased or decreased binding affinity). To design functionally related and functionally variant polypeptides, it is useful to distinguish between conserved and variable amino residues using the homology comparison tables provided herein. To preserve LP function, it is preferable that conserved residues remain unaltered and that the conformational folding ofthe LP functional sites be preserved.
  • alterations of non-conserved residues are carried out with conservative alterations (e.g., a basic amino acid is replaced by a different basic amino acid).
  • conservative alterations e.g., a basic amino acid is replaced by a different basic amino acid.
  • site-specific mutation sites are predetermined, mutants need not be site- specific.
  • Protein mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxyl- terminal fusions, e.g. epitope tags.
  • Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity.
  • Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art (e.g., by Ml 3 primer mutagenesis or polymerase chain reaction (PCR) techniques; see also, Sambrook, et al. (cur. ed.) and Ausubel, et al. (cur. ed., and Supplements).
  • the mutations in the DNA normally should not place coding sequences out of reading frames and preferably do not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins. Additions of peptide moieties to facilitate handling are familiar and routine art techniques.
  • a binding composition (including any fragment thereof, and specifically an epitope) can be combined with parts ofthe constant domain of an immunoglobulin e.g., (IgA, IgE, IgG, IgM) portions thereof (CH 1, CH2, CH3), and any combination thereof including both entire domains and portions thereof), resulting in a chimeric polypeptide.
  • immunoglobulin e.g., (IgA, IgE, IgG, IgM) portions thereof (CH 1, CH2, CH3), and any combination thereof including both entire domains and portions thereof
  • fusion proteins can facilitate purification and often are useful to increase the in vivo half-life ofthe protein. For example, this has been demonstrated for chimeric proteins comprising the first two domains of a human CD4 polypeptide and various domains ofthe constant regions ofthe heavy or light chains of mammalian immunoglobulins (EP 394,827, Traunecker, et al.
  • Fusion proteins with disulfide-linked dimeric structures can also be more efficient in binding and neutralizing other molecules than a monomeric secreted protein or sole protein fragment (Fountoulakis, et al. (1995) J. Biochem.15 270:3958- 3964).
  • Enhanced delivery of an antigen across an epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., WO 96/22024 and WO 99/104813).
  • a fusion protein can comprise various portions ofthe constant region of an immunoglobulin molecule together with a human protein (or part thereof) EP-A-O 464 533 (Canadian counterpart 2045869).
  • EP-A-O 464 533 Canadian counterpart 2045869.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus, can result in, e.g., improved pharmacokinetic properties (EP-A 0232 262.).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified may be desired.
  • the Fc portion may hinder therapy and/or diagnosis if the fusion protein is used as an immunogen for immunizations.
  • human proteins, such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists (Bennett, et al. (1995) I. Molecular Recognition 8:52-58; and Johanson, et al. (1995) J. Biol. Chem. 270:9459-9471).
  • new constructs may be made by combining similar functional domains from other proteins.
  • protein-binding or other segments may be "swapped" between different new fusion polypeptides or fragments (see, e.g., Cunningham, et al. (1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263 : 15985-15992).
  • a binding composition can be fused to a marker sequence, such as a peptide, to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as, e.g., the tag provided in a pQE vector
  • fusion constructions may be generated through the techniques of gene-shuffling, motif-shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling").
  • substantially pure refers to nucleic acid or protein or polypeptide that are removed from their natural environment and are isolated and/or separated from other contaminating proteins, nucleic acids, and other biologicals. Purity, or “isolation” may be assayed by standard methods, and will ordinarily be at least about 50% pure, more ordinarily at least about 60% pure, generally at least about 70% pure, more generally at least about 80% pure, often at least about 85% pure, more often at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in most preferred embodiments, at least 99% pure. Similar concepts apply, e.g., to binding compositions, e.g., such as antibodies ofthe invention.
  • an polypeptide from recombinant cell proteins or polypeptides.
  • Typical exemplary suitable purification procedures include, e.g., without limitation, fractionation on an ion-exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on silica or cation-exchange resins (such as, e.g., DEAE); chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration (using, e.g., Sephadex G-75); protein A Sepharose columns (e.g., to remove contaminants such as IgG); and metal chelating columns (e.g., to bind epitope-tagged forms of a binding composition polypeptide).
  • Such moieties include, without limit, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), cahnodulin binding peptide (CBP), 6- His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His moieties enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • the FLAG, c-myc, and hemagglutinin (HA) moieties enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • “Solubility" of a protein or polypeptide is reflected by sedimentation measured in Svedberg units, which are a measure ofthe sedimentation velocity of a molecule under particular conditions. The determination ofthe sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge (see, Freifelder (1982) Physical Biochemistry (2d ed.) W.H.
  • a soluble particle or polypeptide will typically be less than about 30S, more typically less than about 15S, usually less than about 10S, more usually less than about 6S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S. Solubility of a polypeptide or fragment depends upon the environment and the polypeptide.
  • the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C and more usually greater than about 22° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the size and structure ofthe polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
  • the solvent will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological solvent. Usually the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • a detergent will be added, typically a mild non- denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3-[3- cholamidopropyl)-dimethylammonio]-l -propane sulfonate), or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • CHS cholesteryl hemisuccinate
  • CHAPS 3-[3- cholamidopropyl)-dimethylammonio]-l -propane sulfonate
  • variant refers to a polynucleotide or polypeptide differing from a polynucleotide sequence or a polypeptide ofthe present invention, but retaining essential properties thereof. Generally, variants are closely similar overall in structural and/or sequence identity, and, in many regions, identical to a polynucleotide or polypeptide ofthe present invention.
  • the present invention encompasses nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide sequence that is at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, e.g., a polynucleotide coding sequence of a SEQ ID NO: (or a strand complementary thereto); a nucleotide sequence encoding a polypeptide of a SEQ ID NO:; and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., a fragment as defined herein).
  • Polynucleotides that stably hybridize to a polynucleotide fragment (as defined herein) under stringent hybridization conditions or lower stringency conditions, are also encompassed by the invention, as are polypeptides (or fragments thereof) encoded by these polynucleotides.
  • the present invention is also directed to polypeptides that comprise, or alternatively consist of, an amino acid sequence that is at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, e.g., a polypeptide sequence of SEQ ID NO: Y (or fragments thereof); a polypeptide sequence encoded by a cDNA contained in a deposited clone, and/or a polypeptide fragment of any of these polypeptides (e.g., those fragments as defined herein).
  • a polynucleotide sequence having at least some "percentage identity,” (e.g., 95%) to another polynucleotide sequence means that the sequence being compared (e.g., the test sequence) may vary from another sequence (e.g. the referent sequence) by a certain number of nucleotide differences (e.g., a test sequence with 95% sequence identity to a reference sequence can have up to five point mutations per each 100 contiguous nucleotides ofthe referent sequence).
  • test sequence for a test sequence to exhibit at least 95% identity to a referent sequence, up to 5%) ofthe nucleotides in the referent may differ, e.g., be deleted or substituted with another nucleotide, or a number of nucleotides (up to 5% ofthe total number of nucleotides in the reference sequence) may be inserted into the reference sequence.
  • the test sequence may be: an entire polynucleotide sequence, e.g., as shown in Tables 1-18, the ORF (open reading frame), or any fragment, segment, or portion thereof (as described herein).
  • determining if a particular nucleic acid molecule or polynucleotide sequence exhibits at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a binding composition polynucleotide sequence can be accomplished using known computer programs.
  • one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percentage sequence identity for a test sequence(s) relative to the reference sequence, based on the parameters of a designated program.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel, et al. supra).
  • a typical method for determining , a best overall match (also referred to as a global sequence alignment) between a test and a referent sequence can be determined using , e.g., the FASTDB computer program based on the algorithm of Brutlag, et al. (1990) Comp. App. Biosci. 6: 237-245.
  • the test and referent sequences are, e.g., both DNA sequences.
  • An RNA sequence can be compared by converting U's to T's. The result of a global sequence alignment is given in terms of a percentage identity.
  • the percentage identity is corrected by calculating the number of bases ofthe test sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percentage ofthe total bases ofthe test sequence. Whether a nucleotide is matched/aligned is determined by results ofthe FASTDB sequence alignment. This percentage is then subtracted from the percentage identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percentage identity score. The corrected score is what is used for the purposes of sequence identity for the present invention.
  • bases outside the 5' and 3' bases ofthe subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the test sequence are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base referent sequence is aligned to a 100 base test sequence to determine percentage identity.
  • the deletions occur at the 5' end ofthe referent sequence and therefore, the FASTDB alignment does not show a matched/alignment ofthe first 10 bases at the 5' end.
  • the 10 unpaired bases represent 10% ofthe sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the test sequence) so 10% is subtracted from the percentage identity score calculated by the FASTDB program.
  • a 90 base referent sequence is compared with a 100 base test sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' ofthe subject sequence, which are not matched/aligned with the test. In this case, the percentage identity calculated by FASTDB is not manually corrected. Again, only bases 5' and 3' ofthe subject sequence that are not matched/aligned with the test sequence are manually corrected. No other manual corrections are to made for the purposes ofthe present invention. Especially preferred are polynucleotide variants containing alterations, which produce silent substitutions, additions, or deletions, but do not alter the properties or activities ofthe encoded polypeptide.
  • Nucleotide variants produced by silent substitutions due to the degeneracy ofthe genetic code are preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • a further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described herein.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • a polypeptide exhibiting or having at least about, e.g., 95% "sequence identity" to another amino acid sequence may include, e.g., up to five amino acid alterations per each 100 amino acid stretch ofthe test amino acid sequence.
  • a first amino acid sequence that is at least 95% identical to a second amino acid sequence can have up to 5% of its total number of amino acid residues different from the second sequence, e.g., by insertion, deletion, or substitution of an amino acid residue.
