HK1119183A - Tgf beta 1 specific antibodies - Google Patents

Description

TGF-beta 1-specific antibodies

Technical Field

The present invention relates generally to compositions related to TGF-beta 1-proteins. In particular, the invention provides purified binding compositions useful, for example, as reagents in connection with diagnosing, treating, and preventing cell proliferation, autoimmune/inflammatory, cardiovascular, and fibrotic disorders, and for assessing the effect of exogenous compounds on the expression of nucleic acid and amino acid sequences of such proteins.

Background

TGF-. beta.1, the first member of the transforming growth factor beta (TGF-. beta.) superfamily of secreted polypeptide factors, was discovered approximately 20 years ago. Subsequently members of this family have increased substantially, and now comprise over 30 vertebrate members and a dozen or so structurally and functionally related proteins in invertebrates (e.g., worms and flies) (see, e.g., Attisano and Lee-Hoeflich, 2001Gen. biol.2, review 30101; Moustakas et al 2001J. Cell Sci.114: 4359; Wrana, 2000Cell 100: 189). Members of the TGF β family control many cellular functions, and their activity is critical for the regulation of numerous developmental and homeostatic processes.

As a member of this family, TGF β 1 is involved in a variety of cellular processes such as cell expansion and differentiation, migration, differentiation, apoptosis, embryonic development, extracellular matrix formation, skeletal development, wound healing, hematopoiesis, and immune and inflammatory responses (see, e.g., Roberts and Sporn 1990peptide growth Factors and Their Receptors, pp.419-72, Springer-Verlag, Heidelberg, Germany; Massague, J.1998 Annu.Rev.biochem 67: 753).

In addition, preclinical and clinical data show that TGF β 1 is a major contributor to matrix protein deposition in interstitial fibrosis, and it is also involved in the initiation and progression of a number of related fibrotic disease states, including renal fibrosis, which is prevalent in all forms of chronic kidney disease (CRD). The degree of renal fibrosis is positively correlated with the development of chronic renal failure (CRF, also known as end-stage renal disease (ESRD)) and can lead to death, long-term dialysis, or kidney transplantation.

TGF β is associated with CRF by a complex and diverse event that affects most kidney cells (Bottinger, 2002, j.am. soc. nephrol.13: 2600). These events ultimately lead to tubulointerstitial fibrosis and glomerulosclerosis, and loss of nephron function, ultimately leading to chronic renal failure. Among the three TGF β isoforms, TGF β 1 is dominant in mediating the development of chronic kidney disease not only because it is the most preferentially expressed isoform, but also because TGF β 2 and β 3 both mediate their effects by upregulating TGF β 1 expression (Yu, 2003, kid. int.64, 844). Thus, modulation of TGF β 1 expression is desirable to prevent the deleterious effects of disorders such as chronic kidney disease.

Accordingly, the discovery of novel TGF-beta 1 binding compositions satisfies this need in the art by providing compositions that are useful in the diagnosis, prevention, and treatment of fibrotic disorders, such as chronic kidney disease.

Summary of The Invention

The present invention is based, in part, on the discovery of binding compositions with improved properties that specifically and/or selectively bind mature TGF β 1 and neutralize mature TGF β 1 relative to mature TGF β 2 and/or mature TGF β 3.

These binding compositions comprise: (a) a first part comprising at least one of the following sequences: a) GYRX₁X₂X₃Y[SEQ ID NO：4](ii) a Wherein X₁Is W or A; x₂Is F or L; and X₃Is A, E orY；b)GYX₁FX₂DYNX₃X₄[SEQ ID NO：2](ii) a Wherein X₁Is T or D; x₂Is T, E or F; x₃Is M, I, L or V; and X₄Is H, V or A; or c) X₁X₂YPYDGX₃TGX₄NX₅KX₆KS[SEQ ID NO：3](ii) a Wherein X₁Is Y, Q or S; x₂Is I or V; x₃Is D or E; x₄Is Y, T, H or L; x₅Is Q, K, P or S; and X₆Is F or Y; and/or (b) a second part comprising at least one of the following sequences: x₁QWDX₂X₃X₄PA[SEQ ID NO：7](ii) a Wherein X₁Is Q or S; x₂Is L, D or P; x₃Is N or R; and X₄Is P, F, Y or R; x₁AX₂X₃X₄VX₅YMH[SEQ ID NO：5](ii) a Wherein X₁Is R, Y, E or Q; x₂Is S or T; x₃Is S, V or A; x₄Is S or L; x₅Is S, P, L or Y; or ATSNX₁AX₂[SEQ ID NO：6](ii) a Wherein X₁Is L, N or P; and X₂Is S, K, Y, L, M, F, E, Q, R or H; binding compositions may also be a) said first part comprises at least two of said sequences; b) the first portion comprises three of the sequences; c) the second portion comprises at least two of the sequences; d) the second portion comprises three of the sequences; e) the first portion comprises at least two of the sequences and the second portion comprises three of the sequences; f) the second portion comprises at least two of the sequences and the first portion comprises three of the sequences; or g) the first portion comprises three of the sequences and the second portion comprises three of the sequences; wherein the binding site of the binding composition: specifically immunoreactive with a human TGF β 1 polypeptide; specifically immunoreactive with a murine TGF β 1 polypeptide; produced against purified or recombinantly produced human TGF β 1 protein or fragments thereof; is a monoclonal antibody, Fab, Fv, scFv, F (ab)2 or variable domain of an antibody; having at least one, two or three conservative substitutions; or in the framework of a human or humanized antibody; wherein the binding composition: is an antibodyA molecule; is a monoclonal antibody molecule; is a diabody; is a three-chain antibody; is a four-chain antibody; is a minibody molecule; is monoclonal antibody serum; is detectably labeled; is lyophilized; is sterile; or in a buffered composition; wherein the binding composition is a monoclonal antibody with improved properties.

