MX2008008137A - Tgf-beta binding compositions - Google Patents

Abstract

The present invention provides very high affinity antibodies, or antigen-binding fragments thereof, that neutralize mature human TGF-βl, TGF-β2, and TGF-β3. The antibodies of the invention are useful for treating cell proliferative disorders in a mammal.

Description

LINK COMPOSITIONS TO TGF-BETA FIELD OF THE INVENTION The invention relates to the treatment of cell proliferative diseases, disorders and conditions associated with TGF-β. In particular, the invention provides antibodies and fragments binding to the antigen thereof, which neutralize human, mature TGF-β1, β2 and β3.

BACKGROUND OF THE INVENTION The TGF-β protein family consists of three distinct isoforms (TGF-β1, β2 and β3), whose trajectories activate and regulate multiple gene responses that influence disease states such as, for example, cell proliferative conditions , inflammatory and cardiovascular. The expression of the TGF-β isoform in cancer is complex and variable with different combinations of TGF-β isoforms that have different roles in particular cancers. For example, TGF-ßl and ß3 play a greater role in ovarian cancer and its progress than TGF-ß2; while the expression of TGF-ßl and ß2 is higher in chondrosarcoma tumors of higher degree than ß3. In human breast cancer, TGF-ßl and ß3 are highly expressed with β3 expression that correlates with patients with total survival with Node metastasis and positive TGF-β3 expression have poor diagnostic results. However, in colon cancer, TGF-ßl and ß2 are more highly expressed than ß3 and are present at higher circulation levels than in cancer-free individuals. In glioma cells, TGF-β2 is essential for cell migration. Consequently, there is a need to modulate the expression of multiple TGF-β isoform under conditions of cell proliferation, such as cancer. US 5,571,714, describes the use of anti-TGF antibodies in the treatment of malignancies and metastatic cancer, and in particular, describes a murine antibody designated 1D11.16 (ATCC No. HB9849), which is said to bind both TGF- ßl as ß2. This document states that antibody 1D11.16 binds to TGF-β2 with a Ka of only 3.4 x 108 L / mol (Kd = I / Ka). The treatment of cellular proliferative disorders, such as malignancies and cancer, can be improved by the use of antibodies that neutralize human, mature TGF-βß, β2 and β3, with improved kinetics and binding affinity. Substantial chemical and physical stability, adequate pharmacokinetics and good solubility are also desirable for a pharmaceutical product. Consequently, there remains a need not covered by antibodies having suitable characteristics for the pharmaceutical treatment of cell proliferative disorders.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides monoclonal antibody compositions that neutralize mature human TGF-β1, β2 and β3, with a Kd of less than 4 pM for mature human TGF-β1, and a Kd of less than 8 pM for Mature human TGF-β2, and a Kd of less than 4 pM for mature human TGF-β3. The invention also provides antibodies having the following combinations of light chain variable region (LCVR) and heavy chain (HCVR) sequences: SEQ ID NO: 27 and 51, 27 and 59, 27 and 60, 27 and 61, 27 and 62, 27 and 63, 27 and 64, 28 and 52, 29 and 51, 30 and 53, 31 and 54, 32 and 55, 33 and 56, 34 and 51, 34 and 57, 35 and 58, 36 and 51, 36 and 69, 36 and 75, 37 and 51, 38 and 51, 39 and 51, 40 and 51, 41 and 64, 41 and 67, 42 and 66, 43 and 68, 44 and 66, 45 and 51, 45 and 69, 46 and 70, 46 and 71, 47 and 71, 48 and 72, 49 and 73, 50 and 65, or 50 and 74. The invention also includes antigen binding fragments of such antibodies, as well as antibodies and fragments that have humanized or human structures and constant regions. The antibodies and fragments of the invention are used to treat diseases, disorders and cell proliferative conditions. In one embodiment, antibodies of the present invention neutralize mature human TGF-β1, human TGF-β2 mature and mature human TGF-β3, and have an IC50 of less than or equal to about 100 pM for mature human TGF-β1 and an IC50 of less than or equal to 400 pM for mature human TGF-β2, and an IC50 of less than or equal to about 200 pM for mature human TGF-β3, in a HT-2 cell neutralization assay in vitro. In one embodiment, antibodies of the present invention comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a peptide in CDRH1 with a sequence as shown in SEQ ID NO. : 96 or SEQ ID NO: 100, a peptide in CDRH2 with a sequence as shown in SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 or SEQ ID NO: 102 and a peptide in CDRH3 with a sequence as shown in SEQ ID NO: 98, and wherein said LCVR comprises a peptide in CDRL1 with a sequence as shown in SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 92 or SEC ID NO: 93, a peptide in CDRL2 with a sequence as shown in SEQ ID NO: 89 or SEQ ID NO: 94, and a peptide in CDRL3 with a sequence as shown in SEQ ID NO: 90, or SEQ. ID NO: 95 In one embodiment, antibodies of the present invention further comprise a region of human structure. In another embodiment, antibodies of the present invention comprise an HCVR with a sequence selected from the group consisting of SEQ ID NO: 64, 68, 69, 70 and 74, and an LCVR with a sequence selected from the group consisting of SEQ ID NO: 41, 43, 45, 46 and 50. In another embodiment, antibodies of the present invention comprise an HCVR with a sequence as shown in SEQ ID NO. : 69 and an LCVR with a sequence as shown in SEQ ID NO: 45. In one embodiment, antibodies of the present invention comprise a heavy chain with a sequence selected from the group consisting of SEQ ID NO: 77, 79, 81, 83 and 85, and a light chain with a sequence selected from the group consisting of SEQ ID NO: 76, 78, 80, 82 and 84. In one embodiment, antibodies of the present invention comprise a heavy chain with a sequence as shown in SEQ ID NO: 81, and a light chain with a sequence as shown in SEQ ID NO: 80. The invention provides a method for treating proliferative disorders in a mammal, preferably a primate, and more preferably a human, which comprises administering to a mammal in need of such treatment, an effective amount of an antibody. of the invention or a fragment thereof. The invention provides a method for treating a disease or condition in which fibrogenesis and / or TGF-β mediated angiogenesis are involved in a mammal, preferably a primate, and more preferably a human, including myelodysplastic syndrome (MDS) / myeloproliferative disorder (MPD), breast cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, pancreatic cancer, multiple myeloma, colorectal cancer, other hematological malignancies (hairy cell leukemia, CML, AML, etc.), which comprises administering to a mammal in need of such treatment, an effective amount of an antibody of the invention or a fragment thereof. It may further comprise administering to the mammal an effective amount of a therapeutic agent other than anti-TGF-β antibodies, such as a chemotherapeutic agent, anti-angiogenic agent or cytotoxic chemotherapy. In another embodiment, this invention provides a method for treating a patient with metastatic breast cancer expressing HER2, which comprises administering to the patient, an effective amount of an antibody of the invention and an antibody that blocks signaling through the HE receptor. -2, that is, trastuzumab. The present invention also provides a pharmaceutical composition comprising an antibody of the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient. The present invention also provides the use of an antibody of the invention for the manufacture of a medicament for treating cellular proliferative disorders in mammals. Additionally, this invention provides a pharmaceutical composition adapted to treat cell proliferative disorders comprising an antibody of the invention, in combination with one or more pharmaceutically acceptable excipients, carriers or diluents thereof. The invention also provides the use of an antibody of the invention, for the manufacture of a medicament for treating a disease or condition capable of being improved or prevented by neutralizing TGF-β activities. This invention also provides the use of an antibody of the invention, for the manufacture of a medicament for the treatment of a disease or condition in which, the fggenesis and / or angiogenesis mediated by TGF-β, are involved in a mammal, preferably a primate and more preferably a human, which includes MDS / MPD, breast cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, pancreatic carcinoma, multiple myeloma, colorectal cancer, other hematological malignancies (hairy cell leukemia, CML, AML, etc.), which comprises administering to a mammal in need of such treatment, an effective amount of an antibody of the invention or a fragment thereof. This can furthermore comprise administering to the mammal, an effective amount of an agent Therapeutic administration of anti-TGF-β antibodies, such as a chemotherapeutic, anti-angiogenic or cytotoxic agent, or a cytokine. In addition, this invention provides a pharmaceutical composition adapted for the treatment of conditions in which the neutralization or reduction of TGF-β activities could be beneficial in a mammal, including MDS / MPD, breast cancer, prostate cancer, cancer of ovary, hepatocellular carcinoma, pancreatic cancer, multiple myeloma, colorectal cancer, other hematological malignancies (hairy cell leukemia, CML, AML, etc.), comprising an antibody of the invention in combination with one or more excipients, carriers or diluents pharmaceutically acceptable The invention also provides the use of an antibody of the invention, for the manufacture of a medicament for treating a disease or condition capable of being improved or prevented by neutralizing or reducing activities of TGF-β.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A and B show the amino acid sequences of the light chain and heavy chain variable regions of preferred antibodies of the invention, respectively. The CDR domains are printed in bold. Figures 2A and B show the alignment of the amino acid sequence of the CDR domains of antibodies preferred of the present invention. The variations are printed in bold and underlined. Figure 3 lists the amino acid sequences of the light and heavy chain of a monoclonal antibody of the invention, ie, mAb 12.7.