  • Alterations in amino residues of a polypeptide sequence may occur, e.g., at the amino or carboxy terminal positions or anywhere between these terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous amino residue sections, portions, or fragments within the sequence.
  • whether any particular polypeptide sequence exhibits at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to another sequence for example, such as shown in a Table herein or to an amino acid sequence encoded by a cDNA contained in a deposited clone, can be determined conventionally by using known methods in the art, e.g., a computer algorithm such as ClustalW.
  • a preferred method for determining the best overall match uses the FASTDB algorithm of Brutiag, et al. (1990) Comp. App. Biosci. 6:237-245.
  • the result of such a global sequence alignment is given as a percentage of sequence identity, e.g., with 100% representing complete sequence identity.
  • the percent identity is corrected by calculating the number of residues ofthe test sequence that are N- and C-terminal ofthe subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent ofthe total bases ofthe test sequence. Whether a residue is matched/aligned is determined by results ofthe FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percentage identity score. This final percentage identity score is what is used for the purposes ofthe present invention.
  • FASTDB program If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90-residue subject sequence is compared with a 100-residue test sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini ofthe subject sequence, which are not matched/aligned with the test. In this case, the percent identity calculated by FASTDB is not manually corrected.
  • residue positions outside the N- and C-terminal ends ofthe subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the test sequence are manually corrected for. No other manual corrections are to made for the purposes ofthe present invention.
  • Variants encompassed by the present invention may contain alterations in the coding regions, non-coding regions, or both. Moreover, variants in which 1-2, 1-5, or 5-10 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Naturally occurring variants encompassed herein are "allelic variants," which refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Allelic variants can vary at either the polynucleotide and/or polypeptide level and both types of variants are encompassed by the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis using known methods of protein engineering and recombinant DNA technology.
  • Such variants may be generated to improve or alter the characteristics of a binding composition polypeptide (or fragment thereof).
  • one or more amino acids can be deleted from the N-terminus or C- terminus of a secreted polypeptide ofthe invention (or fragment thereof) without a substantial loss of biological function.
  • Ron, et al. (1993) J. Biol. Chem. 268: 2984-2988 reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-term nal amino acid residues.
  • interferon gamma was shown to exhibit up to ten times increased activity after 8-10 amino acid residues were deleted from the carboxy terminus (Dobeli, et al. (1988) J.
  • polypeptide lacking N- or C-terminal residues of a protein retains such activities can readily be determined by routine methods such as those described herein or known in the art.
  • the invention also encompasses, e.g., polypeptide variants that show biological activity such as, e.g., immunogenicity, or antigenicity.
  • Such variants include, e.g., deletions, insertions, inversions, repeats, and substitutions selected so as have little effect on activity using general rules known in the art. For example, teachings on making phenotypically silent amino acid substitutions are provided, e.g., by Bowie, et al.
  • a technique compares amino acid sequences in different species to identify the positions of conserved amino acid residues since changes in an amino acid at these positions are more likely to affect a protein function. In contrast, the positions of residues where substitutions are more frequent generally indicate that amino acid residues at these positions are less critical for a protein function. Thus, to a first degree, positions tolerating amino acid substitutions typically may be modified while still maintaining a biological activity of a protein.
  • a second technique uses genetic engineering to introduce amino acid changes at specific positions of a polypeptide to identify regions critical for a protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine mutations at every residue in the molecule) can be used.
  • tolerated conservative amino acid substitutions involve replacement ofthe aliphatic or hydrophobic amino acids Ala, Val, Leu and lie; replacement ofthe hydroxyl residues Ser and Thr; replacement ofthe acidic residues Asp and Glu; replacement ofthe amide residues Asn and Gin, replacement ofthe basic residues Lys, Arg, and His; replacement ofthe aromatic residues Phe, Tyr, and Tip, and replacement ofthe small- sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants ofthe present invention include, e.g., but are restricted to, e.g., (i) substitutions with one or more ofthe non-conserved- arnino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol), or (iv) fusion ofthe polypeptide with additional amino acids, such as, e.g., an IgG Fc fusion legion peptide, 0 leader or secretory sequence, or a sequence facilitating purification.
  • additional amino acids such as, e.g., an IgG Fc fusion legion peptide, 0 leader or secretory sequence, or a sequence facilitating purification.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce polypeptides with improved characteristics e.g., such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (Pinckard, et al. (1967) Clin. Exp. Immunol. 2:331-340; Robbins, et al. (1987) Diabetes 36:838-845; Cleland, et al. (1993) Crit. Rev.
  • a further embodiment ofthe invention encompasses a piotein that comprises an amino acid sequence ofthe present invention that contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions, nor more than 15 amino acid substitutions.
  • a peptide or polypeptide it is highly preferable for a peptide or polypeptide to have an amino acid sequence that comprises an amino acid sequence ofthe present invention, which contains at least: one, but not more than: 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.
  • the number of additions, substitutions, and/or deletions in an polypeptide sequence ofthe present invention or fragments thereof is at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 10-50, or 50-150; wherein conservative amino acid substitutions are more preferable than non-conservative substitutions.
  • the present invention provides reagents that will find use in diagnostic and/or therapeutic applications as described herein, e.g., in the description of kits for diagnosis.
  • measuring level of TGF Beta is intended to mean herein measuring or estimating (either qualitatively and/or quantitatively) a level of, e.g., a polypeptide (or fragment thereof) in a first sample (e.g., preferably a biological sample) either directly (e.g., by determining or estimating an absolute piotein oi mRNA level) or relatively (e.g., by comparing to a polypeptide or mRNA level in a second sample).
  • a first sample e.g., preferably a biological sample
  • the level in the first sample is measured or estimated from an individual having, or suspected of having, a disease, syndrome, disorder or condition and comparing that level to a second level, wherein the second level is obtained from an individual not having and/or not being suspected of having a disease, syndrome, disorder or condition.
  • the second level is determined by averaging levels from a population of individuals not having or suspected of having a disease, syndrome, disorder, or condition.
  • a “biological sample” is intended to mean herein any sample comprising biological material obtained from, using, or employing, e.g., an organism, body fluid, exudates, lavage product, waste product, cell (or part thereof), cell line, organ, biopsy, tissue culture, or other source originating from, or associated with, a living cell, tissue, organ, or organism, which contains, e.g., a polypeptide (or fragment thereof), a protein (or fragment thereof), a mRNA (or fragment thereof), or polynucleotide sequence (or fragment thereof) ofthe present invention, including, e.g., without limitation, a sample such as from, e.g., hair, skin, blood, saliva, semen, vomit, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum, urine, fecal matter, a lavage product, etc.
  • a sample such as from, e.g., hair, skin, blood, saliva, semen, vomit, synovi
  • a biological sample can include, e.g., without limitation, body fluids (e.g., such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) that contain a polypeptide (or fragment thereof), mRNA (or fragment thereof), a protein (or fragment thereof), or polynucleotide (or fragment thereof) ofthe present invention, by product, or, waste product; and/or other tissue source found to express a polypeptide (or fragment thereof), mRNA (or fragment thereof), or nucleic acid (or fragment thereof), by product, or, waste product; ofthe present invention.
  • body fluids e.g., such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid
  • tissue source e.g., such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid
  • Methods foi obtaining biological samples e.g., tissue biopsies, body fluids, cells, or waste products from mammals are known in the art.
  • a tissue biopsy is a preferred source.
  • the method(s) provided herein may preferably be applied in a diagnostic method and/or a kit in which a polynucleotide and/or a binding composition polypeptide (or fragment thereof) are attached to a solid support.
  • a binding composition can be used to measure protein or polypeptide expression of a recombinant cell, as a means of assessing the quality and/or quantity of transformation ofthe host cell.
  • Antibodies and other binding compositions di ected towards TGF Beta 1 may be used to purify the corresponding molecule. As described herein, antibody purification of TGF Beta 1 components is both possible and practicable.
  • Antibodies and other binding compositions may also be used in a diagnostic fashion to determine whether TGF Beta 1 protein components are present in a tissue sample or cell population using well-known techniques described herein. The ability to attach a binding composition to a TGF Beta 1 protein provides a means to diagnose disorders associated with TGF Beta 1 misregulation.
  • Antibodies and other binding compositions may also be useful as histological markers. It is likely that TGF Beta 1 protein expression is limited to specific tissue types. By directing a probe, such as an antibody to a TGF Beta 1 protein it is possible to use the probe to distinguish tissue and cell types in situ or in vitro. Diagnosis and Imaging Using an Antibody Labeled antibodies, fragments, derivatives, and analogs thereof that specifically bind TGF Beta 1 can be used for diagnostic purposes to detect, modulate, ameliorate, diagnose, or monitor diseases, disorders, syndromes, and/or conditions associated with aberrant expression and/or activity of TGF Beta 1.
  • a TGF Beta 1 polypeptide or fragment thereof
  • a biological sample such as, e.g., cells or fluids
  • Antibodies ofthe invention can be used to assay polypeptide levels in a sample, e.g., using classical immunoliistological methods known to those of skill in the art (see e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al, J. Cell . Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods typically useful for detecting polypeptide expression include, e.g., immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include, e.g., without limitation, enzyme labels, such as glucose oxidase; radioisotopes, such as, e.g., iodine ( 125 1, 121 1), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and e.g., fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as glucose oxidase
  • radioisotopes such as, e.g., iodine ( 125 1, 121 1), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • One aspect ofthe invention is the detection or diagnosis of a, condition, disease, syndrome, or disorder associated with aberrant expression of a binding composition polypeptide (o fragment thereof) in a mammal, preferably a primate, and most preferably a human primate.