Description of The Preferred Embodiment

I. Overview

It is to be understood that this invention is not limited to the particular compositions, methods and techniques described herein, as such compositions, methods and techniques may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The scope of the invention is limited only by the appended claims.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an organism" includes one or more different organisms, reference to "a cell" includes one or more of such cells, and reference to "a method" includes reference to equivalent steps and methods, etc., known to those of ordinary skill in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate any such disclosure by virtue of prior invention. All publications, patent applications, patents, and other references mentioned herein, including all pictures, drawings, photographs, charts, hyperlinks, and other forms of browser-executed code, are incorporated by reference in their entirety for their teachings as if set forth clearly in the context.

Definition of

"bonding composition"Refers to a molecular entity having selective and/or specific binding affinity for at least one other molecular entity or binding partner. In general, binding refers to interactions that are physiologically related in nature, and may be covalent or non-covalent, and may include members of a multi-protein complex, including but not limited to carrier compounds, dimeric or multimeric partners. The binding composition as a whole or in part may be naturally derived (e.g., isolated and/or purified) or synthetically produced. Generally, the binding composition has at least one region [ such as, by way of non-limiting example, a surface, a shape (e.g., a cavity, a depression, a slit, or a protrusion), a molecular arrangement, or a spatial configuration]Can specifically and/or selectively "fit", "bind" or complement a particular spatial and/or polar configuration of a region or region on a binding partner. Thus, for example, when the binding composition is in close enough proximity to a potential binding partner, the binding composition and the partner specifically and/or selectively bind to each other. Non-limiting examples of binding compositions that pair with binding partners are: antigen-antibody and hapten-binding sites. Non-limiting examples of antibody-binding compositions are: antibodies, diabodies, triabodies, tetrabodies, minibodies, Fab fragments (including, e.g., dimeric or trimeric Fab), Fv fragments, scFv fragments, F (ab)2 fragments, and the like (see, e.g., Hudson for non-limiting examples of antibody binding compositions encompassed by the invention&Souriau 2003Nature Medicine 9: 129-34). A monoclonal antibody binding composition is a monoclonal antibody in the sense that it is derived from a large population of substantially homogeneous antibodies (i.e., the individual antibodies comprising the antibody population are substantially identical (e.g., they may be derived from clones of a single cell type)). But this is not meant to limit its particular origin. Such antibodies can be administered to cells that do not normally produce antibodies, such as CHOEasily produced in NSO or COS cells. In addition, such antibodies can be produced in other types of cells, particularly mammalian cells, and even plant cells, by genetically engineering the cells to express and assemble light and heavy chain polypeptides to form antibody products. Alternatively, such chains may be chemically synthesized, but, because they are specific for a given antigenic determinant, they are still monoclonal antibodies, in the sense of the term "monoclonal antibody" as used herein. Thus, the term monoclonal antibody as used herein is intended to characterize the specificity and purity of an antibody molecule to a greater extent than merely the mechanism by which the antibody is produced.

"binding site"Refers to a particular region, area or configuration of a molecular entity that is involved in specifically and/or selectively binding another molecular entity. A non-limiting example of a binding site is a contiguous amino acid sequence that constitutes a Complementarity Determining Region (CDR) of an antibody. In one embodiment, the binding site of the present invention comprises a sequence having the general formula shown in table 1. In another non-limiting embodiment, the binding site comprises a combination of the sequences in table 1. As another non-limiting example, the binding site is formed by the three-dimensional configuration and spatial configuration of the amino acid sequences that make up the 6 CDR loops of the heavy and light chain variable regions at the edges of the 8 β -folds of the Fab fragment (see, e.g., Chothi and Lesk, 1987J. mol. biol. 196: 901-17). The CDRs disclosed herein can be incorporated/embedded into a framework or molecular structure, as in CDR grafting. In one embodiment, the structure bearing the CDRs of the invention is typically a heavy or light chain sequence of an antibody, or a substantial portion thereof, wherein the CDRs are located at corresponding positions in the CDRs of a naturally occurring VH and VL of an antibody variable domain encoded by a rearranged immunoglobulin gene. The structure and location of immunoglobulin variable domains can be determined by reference (Kabat et al, 1987Sequences of Proteins of Immunological interest, 4 th edition, US department of Health and Human Services, and more recent materials thereof, currently available on the Internet: (available on the Internet as pharmaceuticals for the treatment of diseases and disorders of the Human immune system)http://immuno.b-me.nwu.edu)). Complementarity determining regions are generally defined as Kabat, but can also be according to Chothia (J.mol.biol.196: 901-17) and/or MacCallum (J.mol.Bi.ol.262: 732-45). Given the amino acid sequence in which a particular CDR is embedded, one skilled in the art can routinely determine which amino acid residues make up that particular CDR. In addition, in CDR grafting, can be grafted adjacent to Kabat VH CDR1 by Chothia defined loop residues.

In one embodiment, the CDR sequences of the invention may be introduced into all components of the variable domain lacking the CDR regions by recombinant DNA techniques. For example, Marks et al (1992Biotechnology 10: 779-83) describe methods for producing all of the components of an antibody variable domain. Wherein a consensus primer directed to or adjacent the 5' end of the variable domain is used, together with a consensus primer for the third framework region of the human VH gene, to provide all components of the VH variable domain lacking the CDR 3. Marks et al also describe how this repertoire is combined with the CDR3 sequence of a particular antibody (as described herein). Using similar techniques, the CDR3 sequences of the invention can be shuffled with all components of the VH or VL domain lacking the CDR3, and the shuffled complete VH or VL domain combined with a homologous VL or VH domain to provide specific binding composition members of the invention. Alternatively, the CDR3 may be combined with other CDRs of the invention using similar strategies. Thus, all components may be displayed in a suitable host system, such as the phage display system of W092/01047, in order to screen for suitable specific binding members.