DETAILED DESCRIPTION OF THE INVENTION The term "monoclonal antibody" refers to a composition with a homogeneous antibody population, comprising four peptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids. This variable region of each chain includes three regions that determine the complementarity (CDR) that recognizes and binds a particular antigen. A "monoclonal antibody" can be an intact antibody (comprising a complete heavy chain and a complete light chain), a substantially intact antibody, or a portion or fragment of an antibody that comprises a binding portion to the antigen, eg, a Fab fragment, or F (ab ') 2 fragment of an antibody. However, a "monoclonal antibody" may be a single-chain variable fragment (scFv), which consists of the heavy chain variable region (VH) and the light chain variable region (VL) of a bound antibody together by a flexible peptide linker. A "homogeneous antibody population" refers to a population of homogeneous or substantially homogeneous antibody (i.e., at least about 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, more preferably at least about 97% or 98% or more preferably at least 99% of the antibodies in the population could compete in an ELISA assay for the same antigen or epitope). The antibodies may or may not be glycosylated and still fall within the bonds of the invention. The monoclonal antibodies may be homogeneous if they have identical amino acid sequence, although they may differ in a post-translational modification eg, glycosylation pattern. The term "effective amount" is taken to mean a dose of a compound of Formula I necessary to achieve the desired pharmacological effect. The term "epitope" refers to such a portion of a molecule capable of being recognized by and bound by an antibody in one or more of the binding regions to the antibody antigen. Epitopes often consist of a grouping of chemically active surface molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics, as well as specific loading characteristics.

The term "Kd" refers to the dissociation constant of a particular antibody-antigen interaction. It is calculated by the formula: Kapagado / Kencen? Cio = Kd. The term "kapagado" refers to the association constant or velocity, or specific reaction rate of the reaction that forms the complex or front, measured in units: M-l sec-1. The term "kapagado" refers to the quenched rate constant or dissociation, or specific reaction rate, for dissociation of an antibody from the antibody / antigen complex, measured in units: l / second. The affinity of a monoclonal antibody of the present invention is often correlated with a kapaga or lower rather than a higher kencend, however, without being bound by theory, both improved kapagado as kencend? Do modalities are encompassed. In a more preferred embodiment, monoclonal antibodies of the present invention are high potency antibodies or fragments thereof, which generally exhibit low kapagado values. The term "neutralizes" as used herein with respect to an activity of a monoclonal antibody of the invention, means the ability to substantially antagonize, prohibit, prevent, restrict, retard, interrupt, eliminate, stop, reduce or reverse, for example, the progress or severity which is being inhibited which includes, but is not limited to, activity or biological property, a disease or condition. The inhibition or neutralization is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher. With respect to the antibody neutralization and binding capabilities of the invention, the TGF-β referred to herein is the biologically active, human, mature form of TGF-β1, TGF-β2, and TGF-β3 (SEQ ID NO: 1, 2, and 3). See also for example, NCBI access No: P01137, NP_003229, and NP_003230, which describes the DNA and amino acid sequences of, respectively, human TGF-β1, β2 and β3 (which includes their precursor domains and mature portions). A human homodimeric TGF-β1, β2 or β3 linked to mature disulfide contains two polypeptides of 112 amino acid residues and has a predicted molecular mass of approximately 25 kDa. The antibodies of the invention bind and neutralize such mature TGF-β isoforms, but do not show significant binding of the latent isoforms TGF-β1, β2 and β3 under similar conditions. Improvements in binding and affinity kinetics solve antibody problems, for example, helping to increase safety profiles and pharmacokinetics; reducing the dosage, toxicity and cost of therapy; and improving biological effectiveness. The dissociation constant (Kd) of an antibody can be found using methods of the technique, for example, Kinexa® (see for example, Darling, et al., 2004 ASSAY and Drug Development Technologies 2: 647-57) or BIAcore® AB (Upsala, Sweden), or by adapting to Karlsson et al., 1991 J Immunol. Methods 145, 299-340. The term "kapagado" here refers to the rate constant or association, or specific reaction rate of the reaction that forms antigen: antibody complex, M "1sec ~ 1. The term" kapagad0"here, refers to the constant of dissociation or "off rate" or specific reaction rate, for dissociation of an antibody from an antibody: antigen complex (sec "1). The term "Kd" here refers to the dissociation constant of a particular antibody complex: antigen -calculated as Kd = kapagado / kencendido- Ka is the inverse of Kd. The Kd of an antibody of the invention for mature human TGF-β1 is in the range of about 0.0001 pM to about 5.0 pM. More preferably, it is in the range of about 0.001 pM to about 3.0 pM. More preferably, it is in the range of from about 0.01 pM to about 0.5 pM. The Kd of an antibody of the invention for mature human TGF-β2 is in the range of about 0.01 pM to about 5.0 pM. More preferably, in the range of about 0.05 pM to about 2.0 pM, and more preferably, it is in the range of about 0.1 pM to about 1.5 pM. The Kd of an antibody of the invention for mature human TGF-β3 is in the range of about 0.005 pM to about 3 pM. More preferably, it is in the range of about 0.05 pM to about 2.8 pM. More preferably, it is in the range of about 0.2 pM to about 2 pM. Preferably, the antibodies of the invention are characterized by a Kd of 4.0xl0-13M or less, for mature TGF-βl, a Kd of 8.0x10-12 M or less for mature human TGF-β2, and a Kd of 4.0xl0- 12M or less for mature human TGF-β3. The Kd of an antibody of the invention for TGF-β1 is at least 2 times greater than the Kd of 1D11.16 for TGF-β1, more preferably it is at least 25 times higher, and more preferably it is at least 50 times higher. To be clear, greater affinity could mean a smaller Kd value. The Kd of an antibody of the invention for TGF-β2 is at least 1.5 times greater than the Kd of 1D11.16 for TGF-β2, more preferably it is at least 10 times higher, and more preferably it is at least 20 times higher. The Kd of an antibody of the invention for TGF-β3 is at least 4 times greater than the Kd of 1D11.16 for TGF-β3, more preferably it is at least 6 times higher, and more preferably it is at least 10 times higher. The Kd of a Fab of the invention for human TGF-ßl mature, is in the range of about 0.009 pM to about 75.0 pM, is more preferably in the range of about 0.018 pM to about 50 pM, and is more preferably in the range of about 0.02 pM to about 25 pM. The Kd of a Fab of the invention for mature human TGF-β2 is in the range of about 0.0045 pM to about 60 pM, it is more preferably in the range of about 0.002 pM to about 45 pM, and is more preferably in the range from about 0.02 pM to about 35 pM. The Kd of a Fab of the invention for TGF-β1 is at least 30 times greater than the Kd of 1D11.16 for TGF-β1, more preferably it is at least 300 times higher, and more preferably is at least 1500 times higher. The Kd of a Fab of the invention for TGF-β2 is at least 10 times greater than the Kd of 1D11.16 for TGF-β2, more preferably it is at least 100 times higher, and more preferably it is at least 500 times higher . Antibodies of the invention exhibit kapaga or mature human TGF-βl preferably in the range of about 110 to about 1 (all values for kapagado are x 10 ~ 6 s "1), more preferably in the range of about 50 to about 6 , and even more preferably in the range of about 15 to about 4: for mature human TGF-β2, preferably in the range of about 240 to about 1, more preferably in the range of about 50 to about 2, and even more preferably in the range of about 20 to about 4; and for mature human TGF-β3, preferably in the range of about 90 to about 1, more preferably in the range of about 50 to about 4, and even more preferably in the range of about 35 to about 6. Antibodies of the invention exhibit improvement of kapagado on 1D11.16 for TGF-ßl, -ß2, and -ß3 in the range of about 2 to about 50 times, more preferably in the range of about 2.5 to about 25 times, and even more preferably in the range of approximately 3 to approximately 15 times. In another embodiment, an antibody of the invention has a kencend greater than 5 for mature human TGF-β1, greater than 2.8 for mature human TGF-β2, and greater than 1.56 for mature human TGF-β3 (all values for kencend The kencending for mature human TGF-ßl is preferably in the range of about 1.5 to about 60, more preferably in the range of about 2 to about 45, and even more preferably in the range from about 3 up about 30; for mature human TGF-β2, is preferably in the range of about 1 to about 40, more preferably in the range of about 1 to about 25, and even more preferably in the range of about 1 to about 15; and for mature human TGF-β3, is preferably in the range of about 1.2 to about 20, more preferably in the range of about 1.2 to about 10, and even more preferably in the range of about 1.2 to about 5. Antibodies of the invention exhibit an average kindergarten improvement over 1D11.16 for TGF-β1, β2, and -3 preferably in the range of about 1.5 to about 40 times, more preferably in the range of about 2 to about 15 times, and even more preferably in the range of about 2 to about 10 times. In a preferred embodiment, an antibody of the invention binds to TGF-β1 on TGF-β2 and β3. An antibody is said to "neutralize" its antigen if the antibody that binds the antigen results in complete or partial inhibition or reduction, of a biological function of the antigen. The neutralization of a biological activity of the TGF-ß isoform is assessed by measuring the complete or partial inhibition or reduction, of one or more indicators m vi tro or in vivo TGF-β activity such as, binding to the receptor, an inhibitory effect on cell growth; chemotaxis, apoptosis, intracellular protein phosphorylation or signal transduction. More preferably, the ability to neutralize the activity of TGF-β, as described herein, is assessed by an assay of HT-2 cell proliferation or by measuring the inhibition of Smad2 phosphorylation. Antibodies of the invention neutralize mature human TGF-β1, mature human TGF-β2, and mature human TGF-β3, and have an IC50 of less than or equal to about 100 pM, 75 pM, 50 pM, 25 pM, 17.5 pM , 10 pM, 4 pM, or 3 pM for mature human TGF-β1, an IC50 of less than or equal to about 400 pM, 345 pM, 200 pM, 100 pM, 77.5 pM, 50 pM, 40 pM, or 23 pM Mature human TGF-β2, and an IC50 of less than or equal to about 200 pM, 115 pM, 105 pM, 75 pM, 50 pM, 45 pM, or 35 pM for mature human TGF-β3 in a cell neutralization assay HT-2 in vi tro. The improved neutralizing activity increases the safety and pharmacokinetics profiles; reduces dosage, toxicity and therapy costs; and improves the biological effectiveness. In a preferred embodiment, the IC50 of an antibody of the invention in the HT-2 cell proliferation / neutralization assay, as described herein, is less than about 20 pM for TGF-β1, less than about 325 pM for TGF-β2, and less than about 125 pM for TGF-β3. Against TGF-β1, an antibody of the invention preferably has an IC0 in the range of about 0.1 to about 50 pM, more preferably in the range of about 0.1 to about 30 pM, and more preferably in the range of about 0.1 to about 20 p.m. Against TGF-β2, an antibody of the invention preferably has an IC50 in the range of about 1 to about 400 pM, more preferably in the range of about 10 to about 350 pM, and more preferably in the range of about 15 to about 325 p.m. Against TGF-β3, an antibody of the invention preferably has an IC50 in the range of about 0.1 to about 200 pM, more preferably in the range of about 1 to about 150 pM, and more preferably in the range of about 10 to about 125 p.m. In another embodiment, antibodies of the invention exhibit an average improvement of IC50 over 1D11.16 in an HT-2 cell proliferation assay for TGF-β1 in the range of about 100 to about 600 fold, more preferably in the range of about 200 to about 500 times, and even more preferably in the range of about 300 to about 400 times; for TGF-β2 in the range of about 10 to about 400 times, more preferably in the range of about 25 to about 300 times, and even more preferably in the range of about 50 to about 200; and for TGF-β3 in the range of about 2 to about 200 times, more preferably in the range of about 4 to about 50 times, and even more preferably in the range of about 6 to about 25. The concentration of an antibody required for neutralizing the activity of TGF-β is dependent on several parameters such as, for example, cytokine concentration, cell type, growth conditions and type of activity studied. The neutralization character of an antibody of the invention is assessed by measuring the degree of inhibition of phosphorylation of the Smad2 protein in a U87MG human tumor xenograft model assay, as described herein. In another preferred embodiment, an antibody of the invention has a TED50 (therapeutic effective dose) in such assay, in the range of about 100 to about 200 mg / kg, more preferably in the range of about 50 to about 80 mg / kg; and even more preferably in the range of about 5 to about 25 mg / kg.