  • detection or diagnosis comprises administering to a subject
  • an effective amount of a labeled binding composition that specifically and/or selectively binds a TGF Beta 1 polypeptide (or fragment thereof); detecting the labeled molecule in the subject to determine the amount of labeled molecule in relation to a typical background level to determine that the subject has a particular disease, disorder, condition, syndrome, or state that is associated with an over-expression, under-expression, or miss-expression of TGF Beta 1.
  • Background levels can be determined by various methods, including, e.g., comparing the amount of detected, labeled-molecule with a standard value previously determined for a particular system.
  • the size ofthe subject and the imaging system used are important factors in determining the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected normally ranges from about 5 to 20 militaries of 99 Tc.
  • the labeled antibody or antibody fragment preferentially accumulates at the location of a cell that contains a specific polypeptide (or fragment thereof) of interest.
  • the time interval for permitting a labeled molecule to preferentially concentrate at a site in a subject and for unbound labeled molecule to be cleared is approximately in the range of 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. In another embodiment, the time interval (following administration) is approximately in the range of 5 to 20 days or 5 to 10 days.
  • monitoring of a disease, condition, syndrome, or state of a disorder is carried out by repeating the method for initial diagnosis, e.g., one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • Presence ofthe labeled molecule can also be detected in a subject using methods art known for in vivo scanning. These methods depend, e.g., upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label.
  • Methods and devices that may be used for diagnostic methods ofthe invention include, e.g., without limitation, computed topography (CT), whole body scan such as, e.g., position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed topography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • a molecule associated with a composition of the invention is labeled with a radioisotope and is detected in a subject using a radiation responsive surgical instrument (see, e.g., U.S. Patent No. 5441,050).
  • a molecule is labeled with a fluorescing compound and is detected in the patient using a fluorescence responsive instrument.
  • a molecule is labeled with a positron emitting metal and is detected in a subject using positron emission-tomography.
  • a molecule is labeled with a paramagnetic label and is detected in a subject using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • a binding composition e.g., an antibody
  • a binding composition is used to diagnose a disorder, state, condition, syndrome, or disease associated with the expression of TGF Beta 1.
  • the same binding composition can be used in an assay to monitor a subject being treated for a TGF Beta 1 associated condition.
  • Diagnostic assays include methods that utilize the binding composition and a label to detect it in a sample, e.g., in a human body fluid or in a cell or tissue extract.
  • Binding compositions such as, e.g., antibodies, are used with or without modification, and are labeled by covalent or non-covalent attachment of a reporter molecule.
  • TGF Beta 1 (or fragment thereof), including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of TGF Beta 1 expression.
  • Normal or standard expression values are established using any art known technique, e.g., by combining a sample comprising an TGF Beta 1 polypeptide (or fragment thereof) with, e.g., antibodies under conditions suitable to form an TGF Beta 1 :binding composition (antibody) complex.
  • the amount of a standard complex formed is quantitated by various methods, such as, e.g., photometric means.
  • TGF Beta 1 polypeptide (or fragment thereof) expressed in subject, control, and samples are then compared with the standard values. Deviation between standard and subject values establishes parameters for correlating a particular disorder, state, condition, syndrome, or disease with a certain level of expression (or lack thereof) for an TGF Beta 1 polypeptide (or fragment thereof).
  • assays are repeated on a regular basis to monitor the level of TGF Beta 1 expression. The results obtained from successive assays are used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of TGF Beta 1 (either under- or over expressed) in biopsied tissue from a subject may indicate a predisposition for the development of a disorder, state, condition, syndrome, or disease of cell proliferation or it may provide a means for detecting such a disorder, state, condition, syndrome, or disease prior to the appearance of actual clinical symptoms.
  • a more definitive initial detection may allow earlier treatment thereby preventing and/or ameliorating further progression of cell proliferation.
  • a binding composition ofthe invention e.g., such as an antibody, is useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions, immune/inflammatory, cardiovascular, neurological, fibrotic, and developmental disorders. Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using a composition(s) provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by TGF Beta 1 is a target for an antagonist ofthe TGF Beta 1 protein such as a binding composition ofthe invention.
  • Recombinant and/or isolated binding compositions ofthe invention such as, e.g., antibodies
  • These reagents can be combined for use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients.
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients.
  • These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
  • This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • Another therapeutic approach included within the invention involves direct administration of reagents, formulations, or compositions by any conventional administration techniques (such as, e.g., without limit, local injection, inhalation, or systemic administration) to a subject.
  • the reagents, formulations, or compositions included within the bounds and metes ofthe invention may be targeted by any ofthe methods described herein (e.g., polynucleotide delivery techniques).
  • the actual dosage of reagent, formulation, or composition that modulates a disease, disorder, condition, syndrome, etc. depends on many factors, including the size and health of an organism, however one of one of ordinary skill in the art can use the following teachings describing methods and techniques for detennining clinical dosages (see, e.g., Spilker (1984) Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, pp. 7- 13, 54-60; Spilker (1991) Guide to Clinical Trials, Raven Press, Ltd., New York, pp. 93- 101; Craig and Stitzel (eds. 1986) Modern Pharmacology. 2d ed., Little, Brown and Co., Boston, pp. 127-33; Speight (ed.
  • Interspecies scaling of effective doses can be performed following art known principles (e.g., see, Mordenti and Chappell (1989) "The Use of Interspecies Scaling in Toxicokinetics," in Toxicokinetics and New Drug Development; Yacobi, et al. (eds.) Pergamon Press, NY).
  • Effective doses can also be extrapolated using dose-response curves derived from in vitro or animal-model test systems. For example, for antibodies a dosage is typically 0.1 mg/kg to 100 mg/kg of a recipient's body weight.
  • a dosage is between 0.1 mg/kg and 20 mg/kg of a recipient's body weight, more preferably 1 mg/kg to 10 mg/kg of a recipient's body weight.
  • homo-specific antibodies have a longer half-life than hetero-specific antibodies, (e.g., human antibodies last longer within a human host than antibodies from another species, e.g., such as a mouse, probably, due to the immune response ofthe host to the foreign composition).
  • hetero-specific antibodies e.g., human antibodies last longer within a human host than antibodies from another species, e.g., such as a mouse, probably, due to the immune response ofthe host to the foreign composition.
  • lower dosage of human antibodies and less frequent administration is often possible if the antibodies are administered to a human subject.
  • the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) by using modifications such as, e.g., lipidation.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ofthe ingredients ofthe compositions ofthe invention and instructions such as, e.g., for disposal (typically, in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products).
  • the quantities of reagents necessary for effective treatment will depend upon many different factors, including means of administration, target site, physiological state ofthe patient, and other medicaments administered. Thus, treatment dosages should be titrated to optimize safety and efficacy.
  • dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents.
  • Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage.
  • Various considerations are described, e.g., in Gilman, et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed.) Mack Publishing Co., Easton, PA. Methods for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, NJ. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier. Slow release formulations, or a slow release apparatus will often be utilized for continuous administration.
  • Binding compositions may be administered directly to the host to be treated or, depending on the size ofthe compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin before their administration.
  • Therapeutic formulations may be administered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed.) Mack Publishing Co., Easton, PA; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et al.
  • compositions comprising, e.g., a therapeutically effective amount of a composition ofthe invention in a pharmaceutically acceptable carrier.
  • “pharmaceutically acceptable carrier” means a carrier approved by a federal regulatory agency ofthe United States of America, or a regulatory/administrative agency of a state government ofthe United States or a carrier that is listed in the U.S. Pharmacopoeia or other pharmacopoeia; which is generally recognized by those in the art for use in an animal, e.g., a mammal, and, more particularly, in a primate, e.g., a human primate.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle that is administered with a composition ofthe invention.
  • a pharmaceutical carrier typically can be a sterile liquid, such as water or oils, (including those of petroleum, animal, vegetable, or synthetic origin, e.g., such as peanut oil, soybean oil, mineral oil, sesame oil and the like).
  • sterile water is a preferred carrier when a pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include, e.g., without limit, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a composition ofthe invention if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • a composition ofthe invention can be in a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc., or it can be formulated as a suppository (with traditional binders, and/or carriers, e.g., such as triglycerides).
  • Oral formulations encompassed include, e.g., without limit, standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Additional examples of suitable pharmaceutical carriers are described in the current edition of "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • compositions will contain a therapeutically effective amount of a composition ofthe invention, preferably in purified form, together with a suitable amount of carrier to provide for proper administration to a subject.
  • a formulation will suit the mode of administration.
  • a composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to, e.g., a human.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include, e.g., a solubilizing agent and a local anesthetic such as lidocaine to promote comfort at the injection site.
  • ingredients are supplied either separately or mixed in unit dosage form, e.g., as a dry lyophilized powder or water free concentrate in a hermetically sealed container (such as an ampoule or sachet indicating the quantity of active agent).
  • a composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed before administration.
  • Compositions ofthe invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include, e.g., without limit, anionic salts (such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,) and cationic salts, (e.g., such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc).
  • anionic salts such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.
  • cationic salts e.g., such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc).
  • the amount ofthe composition ofthe invention that will be effective in the modulation treatment, inhibition, amelioration, or prevention of a disease, syndrome, condition, or disorder associated with aberrant expression and/or activity of a polypeptide (or fragment thereof), or a polynucleotide (or fragment thereof) ofthe invention can be determined without undue experimentation by the ordinary artisan using standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges. Dosage requirements in a circumstance typically will depend on, e.g., the route of administration, the seriousness ofthe disease, condition, syndrome, or disorder; and the judgment ofthe practitioner or clinician.
  • compositions of the invention by any conventional administration technique (such as, e.g., without limit, local injection, inhalation, or systemic administration), to a subject with e.g., an infectious, a microbial, a bacterial, a viral or a fungal condition.
  • a composition or formulation may also be targeted to a specific cell or a receptor by any method described herein or known in the art.