A binding composition: TGF-beta 1 complexes

The term "binding composition" as used herein: TGF-beta 1 complex "refers to a complex of a binding composition and a TGF-beta 1 protein formed by the binding composition specifically and/or selectively binding TGF-beta 1 protein. In a preferred embodiment, all references to TGF β 1 refer to the "mature" primate TGF β 1 protein. In a more preferred embodiment, all references to TGF-beta 1 refer to the mature human TGF-beta 1 protein (see, e.g., NCBI accession No.: P01137, which describes the amino acid sequence of the human transforming growth factor-beta 1 precursor (TGF-beta 1) and its mature part). In each case, reference throughout to TGF-beta 1 refers to the mature, biologically active form of TGF-beta 1[ TGF-beta 1 is secreted from cells as an inactive "latent" complex, comprising a TGF-beta 1 homodimer formed by non-covalent complexation with two precursor fragments, to which one of several potential TGF-beta 1 binding proteins is typically attached (see, e.g., Annes et al, 2003J. cell Science 116: 217-24; Miyazono et al, 1993Growth Factors 8: 11-22; Munger et al, 1997 Kidney int.51: 1376-82; Oklu & Hesketh 2000biochem. J.352: 601-10). This latent TGF-beta 1 complex represents an important safety measure against "inadvertent" activation, stabilizing latent TGF-beta 1 and targeting it to the extracellular matrix, thereby concealing it there (Taipal et al, 1998Adv. cancer Res.75: 87-134). Thus, the extracellular matrix acts as a reservoir where TGF-beta 1 can be readily activated without the need for new cellular synthesis. TGF β 1 secretion as a latent complex would necessitate the presence of a step of regulatory activation most likely mediated by an activity that would preferentially degrade TGF β 1 precursor fragments thereby releasing the highly stable mature active TGF β 1 dimeric form of the protease ].

Specific binding of the binding composition means that the binding composition has a binding site that recognizes the TGF β 1 region, usually in its native active conformational form. For example, antibodies raised against TGF β 1 and recognizing the TGF β 1 epitope can form binding compositions by specific binding: TGF-beta 1 complex. Typically, the binding composition: the formation of TGF-beta 1-protein complexes may allow the detection of TGF-beta 1in biological samples (e.g., in admixture with other proteins and biological agents). Epitopes of binding compositions of the invention can be determined using techniques described herein or in the art (see, e.g., G. Tribbick 2002Journal of immunological Methods 267: 27-35; Woods and Hamuro 2001Journal of cellular Biochemistry Supplement 37: 89-98), and/or using Methods of competitive binding described herein. In a preferred embodiment, the epitope of the binding composition of the invention comprises the amino acid sequence of SEQ ID NO: 1 amino acid residue YYVGRK; in another embodiment, the epitope of the binding composition of the invention comprises SEQ ID NO: 1 and amino acid residue yvgrk of SEQ ID NO: 1 YSKV; in a further embodiment, the epitope of the binding composition of the invention comprises a peptide from SEQ ID NO: at least 1, 2, 3, 4, 5, or 6 adjacent or non-adjacent residues of yvgrk in 1 and/or a nucleotide sequence derived from SEQ ID NO: at least 1, 2, 3 or 4 adjacent or non-adjacent residues of YSKV in 1 (embodiments are contemplated to include any and all combinations thereof, such as, but not limited to, YVGRK and KV of SEQ ID NO: 1; or YVGRK and Y and KV of SEQ ID NO: 1 (all such combinations are obtainable using computer algorithms and well-known mathematical formulas to arrange and combine.) for example, in yet another preferred embodiment, an epitope of the invention is functionally defined in accordance with the ability of a binding composition of the invention to prevent competitive binding compositions against the same antigen, such as TGF-beta 1, from subsequently forming a binding complex (such competitive binding is described herein).

Terms used herein"specific binding"Refers to the situation where one member of a specific binding pair does not exhibit significant binding to other molecules than its specific binding partner. This term also applies to the case where the antigen binding domain is specific for a particular epitope carried on a number of antigens, in which case the specific binding member carrying the antigen binding domain is capable of binding to a different antigen carrying said epitope. Thus, a binding composition that is specific for mature TGF β 1 "does not significantly bind to molecules other than the partner to which it specifically binds (i.e., mature TGF β 1").

Phrase for binding composition"specific binding"OrSpecific immune response "Refers to a binding reaction that determines the presence of a binding composition in the presence of a heterologous protein or other population of biological components. Thus, under the specified immunoassay conditions, a specified binding composition is capable of binding a specific protein without significantly binding to other proteins in the sample. Under such conditions, specific binding to a binding composition requires a binding composition that is selected for its specificity for a particular protein. For example, it can be directed against the human active dimeric form of TGF-beta 1 (whose mature amino acid sequence monomers are [ SEQ ID NO: 1]]Said) generating a bondCompositions such as antibodies are then selected to obtain antibodies specifically immunoreactive only with the TGF-beta 1 protein and not with other proteins. Such binding compositions are capable of distinguishing proteins that are highly homologous to human TGF-beta 1 protein, e.g., other human TGF-beta 0 isoforms (e.g., human TGF-beta 12 or human TGF-beta 23). In preferred embodiments, the specificity of a binding composition of the invention for mature TGF β 1 is equal to or greater than 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 100, 125, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10,000 fold higher relative to the specificity of mature TGF β 2 and/or mature TGF β 3. In another preferred embodiment, the specificity of the compositions of the invention has particular specificity values for human TGF-beta 2 as listed above and different specificity values for human TGF-beta 3, e.g., specificity for mature TGF-beta 1 equal to or greater than 100 times its specificity relative to mature TGF-beta 2 and equal to or greater than 900 times its specificity relative to mature TGF-beta 3. Any such combination is encompassed.