Certain antibodies and fragments of the invention have specific CDRs: VHCDR1 XXX2WMN [SEQ ID NO: 10, wherein Xi is either T or S; and X2 is either E, or Y]; VHCDR2 QIFPX? X2GSTNYX3EMX4EG [SEQ ID NO: 11, where Xi is either A, or F; X2 is either S, T, or L; X3 is either N, G, S, D, or A; X4 is either F, or Y]; VHCDR3 GXiGNYALDAMDY [SEQ ID NO: 12, where Xi is either D, I, M, Y, L, V, Q, or F]; VLCDR1 RASESVDX? X2GNSFMH [SEQ ID NO: 13, where Xx is either S, Y, F, or L; X2 is either Y or W]; VLCDR2 XiASNLES [SEQ ID NO: 14, where Xi is either L, or Y]; and VLCDR3 X? QX2X3EDPLT [SEQ ID NO: 15, where X? is either Q, T, or C; X2 is either N, or H; X3 is either N, I, M, D, T, or A]. Also encompassed are antibodies created with CDRs of such formulas after engineered CDRs (in appropriate orientation) into humanized or human antibody structure sequences, to produce compositions of the invention that neutralize human, TGF-β1, β2 and β3, mature . Any method known in the art can be used to incorporate specific CDRs within the structure sequences. As described, humanized or human variable structure sequences can be derived from any germline or variable human domain rearranged, or for example, synthetic variable domain based on consensus sequences of known human variable domains. Variable domain structure sequences Preferred are those that do not significantly affect the biological properties of an anti-TGF-β1 antibody modality, (52 and (53 - that is, the ability to bind with high affinity and neutralize mature human TGF-β1, β2 and β3. Preferably, such structures additionally do not stimulate significant immunogenic reactions when administered to a human.Structure sequences may be naturally occurring human antibody sequences or consensus sequences of several human antibodies.Non-limiting examples of structure sequences for the variable region of Heavy chain antibody modalities of the invention include, the VH DP-5 segment (Tomlmson, et al., 1992 J. Mol. Biol. 227: 776-98) and the J segment of JH4, JH1 or JH5 (Ravetch, et al. al., 1981 Cell 27: 583-91), the Vk Ll segment (Cox, et al., 1994 Eur. J. Immunol., 24: 827-36) and the J segment of k4 (Hieter, et al., 1982 J. Biol. Chem. 10: 1516-22), are non-limiting examples of e structure sequences for the light chain variable region. In a preferred embodiment, the FR1 fragment of HCVR comprises [SEQ ID NO: 16]; FR2 structure of HCVR comprises [SEQ ID NO: 17]; FR3 structure of HCVR comprises [SEQ ID NO: 18]; and the FR4 structure of HCVR comprises [SEQ ID NO: 19]. In another preferred embodiment, the structure FR1 of LCVR comprises [SEQ ID NO: 20]; FR2 structure of LCVR comprises [SEQ ID NO: 21], structure FR3 of LCVR comprises [SEQ ID NO: 22]; and structure FR4 of LCVR comprises [SEQ ID NO: 23]. In another preferred embodiment, an HCVR structure comprises [SEQ ID NO: 86] wherein Xi is either S, Y, F, or L; X2 is either Y or W; X3 is either A, or F; X4 is either S, T, or L; X5 is either N, G, S, D, or A; X6 is either F, or Y; and X7 is either D, I, M, Y, L, V, Q, or F. In another preferred embodiment, an LCVR structure comprises [SEQ ID NO: 87] wherein Xx is either T or S; X2 is either E, or Y; X3 is either it is either L, or Y; X4 is either Q, T, or C; X5 is either N, or H; and X? is either N, I, M, D, T, or A. In another preferred embodiment, the constant regions and structure may contain alterations, deletions, additions, substitutions or any combination thereof, compared to human sequences. The constant regions and structures in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are substituted, deleted or added in any combination are preferred. In another embodiment, the structure has 85-99% sequence identity to a structure described herein. In one embodiment, a preferred heavy chain constant region for use with an antibody binding composition of the invention is an IgG constant region. In a more preferred embodiment, the IgG constant region is an IgG1 constant region or an IgG4 constant region (even more preferably, they are constant regions of [SEQ ID NO: 24]; or [SEQ ID NO: 25]). A preferred light chain constant region sequence of the invention is [SEQ ID NO: 26]. In another preferred embodiment, the antibody binding compositions contain the IgG1 heavy chain constant region or the IgG4 heavy chain constant region and the kappa light chain constant region. Also encompassed are polynucleotide sequences encoding antibodies of the invention. When antibodies are engineered using CDRs of the invention, the identification of residues within antibody fragments probably influences the antigen binding donor and the acceptor sequences can be determined by aligning templates of sequences derived from antibody repertoires. "Invariant residues" (Kabat et al., 1991), and "key residues" (Chothia et al., 1989), can be identified, and canonical class assignments of loops binding to the donor antigen L1-L3, H1, and H2 , respectively, can be determined by selecting a proposed sequence against sequence templates (see for example, Martin &Thornton, 1996 Mol. Biol. 263: 800-15). Residues at the VH / VL interface (Chothia et al., 1985) and residues known to be structurally conserved in Nuclear sites (Chothia et al., 1998) are compared with corresponding donor and acceptor residues. Residues of acceptor and donor structure without pairing in these sites are analyzed based on the information of other antibodies of known structure (Berman, et al., 2000 Nucí Acids Res. 28 (1): 235-42). The selection of human structures as templates for humanization of non-human V regions can define subsequent decisions regarding which residues to humanize. The choice of homologous templates of antibodies with known crystal structure, germline, non-germinal or consensus sequences derived from available databases, can be used (see, for example, Routledge et al. (Routledge, et al., 1993 in Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man (Clark, M., ed) ppl4-44, Academic Titles, Nottingham, UK, and B. Lo, 2004 Antibody Engineering: Methods and Protocols, Humana Press). designing antibodies is to choose the closest human germline sequence as the structure to receive donor CDRs (Tomlinson et al., 1992), using a better fit strategy to look for germline sequences in the databases. of germline is used because human germline sequences do not present somatic hypermutations, which are potentially immunogenic. nucleic acids encoding variant antibodies of the invention from a combination of framework regions and CDRs of the invention included in modified human structures, can be engineered using a consensus human germline strategy, wherein a human subgroup is used as the structure (see for example, Presta et al., 1993 J. Immunol 151: 2623-32, Couto et al., 1994 Hybridoma, 13: 215-9, Couto et al., 1995 Cancer Res. (Suppl.), 55 , 5973s-7s; Werther et al., 1996 J. Immunol., 157: 4986-95; O'Connor et al., 1998 Protein Engng 11: 321-8). Antibody fragments (as described), or part of a sequence or SEQ ID NO. aguí, they are also covered. Such polypeptide and / or protein fragments may be "free standing" or comprise part of a larger polypeptide or protein, of which the fragment forms a portion or region, eg, a single continuous region of a SEQ ID NO. : here, such as, for example, connected in a fusion protein. Polynucleotides encoding such fragments are also contemplated. Antibodies of the invention are engineered by any method known in the art, such as, for example, chemical synthesis; or recombinant, genetic or molecular engineering design, and are not restricted by the creation method. Typically, the nucleic acids encoding antibodies of the invention include a sequence of expression control polynucleotide, operably linked to the coding sequences, which include heterologous or naturally associated promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors that transform or transfect host eukaryotic cells, but control sequences for prokaryotic hosts can also be used. Once the vector is incorporated into an appropriate host cell, it is propagated under conditions suitable for expressing sequences, and, as desired, for the collection and purification of light chains, heavy chains, light / heavy chain dimers, or antibodies intact, link fragments or other forms of immunoglobulin. The human constant region DNA sequences are isolated by methods known in the art from a variety of human cells, but preferably, from immortalized B cells. Suitable source cells for the polynucleotide and host cell sequences for immunoglobulin and secretion expression are obtained from sources known in the art. The invention encompasses sequences of functiopolypeptides with substantial sequence identity or similarity to a sequence here (eg, at least: 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% identical to a sequence of the invention (or fragment)), using methods known in the art, such as, for example, paired waste optimization. Such sequences include any with a functiocharacteristic of an antibody of the invention (eg, binding and neutralization of mature human TGF-β1, β2 and β3 in an assay described herein). Such functioy related embodiments include additions, substitutions and / or deletions of amino acid residues of a sequence of the invention in a CDR or a constant region. Substitutions and / or amino acid additions can be based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues involved. Also, non-classical amino acids or chemical amino acid ags can be substituted or added in the polypeptide sequence. All of such variants are in the field of those skilled in the art of molecular biology, given the teachings of agui, specifying unique formulas or polypeptide sequences and the functiolimitations of the invention. Preferred embodiments of the composition of the invention have one or more of the following characteristics: Kd less than 2 x 10"13 M for TGF-β1, less than 5 x 10 ~ 13 M for TGF-β2, less than 8 x 10" 13 M for TGF-β3; kapagado less than 8 x 10"6 s ~ l for TGF-ßl, less than 11 x 10" 6 s "1 for TGF-ß2, less than 13 x 10 ~ 6 s_1 for TGF-ß3; kencend? 5 x 107 M "1 s" 1 for TGF-ßl, greater than 2 x 107 M "1 s" 1 for TGF-ß2, and greater than 1.5 x 107 M "1 s" 1 for TGF-ß3; Kd improvement of a Fab composition for TGF-β1 with respect to a Fab of 1D11.16 for TGF-β1 of at least 6000-fold; Kd improvement of an antibody composition with respect to 1D11.16 greater than about 34-fold for TGF-β1, greater than about 13-fold for TGF-β2, and greater than about 9-fold for TGF-β3; IC 50 greater than about 300-fold higher than an IC 50 value of 1 D 11.16 in an HT-2 assay; an ED50 value in the range of about 11 to about 17 mg / kg in a U87MG human tumor xenograft model for the inhibition of Smad2 phosphorylation of (as described herein); less than 5.0% aggregation of a composition of the invention as determined by size exclusion chromatography after storage for 30 days at a concentration of lmg / ml, 40 ° C, in standard buffer (such as for example, Citrate ( 20mM); Phosphate (10mM); or PBS (10mM phosphate, containing 150mM NaCl)), and at pH (5.0, 6.5, or 7.4); less than 1% degradation products or non-acidic forms - as determined by cation exchange chromatography (CEX) - after storage for 30 days at 1 mg / ml, less than 35 ° C, in standard buffer (such as example, Citrate (20mM); Phosphate (10mM) or PBS (10mM phosphate, containing 150mM NaCl)), and < pH7.0; and greater than 80% of protein recovery of a composition of the invention using methods known in the art after storage at a concentration of 2 mg / ml at 4 ° C for two weeks and recovery from dialysis at pH 6.0. The compositions of the invention are employed as therapeutics to modulate, treat, inhibit, alleviate or prevent a disease, disorder, condition, syndrome or condition associated with one or more of TGF-β1, and β2 and β3. The antibodies can be administered using any method known in the art and can be combined for use with conventional pharmaceutically acceptable carriers, diluents, stabilizers and excipients. These combinations can be placed in dosage forms such as by lyophilization in sealed dosage vials or under storage in stabilized aqueous preparations. Pharmaceutically acceptable carriers, diluents, stabilizers and excipients are known or described, for example in the Merck Index, Merck & Co. , Rahway, NJ. In particular, antibodies (or fragments) of the invention are used for the treatment of cell proliferative disorders, including any disease, syndrome, disorder, condition or condition, which affects any cell, tissue, any site or any combination of organs, tissues or body parts, which are characterized by a single or multiple local abnormal proliferation of cells, groups of cells or tissues, whether benign or malignant. As defined herein, a cell proliferative disorder encompasses for example, hematological malignancies (such as, for example, MDS or MPD, such as, for example, with megakaryocyte involvement (see, eg, Sakamaki, et al., 1999 Blood 94 (6): 1961-70), hairy cell leukemia and other hematologic malignancies, such as, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) ), etc.)); non-small cell lung cancer; breast cancer; prostate cancer (which includes refractory hormone); ovarian cancer; hepatocellular cancer; pancreatic cancer; multiple myeloma; Colorectal cancer; a neoplasm of the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine system (for example, an adrenal gland, a parathyroid gland, the pituitary, the testicles, the ovary, the thymus or the thyroid), oo, head, neck, nervous system (central or peripheral), the lymphatic system, pelvis, skin, spleen, thorax and urogenital system. Additionally, an antibody of the invention is employed for musculoskeletal diseases, such as for example, treatment of muscle wasting (e.g., cachexia); promotion of muscle growth (for example, after the disease, trauma, reconstruction, relocation).