  • TGF Betas The effect of TGF Betas on immune and inflammatory responses 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) leukocyte recruitment and activation.
  • a binding composition ofthe invention can be useful in ameliorating, treating, preventing, modulating, and/or diagnosing a disease, disorder, syndrome, or condition ofthe immune system, by, e.g., activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis or directed movement) of an immune cell.
  • immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
  • myeloid platelets, red blood cells, neutrophils, and macrophages
  • lymphoid B and T lymphocytes
  • a binding composition can be useful in ameliorating, treating, preventing, modulating, and/or diagnosing a disease, disorder, syndrome, and/or a condition of a hematopoietic cell.
  • a binding composition could be used to increase or inhibit the differentiation or proliferation of a hematopoietic cell, including a pluripotent stem cell such an effect can be implemented to treat, prevent, modulate, or ameliorate a disease, disorder, syndrome, and/or a condition associated with a decrease in a specific type of hematopoietic cell.
  • An example of such an irnmunological deficiency, disease, disorder, syndrome, and/or condition includes, e.g., without limitation, a blood condition (e.g.
  • a binding composition can be used to modulate hemostatic or thrombolytic activity.
  • increasing hemostatic or thrombolytic activity can treat or prevent a blood coagulation condition such as e.g., afibrinogenemia, a factor deficiency, a blood platelet disease (e.g. thrombocytopenia), or a wound resulting from e.g., trauma, surgery, etc.
  • a composition ofthe invention can be used to decrease hemostatic or thrombolytic activity or to inhibit or dissolve a clotting condition.
  • Such compositions can be important in a treatment or prevention of a heart condition, e.g., an attack infarction, stroke, or mycardial scarring.
  • a binding composition may also be useful in ameliorating, treating, preventing, modulating and/or diagnosing an autoimmune disease, disorder, syndrome, and/or condition such as results, e.g., from the inappropriate recognition by a cell ofthe immune system ofthe self as a foreign material.
  • Such an inappropriate recognition results in an immune response leading to detrimental effect destruction on the host, e.g., on a host cell, tissue, protein, or moiety, e.g., a carbohydrate side chain.
  • a binding composition which inhibits a detrimental immune response particularly, e.g., a proliferation, differentiation, or chemotaxis of a T-cell, may be effective in detecting, diagnosing, ameliorating, or preventing such an autoimmune disease, disorder, syndrome, and/or condition.
  • autoimmune conditions examples include, e.g., without limit Addison's Disease syndrome hemolytic anemia, anti-phospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease syndrome, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopafhies, Purpura, Reiter's Disease syndrome, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary jmflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disease.
  • Addison's Disease syndrome hemolytic anemia e.g., without limit Addison's Disease syndrome hemolytic anemia, anti-phospholipid syndrome, rheumatoid arthritis,
  • allergic reactions and conditions such as asthma (e.g., allergic asthma) or other respiratory problems, may also be ameliorated, treated, modulated or prevented, and/or diagnosed by a binding composition (or fragment thereof), or an agonist or antagonist thereto.
  • inventive compositions can be used to effect, e.g., anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • a binding composition may also be used to modulate, ameliorate, treat, prevent, and/or diagnose organ rejection or graft- versus-host disease (GVHD).
  • organ rejection occurs by a host's, immune-cell destruction of a transplanted tissue or cell.
  • a similarly destructive immune response is involved in GVHD, however, in this case, transplanted foreign immune cells destroy host tissues and/or cells.
  • Administration of a composition ofthe invention which ameliorates or modulates such a deleterious immune response (e.g., a deleterious proliferation, differentiation, or chemotaxis of a T cell), can be effective in modulating, ameliorating, diagnosing, and/or preventing organ rejection or GVHD.
  • a binding composition may also be used to detect, treat, modulate, ameliorate, prevent, and/or diagnose an inflammation, e.g., by inhibiting the proliferation and/or differentiation of a cell involved in an inflammatory response, or an inflammatory condition (either chronic or acute), including, e.g., without limitation, chronic prostatitis, granulomatous prostatitis and malacoplakia, an inflammation associated with an infection (such as, e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease syndrome, Crohn's disease syndrome, or a condition resulting from an over production of a cytokine(s) (e.g., TNF or IL-1 .) Proliferative Disorders
  • An binding composition can be used to modul
  • a desired effect using a composition ofthe invention may also be accomplished either by, e.g., enhancing an existing immune response, or by initiating a new immune response.
  • the desired result may be effected either by, e.g., diminishing or blocking an existing immune response, or by preventing the initiation of a new immune response.
  • hyperproliferative states, diseases, disorders, syndromes, and/or conditions include, e.g., without limitation, a neoplasm ofthe colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine system (e.g., an adrenal gland, a parathyroid gland, the pituitary, the testicles, the ovary, the thymus, or the thyroid), eye, head, neck, nervous system (central or peripheral), the lymphatic system, pelvis, skin, spleen, thorax, and urogenital system.
  • a neoplasm ofthe colon abdomen, bone, breast, digestive system, liver, pancreas, peritoneum
  • endocrine system e.g., an adrenal gland, a parathyroid gland, the pituitary, the testicles, the ovary, the thymus, or the thyroid
  • eye head, neck, nervous system (central or peripheral)
  • the lymphatic system pelvis, skin
  • hyperproliferative conditions include, e.g., without limit hypergarnmaglobulinemia, lymphoprolife ative conditions, paraproteinemias, purpura, sarcoidosis, Hamartoma, Sezary Syndrome,
  • TGF-betal is an important regulator ofthe normal and malignant prostate.
  • Prostate cancer cells express high levels of TGF-betal, which seems to enhance prostate cancer growth and metastasis by stimulating angiogenesis and by inhibiting immune responses directed against tumour cells.
  • Prostate cancer cells frequently lose their TGF-beta receptors and acquire resistance to the anti-proliferative and pro-apoptotic effects of TGF-betal. Accordingly, high expression of TGF-betal and loss of TGF-beta receptor expression have been associated with a particularly bad prognosis in human prostate cancer patients.
  • TGF-Betal is also associated with breast cancer.
  • One preferred embodiment utilizes an binding composition to inhibit aberrant cellular division, through a polynucleotide delivery technique.
  • the present invention provides a method for treating, preventing, modulating, ameliorating, preventing, inhibiting, and/or diagnosing cell proliferative diseases, disorders, syndromes, and/or conditions described herein by inserting into an abnormally proliferating cell a composition ofthe present invention, wherein said composition beneficially modulates an excessive condition of cell proliferation, e.g., by inhibiting transcription and/or translation.
  • Local administration to an abnormally proliferating cell may be achieved by any art known method or technique discussed herein including, e.g., without limit to transfection, elecfroporation, microinjection of cells, or in vehicles (such as a liposome, lipofectin, o ⁇ a naked polynucleotide).
  • cell proliferative condition is meant any human or animal disease, syndrome, disorder, condition, or state, affecting any cell, tissue, any site or any combination of organs, tissues, or body parts, which is characterized by a single or multiple local abnormal proliferation of cells, groups of cells, or tissues, whether benign or malignant.
  • binding composition may be administered as long as it has a desired effect on the treated cell, e.g., a biologically inhibiting effect on an abnormally proliferating cell.
  • the present invention also encompasses an antibody-based therapy that involves administering an antibody binding composition to a subject to ameliorate, treat, prevent, modulate, and/or diagnose one or more ofthe described diseases, disorders, syndromes, and/or conditions discussed herein.
  • Methods for producing antibodies are known in the art or described herein. Such antibodies may be provided in a pharmaceutically acceptable formulation as known in the art or described herein.
  • a partial summary ofthe manner in which an antibody may be used includes, e.g., binding polypeptides (o fragments thereof) of TGF Beta 1 locally or systemically in the body or by direct cytotoxicity ofthe antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
  • CDC complement
  • ADCC effector cells
  • an antibody, fragment, or derivative thereof of the present invention is useful for ameliorating, modulating, treating, preventing, and/or diagnosing a subject having or developing a cell proliferative and/or a cell differentiation disease, syndrome, disorder, state and/or condition as described herein. Bringing about an effect on such a condition can include, e.g., administering a single or multiple dose of an Antibody, or its fragment, derivative, or conjugate thereof.
  • An antibody may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors which, e.g., serve to increase the number or activity of effector cells that interact with an antibody.
  • a high affinity and/or potent in vivo inhibiting and/or neutralizing antibody that selectively and/or specifically binds TGF Beta 1 polypeptide (or fragment thereof), or an agonist or antagonist thereto will be used against a composition ofthe present invention for an immunoassay on to effect a disease, disorder, syndrome, and/or condition which is associated with expression of a TGF Beta 1 polypeptide (or fragment thereof) or an agonist or antagonist thereto.
  • An antibody, fragments thereof, or regions thereof preferably will have a binding affinity for the TGF Beta 1 protein or fragment thereof that will be, e.g., with a dissociation constant or Kd less than about 5X10 " 6M, 10 " 6M, 5X10 " 7M, 10 " 7M, 5X10 " 8M, 10 " 8M, 5X10 ' 9M, 10 " 9M, 5X10 0M, 10 0M, 5X101 IM, 101 IM, 5X10 2M, 10 " 12M, 5X10 3M, 10 3M, 5X10 4M, 10 4M, 5X10 5M, or 10 5M.
  • a binding composition may be used to, modulate, ameliorate, effect, treat, prevent, and/or diagnose a cardiovascular disease, disorder, syndrome, and/or condition.
  • cardiovascular abnormalities such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome peripheral artery disease, syndrome, such as limb ischemia.
  • Additional cardiovascular disorders encompass, e.g., congenital heart defects which include, e.g., aortic coarctation, car triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as e.g., aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, and ventricular heart septal defects.