The binding compositions of the invention preferably neutralize TGF β 1, and preferably have a dissociation constant (Kd) for TGF β 1 of less than about 100pM, 95pM, 90pM, 85pM, 80pM, 75pM, 70pM, 65pM, 60pM, 55pM, 50pM, 45pM, 40pM, 35pM, 30pM, 25pM, 20pM, 15pM, 14pM, 13pM, 12pM, 11pM, or 10 pM; more preferably less than about 10pM, 9.9pM, 9.8pM, 9.7pM, 9.6pM, 9.5pM, 9.4pM, 9.3pM, 9.2pM, 9.1pM, 9.0pM, 8.9pM, 8.8pM, 8.7pM, 8.6pM, 8.5pM, 8.4pM, 8.3pM, 8.2pM, 8.1pM, 8.0pM, 7.9pM, 7.8pM, 7.7pM, 7.6pM, 7.5pM, 7.4pM, 7.3pM, 7.2pM, 7.1pM, 7.0pM, 6.9pM, 6.8pM, 6.7pM, 6.6pM, 6.5pM, 6.4pM, 6.5pM, 6.2pM, 6.1pM, 7.0pM, 6.9pM, 6.8pM, 6.5pM, 6, 6.5pM, 6, 6.5pM, 6,5, 6.5, 5pM, 5, 5.5.5, 5pM, 5, 5.; even more preferably less than about 5.0pM, 4.9pM, 4.8pM, 4.7pM, 4.6pM, 4.5pM, 4.4pM, 4.3pM, 4.2pM, 4.1pM, 4.0pM, 3.9pM, 3.8pM, 3.7pM, 3.6pM, 3.5pM, 3.4pM, 3.3pM, 3.2pM, 3.1pM, 3.0pM, 2.9pM, 2.8pM, 2.7pM, 2.6pM, 2.5pM, 2.4pM, 2.3pM, 2.2pM, 2.1pM, 2.0pM, 1.9pM, 1.8pM, 1.7pM, 1.6pM, 1.5pM, 1.4pM, 1.3pM, 1.2pM, 1.1pM, 1.0 pM; and even more preferably less than about 9.9pM, 9.8pM, 9.7pM, 9.6pM, 9.5pM, 9.4pM, 9.3pM, 9.2pM, 9.1pM, 9.0pM, 8.9pM, 8.8pM, 8.7pM, 8.6pM, 8.5pM, 8.4pM, 8.3pM, 8.2pM, 8.1pM, 8.0pM, 7.9pM, 7.8pM, 7.7pM, 7.6pM, 7.5pM, 7.4pM, 7.3pM, 7.2pM, 7.1pM, 7.0pM, 6.9pM, 6.8pM, 6.7pM, 6.6pM, 6.5pM, 6.4pM, 7.2pM, 3.2pM, 3.5pM, 3.4pM, 3.5pM, 3.4pM, 3.5pM, 4pM, 3.5pM, 4, 3.5pM, 4pM, 3.5pM, 4pM, 3.5pM, 4pM, 3.5pM, 4, 3.5pM, 4pM, 4, 3.0, 4, 3.5pM, 4pM, 4, 3.5pM, 3.0, 3.5, 1.8pM, 1.7pM, 1.6pM, 1.5pM, 1.4pM, 1.3pM, 1.2pM, 1.1pM, 1.0pM, 0.9pM, 0.8pM, 0.7pM, 0.6pM, 0.5pM, 0.4pM, 0.3pM, 0.2pM, 0.1pM or 0.01 pM. The dissociation constant (Kd) of a binding composition can be determined by any method known in the art, e.g., BIACore^TMMethods of transformation of Karlsson et al 1991J. Immunol. methods 145, 299-340. Other descriptions are described in Jonsson, et al, 1993 Ann.biol.Clin.51: 19-26; jonsson et al, 1991Biotechniques 11: 620-7; johnsson et al, 1995 j.mol.recognit.8: 125-31 parts of; and Johnnson et al, 1991anal. biochem.198: 268-77.

The term "K" as used herein_on"is intended to mean the binding or rate constant or specific reaction rate of a forward or complex-forming reaction, measured in M^-1sec^-1. The term "K" as used herein_off"is intended to mean the dissociation or dissociation constant or rate constant or specific reaction rate, measured in 1/second, of an antibody from an antibody/antigen complex. The term "Kd" as used herein is intended to denote the dissociation constant of a particular antigen-antibody reaction. It can be calculated according to the formula Koff/Kon ═ Kd. The affinity of the binding composition is generally associated with a lower k_offRather than higher k_onRelated, but not to be bound by theory, improved k_offAnd k_onAre included in the embodiments. In a more preferred embodiment, the binding composition of the invention is a high titer antibody or fragment thereof, typically exhibiting a low k_offThe value is obtained. In an even more preferred embodiment, k of a Fab fragment embodiment of the invention_onThe rate is increased by at least 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3 or 2.4 fold over a comparable Fab (e.g.mAb 2471 as described in PCT/US 2004/018921; US60485,820). In another embodiment, k of the composition of the invention_offThe rate is increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 130, 150, 195, 200, 225, 250, 260, 270, 280, 290, or 300 fold over a comparable binding composition (e.g., Fab or mAb).

Preferably, the antibody binding composition specifically and/or selectively binds TGF β 1 as compared to TGF β 2 and/or TGF β 3; more preferably, the antibody binding composition specifically and/or selectively binds human TGF β 1 as compared to human TGF β 2 and/or human TGF β 3. Preferably such antibodies cross-react with TGF β 2 and/or TGF β 3 by less than about 20% (as measured by the ratio of dissociation constants), more preferably by less than about 15%, even more preferably by less than about 10%, even more preferably by less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

Furthermore, the antibody binding composition also preferably recognizes TGF β 1in an active, but not latent, form; more preferably, human TGF-. beta.1 is recognized in an active but not latent form.

Neutralization

In terms of binding compositions, the terms"neutralization"OrAntagonism "Refers to the situation where a binding composition specifically and/or selectively binds another molecular entity, resulting in the elimination or inhibition of a biological effector function of the molecular entity to which the binding composition is bound. With respect to "TGF-beta 1," the term "neutralizing" or "antagonizing" is intended to mean that the binding composition binds to or interacts with TGF-beta 1 such that inhibition of TGF-beta 1 is causedBeta 1 induced biological activity. Inhibition of TGF β 1 biological activity can be assessed by detecting one or more in vivo or in vitro indicators of TGF β 1 biological activity, such as, but not limited to, receptor binding inhibition, fibrosis inhibition, chemotaxis inhibition, or inhibition of signal transduction in a TGF β 1 binding assay (see, e.g., EP 0945464 for non-limiting examples of neutralization assays contemplated). In non-limiting embodiments, the ability of a binding composition to neutralize or antagonize TGF β 1 activity can be assessed using the assays described in the examples herein.