A "chemotherapeutic agent" is a chemical compound used in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as cyclophosphamide and fluorouracil; antimetabolites, such as fluorouracil and gemcitabine; antibiotics, such as adriamycin; and antimyotic agents, such as vincristine and vinorelbine. An "anti-angiogenic agent" refers to a compound that blocks, or interferes with, to some degree, the development of blood vessels. The anti-angiogenic agent can be, for example, a small molecule or antibody that binds to a growth factor or a growth factor receptor involved in promoting angiogenesis. The term "cytotoxic chemotherapy" as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells, including radioactive isotopes (eg, At.211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. A polynucleotide is "operably linked," when it is placed in a functional relationship with another polynucleotide. For example, a promoter or intensifier it is operably linked to a coding sequence if it affects the transcription of the sequence. A peptide is "operably linked" to another peptide when the polynucleotides encode them are operably linked, preferably they are in the same open reading structure. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked including, but not limited to, plasmids and viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced while other vectors can be integrated into the genome of a host cell at the introduction of the host cell, and therefore, replicate along with the genome host However, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors") and exemplary vectors are well known in the art. As used in this document, expressions "cell", "host cell", "cell line" and "cell culture" are used interchangeably and include a single cell or cell culture that is a container of any isolated polynucleotide of the invention or any of the recombinant vectors comprising a sequence encoding an HCVR, LCVR or monoclonal antibody of the invention. Host cells include the offspring of a single host cell, and the offspring may not necessarily be completely identical (in morphology or in a total DNA complement) to the original precursor cell due to mutations and / or natural, accidental or deliberate changes. A host cell includes transformed, transduced or infected cells in vivo or in vitro with one or more recombinant vectors or a polynucleotide that expresses a monoclonal antibody of the invention or a light chain or heavy chain thereof. A host cell which comprises a recombinant vector of the invention (either stably incorporated into the host chromosome or not) can also be referred to as a "recombinant host cell". Recombinant host cells for use in the invention are CHO cells (e.g., ATCC CRL-1650, CRL-1651); HeLa (ATCC CCL-2). Additional host cells for use in the invention include plant cells, yeast cells, other mammalian cells and prokaryotic cells.