  • congenital heart defects which include, e.g., aortic coarctation, car triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein
  • cardiovascular conditions include, e.g., heart disease syndrome, such as, e.g., arrhythmias, carcinoid heart disease syndrome, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial endocarditis), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous pericarditis), pneumopericardium, post-pericardiotomy syndrome, pulmonary heart disease syndrome, rheumatic heart disease syndrome, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculos
  • cardiovascular disorders include, e.g., arrhythmias including, e.g., sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extra systole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown- Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson- White syndrome, sick sinus syndrome, and ventricular fibrillation tachycardias.
  • arrhythmias including, e.g., sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extra systole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown- Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson- White syndrome, sick sinus syndrome, and ventricular fibrillation t
  • Tachycardias encompassed with the cardiovascular condition described herein include, e.g., paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal re-entry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal re-entry tachycardia, sinus tachycardia, Torsades de Pointes Syndrome, and ventricular tachycardia.
  • Additional cardiovascular disorders include, e.g., heart valve disease such as, e.g., aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
  • heart valve disease such as, e.g., aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenos
  • Myocardial conditions associated with cardiovascular disease include, e.g., myocardial diseases such as, e.g., alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
  • myocardial diseases such as, e.g., alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
  • Cardiovascular conditions include, e.g., myocardial ischemias such as, e.g., coronary disease syndrome, such as e.g., angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasispasm, myocardial infarction, and myocardial stxmning.
  • myocardial ischemias such as, e.g., coronary disease syndrome, such as e.g., angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasispasm, myocardial infarction, and myocardial stxmning.
  • coronary disease syndrome such as e.g., angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasispasm, myocardial infarction, and myocardial
  • Cardiovascular diseases also encompassed herein include, e.g., vascular diseases such as e.g., aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel- Lindau Disease syndrome, Klippel-Trenaunay- Weber Syndrome, Sturge- Weber Syndrome, angioneurotic edema, aortic disease, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive disease, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disease, diabetic angiopathies, diabetic retinopa hy, embolism, thrombosis, erythromeialgia, hemorrhoids, hepatic veno-occlusive disease syndrome, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary
  • Cardiovascular conditions further include, e.g., aneurysms such as, e.g., dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
  • aneurysms such as, e.g., dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
  • Arterial occlusive cardiovascular conditions include, e.g., arteriosclerosis, intermittent claudication, carotid stenosis, f ⁇ bromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease syndrome, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
  • Cerebrovascular cardiovascular conditions include, e.g., carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient cerebral ischemia), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
  • Embolic cardiovascular conditions include, e.g., air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms.
  • Thrombotic cardiovascular conditions include, e.g., coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
  • Ische ic conditions include, e.g., cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.
  • Vasculitic conditions include, e.g., aortitis, arteritis, Behcet's Syndrome, Churg- Strauss Syndrome, mucocutaneous lymph node syndrome, hromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
  • a binding composition can be beneficial in ameliorating critical limb ischemia and coronary disease.
  • a binding composition may be administered using any art known method, described herein
  • a binding composition may administered as part of a therapeutic composition or formulation, as described in detail herein. Methods of delivering a binding composition are also described herein.
  • Anti-Hemopoietic Activity The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences typically predominate (see, e.g.,
  • angiogenesis is stringently regulated, and delimited spatially and temporally.
  • pathological angiogenesis such as, e.g., during solid tumor formation, these regulatory controls fail and unregulated angiogenesis can become pathologic by sustaining progression of many neoplastic and non-neoplastic diseases.
  • a number of serious diseases are dominated by abnormal neovascularization (including, e.g., solid tumor growth and metastases, arthritis, some types of eye conditions, and psoriasis; see, e.g., reviews by Moses, et al., Biotech. 9630-634 (1991); Folkman, et al., N. Engl. J. Med., 333: 1757-1763 (1995); Auerbach, et al., J. Microvasc. Res. 29:401-4 11 (1985); Folkman, "Advances in Cancer Research," eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol.
  • TGF beta 1 and its receptors ALK-5 and ALK-1 are implied in the vascular maturation phase of angiogenesis (see, e.g., Bull Acad Natl Med. 2000;184 (3):537-44).
  • angiogenesis contributes to a disease-state, e.g., for example, significant data have accumulated suggesting that solid tumor formation is dependent on angiogenesis (see, e.g., Folkman and Klagsbrun, Science 235:442-447 (1987)).
  • a binding composition provides for the treatment, amelioration, modulation, diagnosis, and/or inhibition of a disease, disorder, syndrome, and/or condition associated with neovascularization.
  • Malignant and metastatic conditions that can be effected in a desired fashion using a binding composition include, e.g., without limitation, a malignancy, solid tumor, and a cancer as described herein or as otherwise known in the art (for a review of such disorders, syndromes, etc. see, e.g., Fishman, et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).
  • the present invention provides a method of ameliorating, modulating, treating, preventing, and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to a subject in need thereof a beneficially effective amount of a binding composition.
  • cancers that may be so affected using a composition ofthe invention includes, e.g., without limit a solid tumor, including e.g., prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as e.g., leukemia.
  • a binding composition may be delivered topically, to treat or prevent cancers such as, e.g., skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
  • a binding composition may be utilized to treat superficial forms of bladder cancer by, e.g., intravesical administration into the tumor, or near the tumor site; via injection or a catheter.
  • intravesical administration into the tumor, or near the tumor site; via injection or a catheter.
  • the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
  • a binding composition may also be useful in modulating, ameliorating, treating, preventing, and/or diagnosing another disease, disorder, syndrome, and/or condition, besides a cell proliferative condition (e.g., a cancer) that is assisted by abnormal angiogenic activity.
  • a cell proliferative condition e.g., a cancer
  • Such close group conditions include, e.g., without limitation, benign tumors, e.g., such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; atherosclerotic plaques; ocular angiogenic diseases, e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, cornea graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) ofthe eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronar
  • the present invention also provides methods for ameliorating, treating, preventing, and/or diagnosing neovascular diseases ofthe eye, including e.g., corneal graft neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
  • neovascular diseases ofthe eye including e.g., corneal graft neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
  • ocular diseases, disorders, syndromes, and/or conditions associated with neovascularization that can be modulated ameliorated, treated, prevented, and/or diagnosed with a binding composition include, e.g., without limit; neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of premature macular degeneration, corneal graft neovascularization, as well as other inflammatory eye diseases, ocular tumors, and diseases associated with choroidal or iris neovascularization (see, e.g., reviews by Waltman, et al., (1978) Am. J. Ophthal.
  • neovascular diseases ofthe eye such as corneal neovascularization (including corneal graft neovascularization), comprising administering to a patient a therapeutically effective amount of a binding composition to the cornea, such that the formation of blood vessels is inhibited or delayed.
  • the cornea is a tissue that normally lacks blood vessels.
  • capillaries may extend into the cornea from the pericorneal vascular plexus ofthe limbus.
  • corneal neovascularization e.g., corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), irnmunological processes (e.g., graft rejection and Stevens- Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of using contact lenses.
  • corneal infections e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis
  • irnmunological processes e.g., graft rejection and Stevens- Johnson's syndrome
  • alkali burns trauma, inflammation (of any cause)
  • toxic and nutritional deficiency states e.g., as a complication of using contact lenses.
  • methods for treating or preventing neovascular glaucoma, comprising adrninistering to a patient a therapeutically effective amount of a binding composition to the eye, such that the formation of blood vessels is inhibited.
  • the composition may be administered topically to the eye to treat or prevent early forms of neovascular glaucoma.
  • the composition may be implanted by injection into the region ofthe anterior chamber angle.
  • the composition may also be placed in any location such that the composition is continuously released into the aqueous humor.
  • methods for treating or preventing proliferative diabetic retinopathy, comprising administering to a patient a therapeutically effective amount of a binding composition to the eyes, such that the formation of blood vessels is inhibited.
  • Additional, diseases, disorders, syndromes, and/or conditions that can be modulated, ameliorated, treated, prevented, and/or diagnosed with a binding composition include, e.g., without limitation, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
  • diseases, disorders, states, syndromes, and/or conditions that can be modulated, ameliorated, treated, prevented, and/or diagnosed with a binding composition include, e.g., without limitation, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors (e.g., hemangiomas), acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vasculogenesis, granulations, hypertrophic scars (
  • cancers such as, e.g., follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, e.g., but without limit, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancel, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune conditions (such as, e.g., multiple sclerosis, Sjo
  • a binding composition is used to inhibit growth, progression, and/or metastases of cancers such as, in particular, those listed herein.
  • Additional diseases, states, syndromes, or conditions associated with increased cell survival that could be modulated, ameliorated, treated, prevented, or diagnosed by a binding composition include, e.g., without limitation, progression, and/or metastases of malignancies and related disorders such as leukemia including acute leukemias (such as, e.g., acute lymphocytic leukemia, acute myelocytic leukemia, including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) and chronic leukemias (e.g., chronic myelocytic, chronic granulocytic, leukemia, and chronic lymphocytic leukemia)), polycythemia Vera, lymphomas (e.g., Hod
  • fibrosarcoma such as, e.g., fibrosarcoma, myxosareoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotlieliosarcoma, lymphangiosarcoma, l3 nphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocar
  • AIDS diseases associated with increased apoptosis that could be modulated, ameliorated, treated, prevented, and/or diagnosed by a binding composition
  • conditions such as, e.g., Alzheimer's disease syndrome, Parkinson's disease syndrome, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor, or prion associated disease
  • autoimmune conditions such as, e.g., multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease syndrome, Crohn's disease syndrome, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, and rheumatoid arthritis
  • myelodysplastic syndromes such as aplastic anemia
  • ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
  • liver injury such as, e.g., hepatitis related liver injury, ischemia reperfusion injury, cholestosis (bile duct injury), and liver cancer
  • toxin-induced liver disease such as, e.g., that caused by alcohol
  • septic shock such as, e.g., cachexia, and anorexia.