In combination with a compositionNeutralizing ActivityAs in the antibody embodiment, detection can be by art-recognized methods. In a non-limiting example, the assay can be performed using or modified from the TGF assay of Randall et al (1993) (J.Immunol Methods 164, 61-67). This assay is based on the ability of TGF β 1 and TGF β 2 to inhibit interleukin 5(IL-5) induced proliferation of the erythroleukemia cell line TF1, and the ability of TGF β 0 specific binding compositions to reverse inhibition of TGF β 1. This assay is reported to be rapid, reproducible, and sensitive to detect TGF-beta 21 less than 500fg/ml and 5-10pg/ml TGF-beta 2. This assay is reported to be 1000-fold less sensitive to other inhibitory molecules such as interferon-beta, interferon-gamma and TNF-alpha. It has been reported that by incorporating specific neutralizing antibodies against TGF β 1 or TGF β 2, this assay can also be specific for TGF β 1 or TGF β 2, recognizing all readily available recombinant molecular species of these molecules as well as native proteins produced from human and bovine platelets, and detecting TGF β in serum samples.

Other assays reported herein or known in the art are also included. For example, the rat anti-Thy1.1 model is a well established model of mesangial proliferative glomerulonephritis (see, e.g., Morita, et al 1998Am J Kidney Dis 31: 559-73; Bagchus, et al 1986Lab. invest.55: 680-7 and Yamamoto and Wilson 1987Kidney int.32: 514-25), and injection of antibodies against Thy antigen localized on the surface of mesangial cells in this model induces mesangial lysis, causing a period of compensatory proliferation of mesangial cells, resulting in increased levels of urine protein (urokinase protein). The anti-Thy1.1 nephritis model resembles human IgA nephritis or Henoch-Schonein purpura in many ways (O' Donoghue et al 1991J Clin Invest 88: 1522-30) and this model has been used to test potential therapeutic approaches to renal disease by determining the ability of test compositions to achieve dose-related reductions in urinary protein (see, e.g., Burg, et al 1997Lab Invest 76: 505-16; Johnson et al 1995Kidney Int 47: 62-9). In preferred embodiments, the binding compositions of the invention are capable of reducing urine protein in such a model by greater than or equal to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%. Embodiments are also included in such models where the range of urine protein reduction is between any two values so shown, which may or may not include any one or both of the endpoint values, such as a range of > 12% and ≦ 42%.

In addition, chronic inhibition of the biological effects of TGF-beta in the kidney can be demonstrated to be effective in preventing renal failure due to Diabetes using Sharma's db/db mouse model of Diabetes testing (see, e.g., 2000PNAS 97: 8015-20), or the ability of a binding composition to alleviate or prevent diabetic nephropathy can be tested using Sharma STZ mouse model of Diabetes (1996Diabetes 45, 522-30). Furthermore, Ueberham et al (2003Hepatology 37 (5): 1067-78) describe a tetracycline regulated gene expression system in a double transgenic mouse model of liver fibrosis, in which the expression of TGF β 1 is regulated by the addition or removal of doxycycline hydrochloride in the drinking water, thereby enabling the expression of TGF β 1 to be switched on and off as desired. Increased expression of TGF in the liver of such animals can lead to a fibrotic disease state that can be reversed by turning off TGF β 1 expression even after 59% reduction in liver weight. The use of this model to evaluate the effect of a binding composition of the invention in inhibiting TGF β 1 biological function can be assessed by comparing the effect of the binding composition of the invention with the effect of doxycycline hydrochloride in shutting off TGF β 1 expression. In particular embodiments using the HT-2 cell proliferation assay described herein, applicants prefer binding composition embodiments comprising an IC50 ≧ at least 50, 60, 70, 80, 90, 100, 105, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, or 450 times as compared to the IC50 value obtained under the same or similar assay conditions with a similar binding composition (e.g., mAb2471 described in PCT/US 2004/018921; US 60/485,820). Also included are embodiments in which the range of IC50 increase, as defined above, is between such two values (either or both of the boundary points may or may not be included, e.g., the range of IC50 increase > 80-fold and ≦ 100-fold for mAb 2471).

Antibodies

According to the inventionAntibody binding compositionsIncluding, for example, but not limited to, 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 anti-idiotypic antibodies to antibodies of the invention), and epitope-binding fragments of any of the foregoing.

Terms used herein"antibodies"Refers to immunoglobulin compositions and immunologically active portions of immunoglobulin compositions, such as binding composition molecules that contain binding sites that specifically and/or selectively bind to antigens. The immunoglobulin compositions of the invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Preferred antibodies are human antigen-binding antibody fragments of the invention, such as but not limited to Fab, Fab ', F (ab') 2, Fc, single chain fv (scFv), single chain antibodies, disulfide-linkedFv (sdfv) and fragments comprising a VL or VH domain. Antigen-binding antibody fragments, including single chain antibodies, may comprise the variable regions alone or in combination with all or part of: a hinge region, a CH1, a CH2, or a CH3 domain, or a combination thereof. Also included in the invention are, but not limited to, antigen binding fragments, which may also consist of any combination of variable and hinge regions, such as the CH1, CH2, or CH3 domains, or combinations thereof. The antibodies of the invention may be from any animal including birds and mammals. Preferably, the antibody is derived from a human, primate, mouse (e.g., mouse and rat), donkey, rabbit, goat, guinea pig, camel, horse or chicken.

The words used herein"human antibodies"Including but not limited to antibodies having human immunoglobulin amino acid sequences, including, for example, but not limited to, antibodies isolated from a human immunoglobulin library (e.g., a human germline library) or an animal containing one or more human immunoglobulin transgenes and which does not express endogenous immunoglobulins (as described herein or as taught in U.S. Pat. No.5,939,598).

The binding composition may be monospecific, bispecific, trispecific or more multispecific. Multispecific antibodies may be specific for different epitopes of a target protein, polypeptide (or fragment thereof), or for both TGF-beta 1 and heterologous epitopes, such as heterologous TGF-beta isoforms or solid support materials (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. Pat. No.4,474,893; 4,714,681; 4,925,648; 5,573,920; or 5,601,819; or Kostelny et al (1992) J.Immunol.148: 1547-1553).