Expression and Purification of Antibody To express an antibody of the invention, a DNA encoding a full-length light or heavy chain is inserted into an expression vector of such that the gene is operably linked to transcriptional or translational control sequences. The expression vector and the expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods. Additionally, the recombinant expression vector can encode a signal peptide which facilitates the secretion of the light and / or heavy chain of the anti-TGF-β monoclonal antibody from a host cell. The light and / or heavy chain gene of the anti-TGF-β monoclonal antibody can be cloned into the vector such that the signal peptide is operably linked to the amino chain structure of the antibody chain gel. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. In addition to the light and / or heavy chain genes of the antibody, a recombinant expression vector of the regulatory sequences of carriers of the invention that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other elements of expression control (eg, polyadenylation signals), as needed, which control transcription or translation of the antibody chain genes. The design of the expression vector, which includes the selection of regulatory sequences may depend on such factors as the one selected from the host cell to be transformed, the expression level of the seized protein. Preferred regulatory sequences for mammalian host cell expression include viral elements directing light levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (eg, the best late promoter adenovirus (AdMLP)) and polyoma virus. In addition to the light and / or heavy chain genes of the antibody and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate the application of the vector in host cells (e.g., origins of replication) and one or more selectable marker genes. The selectable marker gene facilitates the selection of host cells in which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, or a host cell in which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in negative DHFR host cells with methotrexate selection / amplification), the neo gene (by selection G418), and glutamine synthetase (GS) in a negative cell line (such as NSO) for selection / amplification. For expression of the light and / or heavy chains, the expression vectors encoding the heavy and / or light chains are introduced into a host cell by standard techniques, eg, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, transduction, infection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, eukaryotic cells are preferred, and more preferably mammalian host cells, because such cells are more likely to assemble and secrete an appropriately closed and immunologically competent antibody. active. Preferred mammalian host cells for expressing the recombinant antibodies of the invention, include Chinese Hamster Ovary (CHO) cells (including DHFR-CHO cells, described in Urlaub and Chasin, Proc. Nati. Acad. Sci. USA 77: 4216 -20, 1980, used with a selectable DHFR marker, for example, as described in Kaufman and Sharp, J. Mol. Biol. 159: 601-21, 1982 and GS-CHO cells, described in Enosawa, et al., Cell Transplantation 6: 537-540, 1997, used with a selectable marker of glutamine synthetase (GS), NSO myeloma cells, COS cells and SP2 / 0 cells. When the recombinant expression vectors encoding the antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion. of the antibody in the culture medium in which the host cells are grown. The antibodies can be recovered from the host cells and / or the culture medium using standard purification methods. Host cells can also be used to produce portions or fragments of intact antibodies, for example, Fab fragments or scFv molecules by techniques that are conventional. It will be understood by one skilled in the art that variations in the above method are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain of an antibody of this invention. The recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that do not necessarily bind to TGF-β. The Expressed molecules of the truncated DNA molecules are also encompassed by the antibodies of the invention. In a preferred system for the recombinant expression of an antibody of the invention, a recombinant expression vector encoding both the heavy chain of the antibody and the light chain of the antibody is introduced into GS-CHO cells by electroporation. Within the recombinant expression vector, the light and heavy chain genes of the antibody are operably linked to each other for enhancer / promoter regulatory elements (eg, SV40, CMV, adenovirus derivatives and the like, such as a CMV enhancer regulatory element / promoter AdMLP or an SV40 enhancer regulatory element / AdMLP promoter) to direct high levels of transcription of genes. The recombinant expression vector also carries a DHFR, which allows selection of CHO cells that have been transfected with the vector using selection / amplification of methotrexate. The selected transformant host cells are cultured to allow expression of heavy and light chains of the antibody and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells and recover the antibody from the culture medium.

Antibodies, or antigen binding portions thereof, of the invention can be expressed in an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, et al., Nucleic Acids Res. 20: 6287-95, 1992). Once expressed, the intact antibodies, their dimers, individual light and heavy chains, or other forms of immunoglobulin of the present invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation chromatography, ion exchange. , affinity, reverse phase, hydrophobic interaction column, gel electrophoresis and the like. Substantially pure immunoglobulins of at least about 90%, 92%, 94% or 96% homogeneity are preferred, and 98 to 99% or more preferred greater homogeneity, for pharmaceutical use. Once purified, partially or for homogeneity as desired, the peptides can then be used therapeutically or prophylactically, as discussed in this document.