  • a binding composition could be used to prevent and heal damage to the lungs due to various pathological states, such as, e.g., stimulating proliferation and differentiation to promote repair of alveoli and bronchiolar epithelium.
  • emphysema inhalation injuries, that (e.g., from smoke inhalation) and burns, which cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively ameliorated, -ol- treated, prevented, and/or diagnosed using a polynucleotide or polypeptide ofthe invention (or fragment thereof), or an agonist or antagonist thereto.
  • a bindmg composition could be used to stimulate the proliferation of and differentiation of type II pneumocytes, to help treat or prevent hyaline membrane diseases, such as e.g., infant respiratoiy distress syndrome and bronchopulmonary displasia, (in premature infants).
  • a binding composition could stimulate the proliferation and/or differentiation of a hepatocyte and, thus, could be used to alleviate or treat a liver condition such as e.g., fulminant live failure (caused, e.g., by cirrhosis), liver damage caused by viral hepatitis and toxic substances (e.g., acetaminophen, carbon tetrachloride, and other known hepatotoxins) .
  • a liver condition such as e.g., fulminant live failure (caused, e.g., by cirrhosis), liver damage caused by viral hepatitis and toxic substances (e.g., acetaminophen, carbon tetrachloride, and other known hepatotoxins) .
  • Nervous system diseases, disorders, syndromes, states, and/or conditions that can be modulated, ameliorated, treated, prevented, and/or diagnosed with a binding composition include, e.g., without limitation, nervous system injuries diseases, disorders, states, syndromes, and/or conditions that result in either a disconnection or misconnection of an axon or dendrite; a diminution or degeneration of a cell (or part of a cell) ofthe nervous system (such as, e.g., without limitation, neurons, astrocytes, microglia, macroglia, oligodendroglia, Schwann cells, and ependymal cells); demyelination or improper mylenation; neural cell dysfunction (such as, e.g., failure of neurotransmitter release or uptake); or interference with mylemzation.
  • nervous system injuries diseases, disorders, states, syndromes, and/or conditions that result in either a disconnection or misconnection of an axon or dendrite e.g., without limitation,
  • TGF-beta 1 mRNA and piotein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-beta 1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-beta 1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-beta 1 protein observed in beta
  • TGF-beta 1 A/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims.
  • AD Alzheimer's disease
  • DS Down's syndrome
  • TGF-beta 1 has an organizing role in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues, (see, e.g., Finch, et al., J Cell Biochem. 1993 Dec;53(4):314-22).
  • oo -o - Nervous system lesions that may be modulated, ameliorated, treated, prevented, and/or diagnosed in a subject using a binding composition ofthe invention, include, e.g., without limitation, the following lesions of either the central (including spinal cord and brain) or peripheral nervous system: (1) ischemic lesions, in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, including e.g., cerebral infarction (or ischemia), or spinal cord infarction (or ischemia); (2) traumatic lesions, including, e.g., lesions caused by physical injury or associated with surgery (e.g., lesions that sever a portion ofthe nervous system), or compression injuries; (3) malignant lesions, in which a portion ofthe nervous system is comprised by malignant tissue, which is either a nervous system associated malignancy or a malignancy derived from non- nervous-system tissue; (4) infectious lesions, in which a portion ofthe nervous system is comprised because of infection (
  • motor neuron diseases, disorders, syndromes, and/or conditions that may be modulated, ameliorated, freated, prevented, and/or diagnosed using a binding composition include, e.g., without limitation, infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy (that may affect motor neurons as well as other components ofthe nervous system), as well as conditions that selectively affect neurons such as, e.g., without limitation, Amyotrophic lateral sclerosis progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
  • infarction infection
  • toxin trauma, surgical damage
  • degenerative disease or malignancy that may affect motor neurons as well as other components ofthe nervous system
  • conditions that selectively affect neurons such as, e.g., without limitation,
  • Chemotaxis A binding composition may have an effect on a chemotaxis activity.
  • TGF- ⁇ l has been shown to be chemotactic for fibroblasts; it induces the synthesis of matrix proteins and glycoproteins and inhibits collagen degradation by induction of protease inhibitors and reduction of metalloproteases (see, e.g., Ward & Hunninghake, Am. J. Respir. Crit. Care Med. 1998; 157: S123-S129).
  • chemotactic molecules can attract or mobilize (but may also repeal) cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) or cell processes (e.g., filopodia, psuedopodia, lamellapodia, dendrites, axons, etc.) to a particular site (e.g., such as inflammation, infection, site of hyperproliferation, the floor plate ofthe developing spinal cord, etc.).
  • a particular site e.g., such as inflammation, infection, site of hyperproliferation, the floor plate ofthe developing spinal cord, etc.
  • mobilized cells can then fight off and/or modulate a particular trauma, abnormality, condition, syndrome, or disease.
  • a binding composition may have an effect on a chemotactic activity of a cell (such as, e.g., an attractive or repulsive effect).
  • a chemotactic molecule can be used to modulate, ameliorate, treat, prevent, and/or diagnose inflammation, infection, hyperproliferative diseases, disorders, syndromes, and/or conditions, or an immune system disorder by increasing the number of cells targeted to a particular location in the body.
  • a chemotactic molecule can be used to attract an immune cell to an injured location in a subject.
  • a binding composition that had an effect on a chemotactant could also attract a fibroblast, which can be used to modulate, ameliorate, and/or treat a wound.
  • a binding composition may inhibit a chemotactic activity to modulate, ameliorate, treat, prevent, and/or diagnose a disease, disorder, syndrome, and/or a condition. its This invention also contemplates use of binding compositions in a variety of diagnostic kits and methods for detecting the presence of TGF Beta 1.
  • the kit will have a compartment containing either a defined TGF Beta 1 or a binding composition, which recognizes one or the other, e.g., binding partner fragments or antibodies.
  • a preferred kit for determining the concentration of TGF Beta 1 in a sample would typically comprise a labeled compound, e.g., binding composition or antibody, having known binding affinity for the TGF Beta 1 protein, a source of TGF Beta 1 protein (naturally occurring or recombinant), and a means for separating the bound from free labeled compound, for example, a solid phase for immobilizing the TGF Beta 1 piotein. Compartments containing reagents, and instructions, will normally be provided. Antibodies, including antigen binding fragments, specific for a TGF Beta 1 protein or fragments thereof are useful in diagnostic applications to detect the presence of elevated levels of TGF Beta 1 protein and/or its fragments.
  • a labeled compound e.g., binding composition or antibody, having known binding affinity for the TGF Beta 1 protein
  • a source of TGF Beta 1 protein naturally occurring or recombinant
  • a means for separating the bound from free labeled compound for example, a solid phase for immobilizing the TGF Beta
  • diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the protein in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-TGF Beta 1 or -WDS protein complex) or heterogeneous (with a separation step).
  • Various commercial assays exist, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.
  • unlabeled antibodies are employed by using a second antibody which is labeled and which recognizes an antibody to a TGF Beta 1 protein or to a particular fragment thereof.
  • Similar assays are also extensively discussed in the literature (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Press, NY; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press, Orlando, FL; Price and Newman (eds.) (1991) Principles and Practice of Immunoassay Stockton Press, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press, NY).
  • Anti-idiotypic antibodies may have similar use to diagnose the presence of antibodies against an TGF Beta 1 protein or polypeptide, as such may be diagnostic of various abnormal states, conditions, disorders, or syndromes.
  • ⁇ OJ- overproduction of TGF Beta 1 protein may result in production of various irnmunological or other physiological reactions which may be diagnostic of abnormal physiological states, e.g., in cell growth, activation, or differentiation.
  • the reagents for diagnostic assays are supplied in kits, to optimize the sensitivity ofthe assay.
  • the protocol, and the label either labeled or unlabeled antibody or binding composition, or labeled TGF Beta 1 protein is provided.
  • the kit contains instructions for proper use and disposal ofthe contents after use.
  • the kit has compartments for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • Many ofthe aforementioned constituents ofthe drug screening and the diagnostic assays may be used without modification, or may be modified in a variety of ways.
  • labeling may be achieved by covalently or non-covalently joining a moiety that directly or indirectly provides a detectable signal.
  • the protein, test compound, TGF Beta 1 protein or polypeptide (or antibodies thereto) are labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups such as, e.g., without limitation, radiolabels (e.g., ⁇ ); enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase; and fluorescent labels (U.S. Pat. No. 3,940,475) that are capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to an art known label such as one ofthe above. There are also numerous methods of separating the bound from the free protein, or alternatively bound from free test compound.
  • a TGF Beta 1 protein is immobilized on various matrices followed by washing. Suitable matrices include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the TGF Beta 1 protein to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin-avidin.
  • the last step in this approach involves the precipitation of protein/binding composition or antigen/antibody complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • suitable separation techniques include, without limitation, a fluorescein antibody magnetizable particle method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.
  • a kit may include, e.g., a recombinantly produced or chemically synthesized polypeptide antigen.
  • the polypeptide antigen ofthe kit may also be attached to a solid support.
  • the detecting means ofthe above-described kit includes, e.g., a solid support to which said polypeptide antigen is attached.
  • Such a kit may also include, e.g., a non-attached reporter-labeled anti-human antibody. In this embodiment, binding ofthe antibody to the polypeptide antigen is detected by binding ofthe reporter-labeled antibody.
  • the invention includes, e.g., a diagnostic kit for use in screening a biological sample, e.g., such as serum, containing an antigen of a polypeptide (or fragment thereof) ofthe invention.
  • the diagnostic kit can include, e.g., a substantially isolated antibody specifically and/or selectively immunoreactive with a polypeptide or polynucleotide antigen, and, a means for detecting the binding ofthe polynucleotide or polypeptide antigen to the antibody.