The binding compositions may be described or specified in terms of the epitope or portion of the TGF-beta 1 protein (or fragment thereof) that it recognizes or selectively and/or specifically binds. Epitopes or polypeptide portions may be specified as described herein: such as the N-terminal and C-terminal positions, the sizes of adjacent amino acid residues, or listed in the accompanying tables and/or figures, or as described herein. In addition, antibodies that specifically bind to an epitope, polypeptide, protein, polypeptide, or protein fragment may also be specifically excluded from the present invention. For example, applicants reserve the right to conditionally claim antibodies that specifically bind to an epitope, polypeptide, protein, polypeptide, or protein fragment. Accordingly, the present invention encompasses a first or other antibody that specifically binds to a polypeptide or protein or fragment thereof, while excluding a second or other antibody that also selectively binds to the same protein or polypeptide or fragment thereof, e.g., possibly by binding to a different epitope. In a preferred embodiment, applicants conditionally claim a binding composition that specifically and/or selectively binds the TGF β 1 isoform and neutralizes TGF β 1, relative to TGF β 2 and/or TGF β 3, comprising at least one binding site comprising: from QQWNGNPPA [ SEQ ID NO: 126 ]; from QQWDSNPPA [ SEQ ID NO: 127 ]; from YIYPYNGDTGYNQKFKS [ SEQ ID NO: 128] (wherein one of the at least five adjacent amino acids is D); or from GYTFTDYTMH [ SEQ ID NO: 129 ].

The antibodies of the invention may also be described or specified in terms of their cross-reactivity. Included are antibodies that do not bind to any other analog, ortholog, paralog or homolog of the target protein, polypeptide or fragment thereof.

The invention also includes antibodies that selectively bind to a polypeptide encoded by a polynucleotide that stably hybridizes under stringent hybridization conditions (as described herein) to a human TGF β 1 polynucleotide sequence.

The binding composition may also be characterized or specified in terms of its binding affinity for a protein or polypeptide or fragment or epitope thereof. In another embodiment, the binding affinity of a binding composition (e.g., an antibody or antibody binding fragment) preferably includes, for example, a range that exhibits a dissociation constant or Kd (or a range between two numbers defined below, which range may or may not include either or both) that is lower thanTwo boundary points): 5X 10^-2M、10^-2M、5×10^-3M、10^-3M、5×10^-4M、10^-4M、5×10^-5M、10^-5M、5×10^-6M、10^-6M、5×10^-7M、10^-7M、5×10^-8M、10^-8M、5×10^-9M、10^-9M、5×10^-10M、10^-10M、5×10^-11M、10^-11M、5×10^-12M、4.9×10^-12M、4.8×10^-12M，4.7×10^-12M、4.6×10^-12M、4.5×10^-12M、4.4×10^-12M、4.3×10^-12M、4.2×10^-12M、4.1×10^-12M、4.0×10^-12M、3.9×10^-12M、3.8×10^-12M、3.7×10^-12M、3.6×10^-12M、3.5×10^-12M、3.4×10^-12M、3.3×10^-12M、3.2X 10^-12M、3.1×10^-12M、3.0×10^-12M、2.9×10^-12M、2.8×10^-12M、2.7×10^-12M、2.6×10^-12M、2.5×10^-12M、2.4×10^-12M、2.3×10^-12M、2.2×10^-12M、2.1×10^-12M、2.0×10^-12M、1.9×10^-12M、1.8×10^-12M、1.7×10^-12M、1.6×10^-12M、1.5×10^-12M、1.4×10^-12M、1.3×10^-12M、1.2×10^-12M、1.1×10^-12M、1.0×10^-12M、0.9×10^-12M、0.8×10^-12M、0.7×10^-12M、0.6×10^-12M、0.5×10^-12M、0.4×10^-12M、0.3×10^-12M、0.2×10^-12M、0.1×10^-12M、10^-12M、5×10^-13M、10^-13M、5×10^-14M、10^-14M、5×10^-15M or 10^-15M。

The invention also encompasses the use of any art-known method of determining competitive binding, such as the immunoassays described or referenced herein, to determine antibodies that competitively inhibit binding of a binding composition to a TGF-beta 1 epitope. In preferred embodiments, the antibody competitively inhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92% or 91%, at least 90%, 89%, 88%, 87%, 86%; at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% (or a range between two such values, which may or may not include any one or two boundary points) of epitope binding.

The antibodies of the invention may act as antagonists of TGF-beta 1 (or fragments thereof). For example, the antibodies or binding compositions of the invention can partially or completely disrupt the interaction between TGF β 1 and a cognate receptor/ligand. Preferably, the antibodies of the invention bind to an epitope of TGF-beta 1 or a portion thereof.

The invention also encompasses antibodies that bind to a ligand and prevent it from binding to a receptor (e.g., by steric hindrance). Ligand-binding antibodies that inhibit receptor activation but do not inhibit receptor binding are likewise included.

Antibodies of the invention may be used, but are not limited to, purifying, detecting or targeting TGF-beta 1 or fragments thereof in vivo or in vitro diagnostic and therapeutic methods. For example, in immunoassays, the Antibodies of the invention are used to qualitatively and/or quantitatively determine the level of TGF β 1 or fragments thereof in a biological sample (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, (Cold spring harbor Laboratory Press, 1999).

The combination composition can be used alone, also can be combined with other compositions. Furthermore, binding compositions such as antibodies can be recombinantly fused to the N-terminus or C-terminus of a heterologous polypeptide, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the antibodies of the invention can be recombinantly fused or conjugated to molecules that can be used as detection assay labels as well as effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins (see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387).

Binding compositions include, for example, derivatives of any type of molecule that have been modified by covalent attachment, for example, to an antibody, such that the covalent attachment does not prevent the antibody from producing an anti-idiotypic response. For example, antibody derivatives include, but are not limited to, antibodies modified by glycosylation, acylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be performed using known techniques, including, but not limited to, specific chemical cleavage, acylation, formulation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-canonical amino acids. The binding composition may be produced by any suitable method known in the art. For example, monoclonal Antibodies can be prepared by any technique known in the art, such as Harlow and Lane, Antibodies: ALABORT Manual, (Cold Spring Harbor Laboratory Press, 1988); harlow and Lane, Using Antibodies: a Laboratory Manual, (Cold spring harbor Laboratory Press, 1999); breitling and Dubel 1999 recombining antibodies (John Wiley and Sons, New York); zola, MonoclonalAntibodies (Garland Press); and James w.goding, MonoclonalAntibodies: principles and Practice (Academic Press).