Composition An antibody of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. The compounds of the invention can be administered alone or in combination with a carrier, diluent and / or pharmaceutically acceptable excipients, in single or multiple doses. The compositions for administration are designed to be appropriate for the selected form of administration, and pharmaceutically acceptable diluents, carriers and / or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like. they are used as appropriate. Said compositions are designed in accordance with the aforementioned techniques as in, for example, Remigton, The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995 which provides a compendium of formulation as they are generally known by doctors. A composition comprising an antibody of the invention can be administered to a subject exhibiting pathologies or disorders as described herein using standard administration techniques including intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository. The route of administration of an antibody of the invention can be parenteral. Preferably, antibodies of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. He Parenteral term as used herein includes intravenous, intramuscular, subcutaneous, rectal vaginal or intraperitoneal administration. Peripheral systemic delivery is preferred by intravenous or intraperitoneal or subcutaneous injection. The composition can typically be sterile and stable under the conditions of manufacture and storage in the provided container, which includes, for example, a sealed vial or syringe. Therefore, the compositions can be sterile filtered after elaborating the formulation, or otherwise elaborated microbiologically acceptable. A typical composition for intravenous infusion can have a volume as much as 250-1000 ml of fluid, such as a sterile Ringer's solution, physiological saline, dextrose solution and Hank's solution and a therapeutically effective dose (eg, 1 to 100 mg / ml, or more) of antibody concentration. The dose may vary depending on the type and severity of the disease. As is well known in medical techniques, the dosage for any subject depends on many factors, including the patient's size, body surface area, age, particular compound to be administered, sex, time and route of administration, general health and other drugs to be administered simultaneously. A typical dose may be, for example, in the range of 0.001 to 1000 μg; without However, doses below or above this exemplary interval are foreseen, especially considering the aforementioned factors. The daily parenteral dosage regimen may be about 0.1 μg / kg to about 100 mg / kg of total body weight, preferably about 10 μg / kg to about 5 mg / kg and more preferably about 10 μg / kg to 3 mg / kg. kg of body weight per day. Progress can be monitored for periodic evaluation. For repeated administration for several days or longer in the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and are not excluded therefrom. The desired dosage can be supplied by a single administration bolus, by multiple administration boluses, or by administration of continuous infusion of the antibody, depending on the pattern of pharmacokinetic decomposition that the physician wishes to achieve. These suggested amounts of the antibody are subject to a large amount of therapeutic discretion. The key factor in the selection of an appropriate dose and scheme is the result obtained. Factors for consideration in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of supply of the antibody, the particular type of antibody, the method of administration, the administration schedule and other factors known to medical professionals. The therapeutic agents of the invention can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier before use. The lyophilization and reconstitution can lead to varying degrees of loss of antibody activity. The dosages can be adjusted to compensate. Generally, pH is preferred between 6 and 8.

Articles of Manufacture In another embodiment of the invention, there is provided an article of manufacture containing materials useful for the treatment of the disorders or conditions described above. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container houses a composition of the invention, which is effective for treating the disorder or condition and can have a sterile access port (for example the container can be a bag or vial of intravenous solution having a plug that can be punctured by a hypodermic injection needle). The active agent in the composition is an anti-TGF-β antibody of the invention. The label on, or associated with, the container indicates that the composition is used to treat the selection condition. The processed article may further comprise a second container comprising a pharmaceutically acceptable buffer, such as buffered phosphate salt, Ringer's solution and dextrose solution. It may also include other desirable materials from a commercial and useful point of view, which includes other buffers, diluents, fillers, filters, needles, syringes and containers inserted with instructions for use. The following examples are offered for illustrative purposes only, and it is not intended to limit the scope of the present invention by any means.

EXAMPLES Example 1: Production and Purification of the Antibody For the simultaneous expression of the light chain and heavy chain of antibodies of the invention, both the genes encoding the heavy chain and the light chain are cloned to form a vector of the double gene with each gene under control of a separate hCMV-MIE promoter. Correct coding sequences are confirmed by DNA sequencing of both heavy and light chain coding sequences. The expression plasmid for the simultaneous expression of the light chain and heavy chain of an antibody of the invention is linearized by the enzyme of single cut Sal I, precipitated with sodium acetate and ethanol, washed with 70% ethanol cooled on ice, and air dried in a sterile biosafety cabinet. The DNA pellet is then dissolved again with the transfection medium and used for transfected CHO cells. Transfection was performed by electroporation of the cell-DNA mixture using Gene Pulsar (BioRad, Hercules, CA) adjusted to 300 V and 1120 uFd. Clones expressing an antibody of the invention are then generalized by limiting dilution and further amplified for antibody production and purification. Antibodies of the invention are purified in accordance with standard procedures of the art, including chromatography of ammonium sulfate precipitation, ion exchange, affinity, reverse phase, hydrophobic interaction column, gel electrophoresis and the like.

Example 2: ELISA Run ELISA using microtitretor plates coated with Costar 3366 (overnight at 4 ° C with 0.4 μg / ml of TGF-β1, TGF-β2 or TGF-β3). The plate is then washed (2X) before adding blocking solution (100 μL) BSA (10 mg / ml) in wash buffer) per well. The dilutions Fab are incubated in coated cavities (1.5 h, 22 ° C). After washing, kappa-alkaline conjugate is added anti-human phosphatase and incubated (1 h, 22 ° C). A colorimetric substrate is added after extensive washing and absorbance measurement (A560). In another example, binding compositions are tested in a competitive ELISA assay. Typically, a solution phase test is performed in which a compound that can compete with an antigen to bind to an antibody, such an antibody is combined with the antibody in the solution phase, then the degree of binding of an antibody to the antigen. is subsequently measured. Materials: Shock absorber containing carbon (50 mM sodium carbonate, pH 9.6). Antigens: TGF-ßl (R & amp; amp;; D Systems, Cat # 240-B / CF, 239 μg / ml), TGF-β2 (RD1, Cat # RDI-1035, 50 μg / ml) and TGF-β3 (RD1, Cat # RDI-1036- / CF , 50 ug / ml) diluted at 0.4 ug / ml in coating buffer. The wash buffer (0.02 M Tris, pH 7.4, 0.15 M NaCl, 0.1% Tween 20 and blocking solution of 10 mg / ml BSA (Sigma A-4503) was dissolved in wash buffer). Proteins used as positive controls are mouse anti-human TGF-β1, β2 or β3 (R & D Systems, cat # 1D11), mouse anti-human TGF-β2 (R & D Systems, cat # BAF302) and TGF -β3 anti-human mouse (R & D Systems, cat # BAF243), which is diluted at 1 ug / ml in blocking buffer. The detection antibody conjugate is conjugated kappa-peroxidase anti-mouse (Southern Biotech, cat # 1050-05), at a working concentration of 1: 2000 in blogging solution. The colored reaction substrate is O-phenylenediamine (OPD) tablets (Sigma cat # P-6912) dissolved in substrate buffer: 0.1 M Na2HP04, pH at 5.0 with 0.05 M citric acid. The OPD working solution (ie, the volume for a 196-well plate) is freshly made before each plate development by dissolving 1x5 mg of OPD tablets in 12.5 ml of the substrate buffer, then by 5 ul of 30% H202 . Protocol: A single 96-well plate was coated with antigen (TGF-ßl, ß2 or (53 to 0.4 ug / ml and dispersed at 50 ul per well) the tape is sealed and stored (16-20 h, 4 ° C The plate is washed (2X) in wash buffer before adding blocking solution (100 ul per 10 mg / ml BSA cavity in wash buffer) After incubation (~ 1 h, 22 ° C) , the plate is washed (2X) with wash buffer, then 100 ul of either sample (diluted in buffer) or control (diluted in PBS) is added per cavity and incubated (1.5 h-22 ° C). incubation, the plate is washed (6X) with wash buffer, then any anti-mouse kappa-peroxidase conjugate (diluted 1: 2000 in blocking solution) or SA-HPR (diluted) is added (100 ul / well). 1: 10,000 in blocking solution.) The test samples are allowed to incubate (1 h, 22 ° C)) before adding 100 ul of the OPD / cavity substrate. After developing color (-10 minutes), the 96-well plate was measured at an absorbance of 490 nm.