  • the antibody is attached to a solid support.
  • the antibody may be a monoclonal antibody.
  • the detecting means ofthe kit may include, e.g., a second, labeled monoclonal antibody.
  • the detecting means may include, e.g., a labeled, competing antigen.
  • test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by an art known method or as described herein. After binding with specific antigen antibody to the reagent and removing unbound serum components, e.g., by washing; the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound, labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, MO).
  • a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, MO).
  • the solid surface reagent in the above assay is prepared by art known techniques for attaching proteinaceous material to a solid support, such as, e.g., polymeric beads, dip sticks, 96-well plate, or filter material.
  • Methods for attachment generally include, e.g., non-specific adsorption of a protein or polypeptide (or fragment thereof) to a solid support or covalent attachment of a polypeptide, protein (or fragment thereof), typically, e.g., through a free amine group, to a chemically reactive group, such as, e.g., an activated carboxyl, hydroxyl, or aldehyde group on the solid support.
  • streptavidin coated plates are used in conjunction with biotinylated antigen(s).
  • TGF Beta 1 Binding Composition Synthesis The CDR and framework sequences of antibody bindmg compositions disclosed herein were identified from clones of Fab fragments, which were isolated from antibody libraries generated from an array of antibody RNA created by immunized C57/Black wild-type mice using OmniclonalTM antibody technology (Biosite®, San Diego, CA). The mice were immunized with human TGF Beta 1 using conjugated keyhole limpet hemoncyanin according to standard methods to improve immunogenicity.
  • Example 2 Fabs of he mAb Anti-TGF Beta 1 Binding Compositions Table 1: Murine, e.g., mouse, mAb 1021 polynucleotide sequence (SEQ ID NO: 1) and corresponding polypeptide (SEQ ID NO: 2) ofthe light chain Fab ofthe mAb 1021 binding composition ofthe invention.
  • SEQ ID NO: 2 The predicted polypeptide sequence ofthe light chain Fab ofthe mAb 1021 binding composition encoded by the polynucleotide sequence of SEQ ID NO: 1.
  • the three putative CDR regions are indicated in bold typeface and underlined starting with CDR 1 (RASSSVSYMH) through CDR2 (ATSNLAS ) until CDR 3 (QQWNGNPPA) .
  • mAb 1021 Heaw Chain DNA Se ⁇ iaenee (1380 bp):
  • SEQ ID NO: 3 The first (5 5 ) codon (ATG coding for methionine) and last (3 3 ) codon (AAA coding for lysine) are indicated in bold typeface and underlined).
  • SEQ ID NO: 4 The predicted polypeptide sequence ofthe heavy chain Fab ofthe mAb 1021 binding composition encoded by the polynucleotide of SEQ ID NO: 3.
  • the three putative CDR regions are indicated in bold typeface and underlined starting with CDR 1 (GYTFTDYNMH) through CDR2 (YIYPYNGDTGYNQKFKS) until CDR 3 (GYYWFAY) .
  • CDRs ofthe invention may include up to 1, 2, 3, 4, or 5 additional amino acids (at either the 5' or 3' end of an identified CDR region) as identified in a Table herein.
  • mAb 2471 Light Chain DNA Sequence (699 bp): SEQ ID NO: 5: The first (5') codon (ATG coding for methionine) and last (3') codon (TGT coding for cysteine) are indicated in bold typeface and underlined.
  • SEQ ID NO: 6 The predicted polypeptide sequence ofthe light chain Fab ofthe mAb 2471 binding composition encoded by the polynucleotide sequence of SEQ ID NO: 5.
  • the three putative CDR regions are indicated in bold typeface and underlined starting with CDR 1 (RASSSVSYMH ) through CDR2 (ATSNLAS ) until CDR 3 (QQWDSNP ) .
  • mAb 2471 polynucleotide sequence (SEQ ID NO: 7) and corresponding polypeptide (SEQ ID NO: 8) ofthe heavy chain Fab ofthe mAb 2471 binding composition ofthe invention.
  • SEQ ID NO: 7 The first (5') codon (ATG coding for methionine) and last (3') codon (AAA coding for lysine) are indicated in bold typeface and underlined.
  • mAb 2471 Heaw Chain Amino Acid Sequence (460 aa): SEQ ID NO: 8: The predicted polypeptide sequence ofthe heavy chain Fab ofthe mAb 2471 binding composition encoded by the polynucleotide of SEQ ID NO: 3. The three putative CDR regions are indicated in bold typeface and underlined starting with CDR 1 (GYTFTDYNMH) through CDR2 (YIYPYNGDTGYNQKFKS ) until CDR 3 (GYYWFAY) .
  • SEQ ID NO: 9 The first (5 5 ) codon (ATG coding for methionine) and last (3 5 ) codon (TGT coding for cysteine) are indicated in bold typeface and underlined.
  • SEQ ID NO: 10 The predicted polypeptide sequence ofthe light chain Fab ofthe mAb 3821 binding composition encoded bythe polynucleotide sequence ofSEQ ID NO: 9. The three putative CDRregions are indicated in boldtypeface and underlined starting with CDR 1 (RASQEISGYLS) through CDR2 (ATSSLDS) until CDR 3 (LQYASSPYT) .
  • mAb 3821 Heaw Chain DNA Sequence 1392 bp: SEQ ID NO: 11 : The first (5 ') codon (ATG coding for methionine) and last (3 ') codon (AAA coding for lysine) are indicated in bold typeface and underlined).
  • SEQ ID NO: 12 The predicted polypeptide sequence ofthe heavy chain Fab ofthe mAb 3821 binding composition encoded by the polynucleotide of SEQ ID NO: 11. The three putative CDR regions are indicated in bold typeface and underlined starting with CDR 1 (GYTFTDYTMH) through CDR2 (LITPFYGDAIYNQKFKG) until CDR 3 (GGLRRGPPFAY) .
  • Consensus ( 51 ) YMHWYQQKPGSSPKP IYATSNLASGVPARFSGSGSGTSYSLTISRVEA 101 CDR3 150 1021LC (100) EDAATYYC ⁇ r ⁇ GNPPAFGGGTKLEI RADAAPTFSXFPPSSEQLTSGGA 2471LC (100) EDAATYYCQQI ⁇ gSl ⁇ PAFGGGTKI ilKtlADAAP'- ⁇ SIFPPSSEQIia'SGGA 3821LC (101) EDFVDYYCI ⁇ rASS r ⁇ G GTI ⁇ s Kil D P'iySX pSSEQ 'ISGG
  • 1021 and 2471 HC's differ by 1 aa after CDR3 1 CDRl 50 1021HC (1) ETCTLLL VLLI- yPGSTGEVQ QQSGPErjVKPGASVKISCKASgjfyF-" 2471HC (1) METDTLLLWVLL]ltlWPGSTGEVQl-QQSGPEL ⁇ 7KPGASyKI SC AS GY FT 338822 l1HHcC (( l1 )) METDTLLLIWLLLIWPGST'GEVQLQQSGAALMRPGVSVKISCKGSG ⁇ F ⁇
  • CCoonn 251 __ 300 1021HC 247) yPBVSSWIFPPKPM)VLgIT T:PiyTCVVVBXS DDPEV FS FvPDVl! 2471HC (247) VPEVSSWIFPPKPKDVLTlTLTPWTCVVvTJISKDDPEVQFS FyDDVE 33882211HHCC ( (225511)) VPEVSS ⁇ IFPPKPKDVLTlTLTP VTCyVVTJISl >DPEVQF5WFVDDVE
  • VHTAQTQPREEQffNSTFRSVSELPIMH DWLNG EFKCRVNSAAFPAPIE 22447711HHCC (229977)
  • TOTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVKSAAFPAPXE 3821HC
  • TGF Beta 1 ELISA Binding compositions ofthe invention (e.g., anti-TGF Beta Fabs designated 1021, 2471, and 3821) were tested in a competitive ELISA assay as described herein. Typically, a solution phase assay is performed in which a compound that might compete with an antigen for binding to a binding composition, such as an antibody, is combined first with the antibody in solution phase, then the degree of binding ofthe antibody with the antigen is subsequently measured.
  • a binding composition such as an antibody
  • Antigens used were TGF- ⁇ 1 (R&D Systems, Cat # 240-B/CF, 239 ug/ml), TGF- ⁇ 2 (RDI, Cat # RDI-1035, 50 ug/ml) and TGF- ⁇ 3 (RDI, Cat # RDI-1036/CF, 50 ug/ml) diluted to 0.4 ug/niL in coating buffer.
  • Wash buffer consisted of 0.02 M Tris pH 7.4, 0.15 M NaCI, 0.1% Tween 20 and blocking solution of 10 mg/ml BSA (Sigma A-4503) dissolved in wash buffer.
  • Proteins used as positive control were mouse-anti-human TGF- ⁇ 1, 2, or 3 (R&D Systems, cat# 1D11), mouse-anti-human TGF- ⁇ 2 - B (R&D Systems, cat#
  • the detection antibody conjugate used was anti-mouse kappa - peroxidase conjugate (Southern Biotech, cat# 1050-05), at a working concentration of 1 :2000 in blocking solution.
  • the subsfrate used for the color reaction was O-phenylenediamine (OPD) tablets (Sigma cat# P-6912), which were dissolved in subsfrate buffer: 0.1 M Na j HPO ⁇ pH to 5.0 with 0.05 M citric Acid.
  • the OPD substrate working solution (i.e., the volume for one 196-well-plate) was freshly made prior to each plate development by dissolving 1 x 5mg OPD tablet in 12.5 mL of substrate buffer followed by the addition of 5 ul of 30% H 2 O 2 .