Terms used herein"monoclonal antibody"And is not limited to antibodies produced by a particular technique, such as hybridoma technology. Methods for producing and screening specific antibodies using hybridoma technology are routine and known in the art. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not to the method used to produce the antibody.

Capable of recognising specific epitopesAntibody fragmentsCan be produced using known techniques. For example, Fab and F (ab ') 2 fragments of the invention can 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). The F (ab') 2 fragment contains the variable region, the constant region of the light chain and the CH1 junction of the light chainAnd (4) domain formation. For example, the binding composition can also be generated using a variety of phage display methods known in the art, wherein a phage particle carrying a functional antibody domain encoding polynucleotide sequence displays the functional antibody domain on the surface of the phage particle.

In specific embodiments, phage display methods are used to display antigen binding domains expressed by repertoires or combinatorial antibody libraries (e.g., human or murine). Phage expressing an antigen binding domain that binds an antigen of interest can be screened or identified with the antigen, such as by using a labeled antigen, or by binding or capturing the antigen on a solid surface or bead. After phage selection, the antibody coding regions on the phage are isolated and used to generate whole antibodies, including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria as described herein or in the literature.

Techniques for the recombinant production of Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art, e.g., WO 92/122324; mullinnax et al BioTechniques 199212 (6): 864-9; and Sawai et al 1995AJRI 34: 26-34; and Better et al 1988Science 240: 1041-3. Examples of the production of single chain Fv's and antibodies include, for example, U.S. Pat. Nos. 4,946,778 and 5,258,498; huston et al 1991Methods in Enzymology 203: 46-88; shu et al 1993PNAS USA 90: 7995-9; and Skerra et al 1988Science 240: 1038-40. In some applications, including in vivo in humans as well as in vitro detection assays, antibodies, preferably humanized or human antibodies, produced according to techniques known in the art are used.

Herein, theHumanized antibodiesRefers to a binding composition that binds a desired antigen, with one or more Complementarity Determining Regions (CDRs) described herein embedded within the framework regions, with little possibility of generating a deleterious immunogenic response upon in vivo introduction into a human host system, such as after parenteral administration. Typically, the CDR donor regions are suitably embedded within a human framework region to enhance antigen binding and/or reduce immunogenicity. Useful inFramework regions can be identified using methods known in the art, such as by (1) modeling the interaction of CDRs with framework residues to identify framework residues important for antigen binding; (2) identification of unusual framework residues at specific positions by sequence comparison (see, e.g., U.S. Pat. No.5,585,089, Riechmann et al Nature 332: 323 (1988)); or (3) empirically identified.

Shown in Table 1 below (a and b) are the variable heavy and light chain Complementarity Determining Regions (CDRs) for a particular monoclonal antibody binding composition embodiment of the invention. CDR regions are represented by standard amino acid single letter codes and standard CDR numbering (i.e., as the CDR values increase, corresponding to closer to the constant domain in a typical IgG heavy or light chain structure; e.g., VH CDR3 is closer to the CH1 domain than VH1CDR 1).

Specific CDR embodiments are generally represented by amino acid formulae that describe the class of CDRs. Similarly, standard single letter amino acid codes are used, and the letter "X" denotes a substitutable amino acid residue whose position in a particular CDR is indicated by a subscript numeral that is arranged numerically from lowest (most amino terminal to CDR) to highest (most carboxyl terminal to CDR). As in VH CDR2, X₁Is the residue most amino terminal to the CDR and the substitutable residue most carboxy terminal is X₆. Using these general formulas, one of ordinary skill in the art will be able to determine the variable heavy or light chain region (V) encompassed by the present invention_LOr V_H) Each of the embodiments specifies all possible CDR implementations at a location. Any particular example of a possible amino acid sequence can be determined simply by using the amino acid code to generate all possible substitutions at each particular X residue in a particular CDR formula. All such embodiments are encompassed by the present invention.

Furthermore, the invention also encompasses equally all possible V's in possible CDR combinations_LOr V_HEmbodiments are described. As with the information provided, there are 72 possible VHCDRs 1, 384 possible VH CDRs 2 and 12 possible VH CDRs 3 that can be readily calculated to bind to a particular composition of the inventionA total of 72X 384X 12 possible V's are produced in the variable heavy chain antibody embodiment_HAnd (4) CDR combination. The present invention also encompasses all such combinations. Similar reasoning applies to any VL CDR and light chain variable domain V_LAll possible combinations are included. For example, there are 192 possible VL CDRs 1, 12 possible VL CDRs 2, and 48 possible VL CDRs 3, resulting in a total of 192 × 12 × 48 possible V's in a particular variable light chain antibody embodiment of the compositions of the invention_LAnd (4) CDR combination. Similarly, the invention also encompasses certain combination composition embodiments of V_HAnd V_LAll possible combinations of pairs.

TABLE 1CDR heavy chain generic formula for binding compositions

●^＊For VH CDR 1: x₁Is T or D; x₂T, E or F; x₃M, I, L or V; and X₄H, V or A.

●^＊＊For VH CDR 2: x₁Y, Q or S; x₂Is I or V; x₃Is D or E; x₄Y, T, H or L; x₅Q, K, P or S; and X₆Is F or Y.

●^＊＊＊For VH CDR 3: x₁Is W or A; x₂Is F or L; and X₃A, E or Y.

TABLE 1b CDR light chain general formula for binding compositions

●^＊For VL CDR 1: x₁R, Y, E or Q; x₂Is S or T; x₃S, V or A; x₄Is S or L; x₅S, P, L or Y.

●^＊＊For VL CDR 2: x₁L, N or P; and X₂S, K, Y, L, M, F, E, Q, R or H.

●^＊＊＊For VL CDR 3: x₁Is Q or S; x₂L, D or P; x₃Is N or R; and X₄P, F, Y or R.