Example 3: Kinetic constants for Fabs A KinExA 3000 instrument (Sapidyne Inst. Inc.) measures the binding kinetics. Briefly, the antigen is covalently coupled to azlactone beads and the binding of a Fab-free invention to the perlillas in the instrument is detected. To measure Kd, they are incubated (l-6d-25 ° C in PBS containing 1% BSA, 0.02% azide and 0.01% Tween 20) individual tubes containing 20 pM Fab (200 pM for antibody) with antigens serially diluted decreased (0-250 nM). After incubation, free Fab is determined in each sample equilibrated in KinExA 3000 by the manufacturer's instructions. Kd values are determined using the KinExA 3000 software. To measure individual kinsense Fabs at 2 nM, 0-240 nM of the antigen is mixed using the injection method in accordance with the manufacturer's instructions, and unbound Fab is detected. The resulting data are used to calculate the kinetics with the KinExA software. The kapagad0 is calculated using the formula K = kapagado / kencendido- Table 1 shows affinity data obtained under KinExA © conditions for Fab modalities linked to TGF Beta 1.

Table 1 Similarly, Fab data for affinity binding of TGF-β2 under KinExA © conditions show Kd < 35 pM.

Example 4: Kinetics Constants for Antibodies Alternative methods for measuring kinetic constants are known, for example: affinity of an antibody of the invention for TGF-β1 (R & D Systems, Cat # 240-B / CF), TGF-β2 (RD1, Cat # RDI-1035) and TGF-β3 (RD1, Cat # RDI-1036 / CF) is measured by BIAcore® 2000. Link affinity measurements for full-length monoclonal antibodies of the invention are determined using Biacore. Except as noted, all reagents and materials were purchased from BIAcore® AB (Upsala, Sweden). All measurements are made at room temperature. The samples are dissolved in HBS-EP buffer (150 mM sodium chloride, 3 mM EDTA, 0.01% surfactant P-20 (w / v) and 10 mM HEPES, pH 7.4). The recombinant protein A it is immobilized in all four flow cells of a CM4 sensor chip at a level of 400-450 response units (RUs) using an amine coupling kit. The link is evaluated using multiple analytical cycles g. Each cycle is carried out at a flow rate of 50 μl / rnin consisting of the steps: injection of 12 μl of the antibody at 0.5 μg / ml, injection of 250 μl of TGF-βl (starting at 5 nM and using serial dilutions of twice at 0.13 nM for each cycle, with two injections for each concentration) followed by any short delay (5 minutes) or long (120 minutes) for dissociation, and regeneration using two injections of 50 μl of 10 mM glycine hydrochloride, pH 1.5. Association and dissociation ratios are performed per cycle by adjusting the biosensor data from a simple association model using ClampXP (Center for Biomolecular Interaction Analysis, University of Utah) to extract the kencend? Do and kapagad0 ratio constants.; the equilibrium bond constant Kd is calculated from Kd Full-length monoclonal antibodies of the invention are constructed by Fabs operably linked to an Fc IgG region using standard techniques: mAb 3. A comprising LC of SEQ ID NO: 76 & HC of SEQ ID NO: 77; mAb 4.17 LC of SEQ ID NO: 84 & HC of SEQ ID NO: 85; mAb 12.4 LC of SEQ ID NO: 78 & HC of SEQ ID NO: 79; mAb 12. 7 LC of SEQ ID NO: 80 & HC of SEQ ID NO: 81 and mAb 12. 8 LC of SEQ ID NO: 82 & HC of SEQ ID NO: 83. When mAb is measured using the assay described, the results are as reported in Table 2 below.

Table 2 Values Average ^ on Mabs Isoform (M_1 s "1) ^ off S J KD (M) Mab 12.4 TGF-bl > 4 E + 07 6.71E-06 < 2.0 E-13 TGF-b2 2.42E + 07 6.00E-06 2.65E-13 TGF-b3 1.76E + 07 15.3E-06 8.51E-13 Mab 12.7 TGF-bl > 5E + 07 7.93E-06 < 2.0 E-13 TGF-b2 2.80E + 07 11.1E-06 4.73E-13 TGF-b3 1.56E + 07 12.8E-06 8.20E-13 Mab 12.8 TGF-bl > 5 E + 07 7.92E-06 < 2.0 E-13 TGF-b2 2.29E + 07 1.22E-05 1.00E-12 TGF-b3 1.85E + 07 1.12E-05 5.40E-13 Mab 3. A TGF-bl > 5 E + 07 1.07E-04 < 2.0 E-12 TGF-b2 > 5 E + 07 2.18E-04 < 4 E-12 TGF-b3 2.90E + 07 2.38E-05 9.26E-13 Mab 4.17 TGF-bl > 5 E + 07 9.71E-06 < 2.0 E-13 TGF-b2 2.37E + 07 9.86E-06 5.29E-13 TGF-b3 2.17E + 07 2.84E-05 1.53E-12 Example 5: Specificity BIAcore was used to assess the specificity of antibodies for entities, such as, for example, the latent form of TGF-β1, β2 or β3. All measurements are made at room temperature. The samples dissolve in buffer HBS-EP (150 mM sodium chloride, 3 mM EDTA, surfactant P-20 0.01% (w / v) and 10 mM HEPES, pH 7.4). Recombinant protein A is immobilized in all four flow cells of a CM4 sensor chip at a 400-450 response unit (RUs) level using an amine coupling kit. The link was assessed using multiple analytical cycles. Each cycle was carried out at a flow rate of 100 μl / minute which consisted of the following steps: injection of 15 μl of an antibody binding composition at 1 μg / ml, injection of 250 μl of either 5 nM of TGF- ßl, 5 nM of latent TGF-ß2 or 5 nM of TGF-ß3 followed by a short delay (5 minutes) for dissociation, and regeneration using two injections of 50 μl of 10 mM glycine hydrochloride, pH 1.5. The amount of signal is determined after capturing the antibody then ligated using the instrument control software. As the signal is provided to the captured protein mass, the stoichiometry of the captured ligand is easily calculable. Under such conditions, the data for antibodies of the invention do not show significant specific binding of the latent TGF isoforms.

Example 6: HT-2 Cell Neutralization Assay To test the ability of an antibody to neutralize TGF-β bioactivity, the assay can be adapted of HT-2 cell proliferation from Tsang, et al., (1995 Citokine 7: 389-97). The HT-2 assay assesses the neutralization characteristics of an antibody to the bioactivity of TGF-β by inhibiting and / or significantly decreasing the cell proliferation of the IL-4-dependent HT2 cell line. Briefly, HT-2 cells proliferate in a dose-dependent manner by IL-4 but suffer from apoptosis by TGF-β. The inhibition of TGF-β proliferation is blocked by adding an anti-TGF-β antibody. Human HT-2 cells proliferate in response to IL-4 but TGF-β1, β2 or β3 inhibit proliferation induced by IL-4. Consequently, an antibody is neutralized if it prevents the normal inhibitory effect of TGF-β on HT-2 cells induced by IL-4. Therefore, cell proliferation induced by IL-4 should proceed spontaneously if sufficient amount of a specific binding composition to TGF-β1, β2 and β3 is added to a mixture of HT-2 cells containing an inhibitory amount of cell proliferation of TGF-ßl, ß2 or ß3. The neutralizing capacity of the dose response is performed using the HT-2 assay in the presence of particular TGF-β isoforms and the proliferation signal IL-. The degree of cell proliferation is determined using a commercial cell proliferation assay (e.g., A CellTiter 96® Cell Proliferation Assay Aqueous Solution from Promega). The HT-2 cells maintained in RPMI 1640 supplemented with 10% FBS, penicillin / streptomycin (100 U / ml and 100 ug / ml respectively), 50 uM of beta-mercaptoethanol and 10 ng / ml of hIL-2 (R &D Systems). The cells are centrifuged at 1000 RPM in a Jouvan CR422 centrifuge and resuspended in PBS. After washing (2X) with PBS, the cells are finally resuspended (0.15xl06 cells / ml in Test Medium (RMPI 1640 red phenol-free supplemented with 2% FBS, penicillin / streptomycin (100 U / ml and 100 ug / ml respectively) and 50 uM of beta-mercaptoethanol.) 50 ul of cells in Test Medium is added to each well of a 96-well plate Variable concentrations of an antibody of the invention are pre-incubated with TGF-β1, recombinant ß2 or ß3 (300 ug / ml in Test Medium) After 30 minutes of pre-incubation, 50 ul of the TGF-β / antibody mixture is added to the HT-2 cells, followed immediately by 50 ul of medium Assay containing 6.0 ng / ml murine IL-4 (2.0 ng / ml final) After incubation with the assay medium (20-48 hours, 37 ° C in a 5% C02 atmosphere, humidified) , 35 ul of Acuosa CellTiter 96 solution (Promega Corp) is added, after the additional incubation (2-3 hours, as above), the assay is quantified by analysis in a 490 nM ELISA plate reader using the CellTiter 96® colorimetric assay (the amount of the formazan product, as measured by the amount of absorbance 490 nm, is directly proportional to the number of living cells). Compared to 1D11.16; ATCC-HB9849, antibodies of the invention exhibit enhanced neutralization of cell death induced by TGF-β1, β2 and (53 and neutralization potency (e.g., IC50 <0.1 mg / ml or <125 pM) as shown in Table 3 below.