  • the plate was washed (2X, twice) with wash buffer. Then, 100 uL of either sample (diluted in buffer) or control (diluted in PBS) was added to each well and incubated (1.5 hours at 22 °C). After incubation, the plate was washed (6X, six times) with wash buffer (described above) before adding 100 uL per well of either anti-mouse kappa - peroxidase conjugate (diluted to 1 :2000 in Blocking solution) or SA-HRP (diluted 1:10,000 in blocking solution). The test samples were left to incubate (1 hour at 22 °C) before adding 100 uL of OPD substrate to each well. After color development (approximately 10 minutes), the 96-well plate was measured at an absorbance of 490nm.
  • TGF- ⁇ ELISA was used to screen Fab binding compositions ofthe invention to determine their specificity and/or selectivity for a particular TGF- ⁇ isoform.
  • Results from these experiments show that the Fabs designated 1021 and 2471 produced an absorbance greater than 1.6 units at 490 nm with TGF- ⁇ l but gave significantly lower values with TGF- ⁇ 2 and TGF- ⁇ 3 thus demonstrating that the 1021 and 2471 binding compositions comprise binding sites that are specific and/or selective for TGF- ⁇ 1. Support for this conclusion was provided by the similarity of the absorbance readings obtained with the known TGF- ⁇ l specific control mAb (designated mAb240), which was obtained from R&D Systems.
  • control mAb240 When the control mAb240 is used in combination with the biotinylated anti-TGF- ⁇ l detection antibody (R&D # BAF240) in sandwich ELISAs, less than 2% cross- reactivity is observed with TGF- ⁇ 3 and TGF- ⁇ 5 and no cross-reactivity is observed with the following cytokines: rhAng, ihCNTF, ih ⁇ -ECGF, rmEGF, rhEGF, rmEGF, rhEpo, bFGF acidic, rhFGF acidic, bFGF basic, rhFGF basic, rf ⁇ FGF-5, rhFGF-6, rhG-CSF, rmGM-CSF, rhGRO ⁇ , rhHB-EGF, rhHGF, rhIFN- ⁇ , ⁇ hIGF-I, rhIGF-II, rhLIF, rmLIF, rhM
  • control readings from a known non-selective mAb, designated 1D11
  • TGF- ⁇ isoforms i.e., TGF- ⁇ 1, 2, and 3
  • Fab 1021 and 2471 are specific and/or selective for TGF-Beta 1 (see Table 10 below).
  • Anti mouse-HRP 0.046 0.078 0.037 SA-HRP 0.046 0.052 0.04 SA-HRP 0.041 0.041 0.041 Values of Table 10 represent the absorbance at 490 nm from duplicate determinations with the Fabs 1021 and 2471 used at a concentration of 10 ug/ml. Assay controls included Anti mouse-HRP, SA-HRP and SA-HRP.
  • Example 3 Neutralization of TGF- ⁇ Activity
  • a binding composition ofthe invention to neutralize TGF beta bioactivity and to neutralize a particular TGF Beta isoform
  • applicants adapted the HT-2 cell proliferation assay of Tsang, et al, (1995 Cytokine 7:389-97). Briefly, the human cell line HT-2 proliferates in response to IL-4 but that proliferation is inhibited by TGF- ⁇ 1 , TGF- ⁇ 2, or TGF- ⁇ 3. Consequently, a binding composition that is specific and/or selective for TGF- ⁇ l can be determined to be neutralizing if it can be shown to prevent the normal inhibitory effect that TGF- ⁇ l has on IL-4 induced HT-2 cells.
  • IL-4-induced cell proliferation should proceed unconstrained if sufficient TGF Beta 1 -specific binding composition is added to a mixture of HT-2 cells and TGF Beta 1. Consequently, the dose response neutralizing capability of binding compositions ofthe invention was assessed in the HT-2 assay in the presence of particular TGF isoforms and the IL-4 proliferation signal. Degree of cell proliferation was assessed using a commercial colorimetric cell proliferation measure (CellTiter 96® AQueous One Solution Cell Proliferation Assay from Promega).
  • HT-2 assay HT-2 cells were maintained in RPMI 1640 supplemented with 10% FBS, 50 ⁇ M ⁇ -Mercaptoethanol and 10 ng/ml human IL-2 (R&D Systems).
  • Cells were centrifuged (1000 RPM in a Jouan CR422 centrifuge) and re-suspended in PBS. After washing (2X), the cells were finally re-suspended (1.5 x IO 6 cells/ml) in assay media (phenol red free RPMI 1640 supplemented with 2% FBS, 50 ⁇ M ⁇ -Mercaptoethanol). The cells were subsequently added (50 ⁇ l) to each well of a 96 well plate.
  • assay media phenol red free RPMI 1640 supplemented with 2% FBS, 50 ⁇ M ⁇ -Mercaptoethanol
  • Varying concentrations (0.0, 3.0, 1.0, 0.3, 0.1, 0.03, 0.01 ug/ml) ofthe binding compositions were pre-incubated with TGF- ⁇ l (300 pg/ml) before they were added to separate wells ofthe 96 well plate. After incubation (30 min at RT), 50 ⁇ l of pre-incubation mixture was added to the cells in a test well, followed immediately thereafter by 50 ⁇ l of assay media containing IL-4 (45 ng/ml murine; R&D Systems).
  • Assays are performed by adding a small amount ofthe CellTiter 96® AQueous One Solution Reagent directly to culture wells, incubating for 1—4 hours and then recording absorbance at 490nm with a 96-well plate reader. Specifically, after an incubation of 20 to 48 hr, 35 ⁇ l of CellTiter 96 Aqueous solution (Promega Corp) was added to each test well.
  • results The neutralization results ofthe TGF Beta 1 specific binding compositions ofthe invention (mAb 2471, mAb 1021 and mAb 3821) compared to the non-specific mAb 1D11 are shown below in Table 11.
  • the values ofthe table show the extent ofthe neutralization as a measure of cell proliferation that has been rescued from inhibition by TGF Beta 1. Each value represents the mean of at least three data points with standard deviation (SD) indicated.
  • TGF- ⁇ l, TGF- ⁇ 2, and TGF- ⁇ 3 can inhibit IL-4 induced cell proliferation of HT-2 cells.
  • the HT-2 assay described above was used to assess the specificity ofthe neutralizing ability of a binding composition ofthe invention (mAb 3821).
  • the HT-2 assay was performed as described above except that TGF- ⁇ 2 and TGF- ⁇ 3 were additionally tested. Consequently, blocking inhibition ofthe IL-4 induced cell proliferation response by mAb 3821 with TGF- ⁇ 2 and TGF- ⁇ 3 would indicate no specificity of its neutralizing capability.
  • results The data below in Table 12 show that the binding composition (mAb 3821) specifically and/or selectively neutralizes only TGF- ⁇ l inhibition of IL-4 induced cell proliferation, whereas the control mAb 1D11 non-specifically blocks all three TGF- ⁇ l isoforms (TGF- ⁇ l, TGF- ⁇ 2, and TGF- ⁇ 3) from inhibiting IL-4 induced cell proliferation.
  • the values ofthe table show the extent ofthe neutralization as a measure of cell proliferation that has been rescued from inhibition by TGF Beta 1, 2, or 3.
  • Column 1 is a control showing the effect of no inhibitory TGF Beta isoform added to IL-4 induced HT-2 cell proliferation (note, the average colorimetric value indicating normal cell proliferation is approximately 100).
  • Column 4 shows the unmitigated inhibitory effect of lOOpg/ml of each inhibitory TGF Beta isoform (indicated by the row on the far left) on IL-4 induced cell proliferation (note the average degree of cell proliferation is below 5).
  • Column 2 shows the lack of any neutralizing effect of mAblDl 1 on lOOpg/ml of each inhibitory TGF Beta isoform.
  • Example 3 Affinity Measurement The affinity of mAb 2471, mAb 1021 and mAb 3821 for human TGF- ⁇ 1 (R&D Systems, Cat # 240-B/CF), TGF- ⁇ 2 (RDI, Cat # RDI-1035) and TGF- ⁇ 3 (RDI, Cat # RDI-1036/CF) is measured using a BIAcore® 2000 instrument.
  • the BIAcore® utilizes the optical properties of surface plasmon resonance to detect alteration in protein concentration of interacting molecules within a dextran biosensor matrix. Except where noted, all reagents and materials are purchased from BIAcore® AB (Upsala, Sweden). All measurements are performed at room temperature.
  • HBS-EP buffer 150mM sodium chloride, 3mM EDTA, 0.005% (w/v) surfactant P-20, and lOmM HEPES, pH7.4
  • Goat anti-human Fc antibody is immobilized on flow cells 1 and 2 of a Bl sensor chip at a level of 500 response units (RUs) using an amine coupling kit. Binding is evaluated using multiple analytical cycles.
  • Each cycle is performed at a flow rate of 50 ⁇ L/minute and consists ofthe following steps: injection of lO ⁇ L of an mAb 2471, mAb 1021 and mAb 3821 antibody at 1 ⁇ g/mL, injection of 240 ⁇ L of TGF- ⁇ 1, TGF- ⁇ 2 3 and TGF- ⁇ 3 (starting at lOOnM and using two-fold serial dilutions for each cycle) followed by 20 minutes for dissociation, and regeneration using 50 ⁇ L of lOmM glycine hydrochloride, pH1.5. Association and dissociation rates for each cycle are evaluated using a "Langmuir 1:1 with mass transport" binding model in the BIAevaluation software.

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Abstract

L'invention concerne des compositions de liaison isolées et/ou recombinantes, ainsi que des réactifs associés à ces compositions. L'invention concerne également des méthodes d'utilisation desdits réactifs et des nécessaires de diagnostic.
EP04776554A 2003-07-09 2004-07-06 Ligands de tgf-beta1 Withdrawn EP1646655A2 (fr)

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