Table 3 Example 7: Heterologous Graft Neutralization Assay In binding TGF-βRI and TGF-βRII receptors, TGF-β ligands activate a signaling cascade, in which the Sbad-2 proteins are phosphorylated to produce upstream biological effects such as , for example, in cancer. The inhibition and / or significant decrease of Smad2 phosphorylation reveals the neutralization of biological activities of TGF-β via transcriptional activation (see, for example, Li et al., 2005 World J Surg 29 (3): 306-11). To assess the effectiveness of In vivo neutralization of an antibody, Fosfo-Smad2 levels are performed in a heterologous graft model and / or in multiple organs or tissue after exposing an antibody of the invention to provide evidence of its neutralization efficacy in cell proliferative conditions such as, for example, cancer. The in vivo efficacy tested is assessed by measuring the degree of phospho-Smad2 inhibition using a highly vascularized U87MG human tumor heterologous graft model (see, for example, Plowman et al., 1997"Human tumor xenograf models" in Anticancer Drug Development Guide : Preclinical Screening Clinical Triais and Approval: Teicher B (ed) pp 101-25. Humana Press: Totowa NJ). Pure female nu / nu nude mice (Charles River, -22-24 g) were quarantined and maintained (water and food 7d ad libi tum) before experimental manipulation. Initiating tests for side injections (cs) of subconfluent human U87MG glioblastoma cells (~ 5xl06 / per animal in 0.2 ml of culture medium mixed with Matrigel (BD Biosciences, 1: 1 v / v)) to promote growth of tumor. The heterologous grafts are then monitored until the tumor volume reaches ~ 300 mm3 after the animals were randomly divided into treatment groups (10 / group) with dosing initiated. Initiating post-tumor implantation therapy when mAb 12.7 (i.p.) is administered at various dosages (eg, 1, 10, 100 ug / animal) in a saline vehicle twice a week (q4d) for a dosing duration of two weeks. Controls (100 ug) human and saline IgG4 are dosed in parallel. The animals are sacrificed 48 hours after the tumor dose and the lung samples are collected and divided into two frozen in liquid nitrogen. Samples are subsequently ground or lysed for Smad2 phosphorylation analysis by ELISA using antibodies against phosphorylated or total Smad2. Blood is also collected in tubes treated with EDTA via cardiac puncture. Blood samples are centrifuged to obtain plasma samples (800 rpm, 4 ° C, 30 minutes, then 3000 rpm, 4 ° C, 10 minutes), which is stored (~ 80 ° C) until analysis. The statistical comparison of Smad2 phosphorylation is performed using JMP5.1 (SAS Institute). Dunner and one-way ANOVA tests are also used with a control. The phospho-Smad2 levels are tested to evaluate the target inhibition induced by treatment with compositions of the invention. The phospho-Smad2 level is normalized to total Smad (or total protein) to minimize the variation introduced by tissue size and manipulation process. Data from this heterologous graft model U87MG shows a dose-dependent inhibition of Smad2 phosphorylation, thus demonstrating the in vivo neutralization efficiency in TGF-β modulation effects on cell proliferation. The data show that a dose of 10 μg decreases Smad2 phosphorylation by 60% (p = 0.012), with 72% inhibition achievable at a dose of 100 μg (p <0.0001). In addition, a 75% decrease in phosphorylation Smad2 is observed in lung tissue at a dose of 100 μg of a composition of the invention (p <0.001). There is also a dose-dependent decrease in Fosfo-Smad2 levels (in relation to total Smad (T-Smad) and decreased Fosfo-Smad2 levels in lung tissue (at a dose of 100 ug).

Similar data are obtained when phospho-Smad levels are normalized to either total Smad levels or the square root of total Smad levels (tSmad), thus, it also indicates that the percentage of inhibition of tumor growth correlates with increased dosage delivery of a binding composition of the invention.

Claims (11)

1. A humanized monoclonal antibody, characterized in that it neutralizes mature human TGF-β1, mature human TGF-β2 and mature human TGF-β3, and has a Kd of 4.0 x 10 ~ 12 M or less for mature human TGF-β1, and a Kd of 8.0 xl0"12M or less for mature human TGF-β2, and a Kd of 4.0 xlO" 12M or less for mature human TGF-β3.

2. A monoclonal anti-TGF-β antibody characterized in that it neutralizes mature human TGF-β1, mature human TGF-β2, and mature human TGF-β3, having an IC50 of less than or equal to about 100 pM for human TGF-β1 mature, and an IC50 of less than or equal to about 400 pM for mature human TGF-β2, and an IC50 of less than or equal to approximately 200 pM for mature human TGF-β3 in a HT-2 cell neutralization assay vi tro.

3. The monoclonal antibody according to claim 1 or claim 2, characterized in that said antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a peptide in CDRH1 with a sequence as shown in SEQ ID NO: 96 or SEQ ID NO: 100, a peptide in CDRH2 with a sequence as shown in SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, or SEQ ID NO: 102, and a peptide in CDRH3 with a sequence as shown in SEQ ID NO: 98 and wherein said LCVR comprises a peptide in CDRL1 with a sequence as shown in SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93, a peptide to CDRL2 with a sequence as shown in SEQ ID NO: 89 or SEQ ID NO: 94, and a peptide to CDRL3 with a sequence as shown in SEQ ID NO: 90, or SEQ ID NO: 95.

4. The monoclonal antibody according to claim 2, characterized in that it also comprises a region of human structure.

5. The monoclonal antibody according to any of claims 1 to 3, characterized in that it comprises a HCVR with a sequence selected from the group consisting of SEQ ID NO: 64, 68, 69, 70 and 74, and an LCVR with a sequence selected from the group consisting of SEQ ID NO: 41, 43, 45, 46 and 50.

6. The monoclonal antibody according to claim 4, characterized in that it comprises an HCVR with a sequence as shown in SEQ ID NO. : 69 and an LCVR with a sequence as shown in SEQ ID NO: 45.

7. A monoclonal antibody, characterized in that it comprises a heavy chain with a sequence selected from the group consisting of SEQ ID NO: 77, 79, 81 , 83 and 85, and a light chain with a sequence selected from the group consisting of SEQ ID NO: 76, 78, 80, 82 and 84.

8. The monoclonal antibody according to claim 7, characterized in that it comprises a heavy chain with a sequence as shown in SEQ ID NO: 81 and a light chain with a sequence as shown in SEQ ID NO: 80.

9. A composition, characterized in that it comprises said antibody according to any of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A monoclonal antibody according to any of claims 1 to 8, for use as a medicament.

11. Use of said antibody according to any of claims 1 to 8, in the manufacture of a medicament for treating cellular proliferative disorders in a subject in need thereof.