The invention also encompasses antibody binding compositions in which the CDRs encompassed by the invention are embedded or carried within the framework regions of a human antibody in an appropriate orientation to enable the binding composition to specifically and/or selectively bind mature TGF β 1 and neutralize mature TGF β 1 relative to mature TGF β 2 and/or mature TGF β 3. The particular CDRs can be embedded or placed within an appropriate framework using techniques known in the art. The variable domains employed in the present invention may be from any germline or rearranged human variable domain, or may be variable domains synthesized from consensus sequences of known human variable domains.

Preferably, the variable domain framework does not significantly affect the biological properties of the anti-TGF β 1 antibodies in the binding composition embodiments, i.e., the ability to specifically and/or selectively bind and neutralize mature TGF β 1 relative to mature TGF β 2 and/or TGF β 3. More preferably, the framework does not elicit an additional significant immunogenic response when administered (e.g., parenterally) to a human subject. Preferred framework sequences may be sequences naturally occurring in a human antibody or a consensus sequence of several human antibodies. Non-limiting examples of heavy chain variable region framework sequences for antibodies in embodiments of the invention include the VH segment DP-5(Tomlinson et al 1992 J.mol.biol.227: 776-98) and the J segment JH4, JH1, or JH5(Ravetch et al 1981Cell 27: 583-91). Vk segment L1(Cox et al 1994Eur. J. Immunol.24: 827-36) and J segment Jk4(Hieter et al 1982J. biol. chem.10: 1516-22) are non-limiting examples of light chain variable region framework sequences. In a preferred embodiment, the HCVR ER1 framework comprises QVQLVQSGAEVKKPGASVKVSCKAS [ SEQ id no: 8 ]; the HCVR FR2 framework comprises WVRQAPGQGLEWMG [ SEQ ID NO: 9 ]; the HCVR FR3 framework comprises RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR [ SEQ ID NO: 10 ]; the HCVR FR4 framework comprises WGQGTLVTVSS [ SEQ ID NO: 11]. In another preferred embodiment, the LCVR FR1 framework comprises DIQMTQSPSSLSASVGDRVTITC [ SEQ ID NO: 12 ]; the LCVR FR2 framework comprises a sequence selected from [ SEQ ID NO: 13-36 ]; the LCVR FR3 framework comprises GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC [ SEQ ID NO: 37 ]; the LCVRFR4 framework comprises FGQGTKLEIK [ SEQ ID NO: 38]. In more preferred embodiments, such framework regions may contain alterations, deletions, additions, substitutions, or any combination thereof. Furthermore, a framework of 1, 2, 3, 4, 5,6, 7,8, 9 or 10 amino acids is preferably substituted, deleted or added in any combination.

In one embodiment, preferred heavy chain constant regions for embedding the CDRs of the antibody binding composition of the invention include, for example, IgG constant regions. In more preferred embodiments, the IgG constant region is an IgG1 constant region or an IgG4 constant region as shown below.

IgG1[SEQ ID NO：39]：

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, respectively; or

IgG4[SEQ ID NO：40]

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

Preferred light chain constant region sequences of the invention are kappa chain constant regions as shown below:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO：41]

in another preferred embodiment, the antibody binding composition comprises an IgG1 heavy chain constant region or an IgG4 heavy chain constant region and a kappa light chain constant region.

Using the information provided herein, one of ordinary skill will be able to establish mAb embodiments of the invention, such as No.46P-L1-6, having a light chain comprising:

DIQMTQSPSSLSASVGDRVTITCEASSSVSYMHWYQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWDLNPPAFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [ SEQ ID NO: 130] wherein:

LCVR FR1 framework DIQMTQSPSSLSASVGDRVTITC;

VL CDR1 ═ X₁AX₂X₃X₄VX₅YMH (wherein X₁R, Y, E or Q; x₂Is S or T; x₃S, V or A; x₄Is S or L; and X₅S, P, L or Y) EASSSVSYMH;

LCVR FR2 framework WYQQKPGKAPKPLIY;

VL CDR2 ═ formula ATSNX₁AX₂(wherein X₁L, N or P; and X₂S, K, Y, L, M, F, E, Q, R or H);

LCVR FR3 framework GVPSRFSGSGSGTDFTLTISSLQPEDEATYYC;

VL CDR3 ═ X₁QWDX₂X₃X₄PA (where X₁Is Q or S; x₂L, D or P; x₃Is N or R; and X₄P, F, Y or R) QQWDLNPPA;

LCVR FR4 framework FGQGTKLEIK; and is

Light chain constant region ═

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；

Having a heavy chain comprising:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQGLEWMGYIYPYDGDTGYNQKFKSRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGYRWEAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG[SEQ ID NO：131]wherein:

HCVR FR1 framework QVQLVQSGAEVKKPGASVKVSCKAS;

VH CDR1 (formula GYX)₁FX₂DYNX₃X₄(wherein X₁Is T or D; x₂T, E or F; x₃M, I, L or V; and X₄GYTFTDYNMH at H, V or A);

HCVR FR2 framework WVRQAPGQGLEWMG;

VH CDR2 ═ formula X₁X₂YPYDGX₃TGX₄NX₅KX₆KS (wherein X₁Y, Q or S; x₂Is I or V; x₃Is D or E; x₄Y, T, H or L; x₅Is Q, K, P or S, and X₆YIYPYDGDTGYNQKFKS for F or Y);

HCVR FR3 framework RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR;

VH CDR3 ═ formula GYRX₁X₂X₃Y (wherein X)₁Is W or A; x₂Is F or L; and X₃A, E or Y) GYRWFAY;

HCVR FR4 framework WGQGTLVTVSS; and is

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP for the constant region of heavy chainPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

Nucleotides encoding antibodies

The invention also encompasses nucleic acid molecules comprising a polynucleotide sequence encoding a binding composition of the invention or a fragment thereof or a polypeptide sequence of the invention. Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides determined by any method known in the art. For example, if a polypeptide sequence (e.g., an antibody or fragment thereof) is known, the polynucleotide encoding the polypeptide can be determined simply by taking advantage of the degeneracy of the genetic code in any computer algorithm, and the resulting sequence information can be used to assemble, for example, chemically synthesized oligonucleotides (as described in Kutmeier et al (1994) BioTechniques 17: 242), briefly, such a method comprising synthesizing overlapping oligonucleotides comprising a partial sequence encoding the polypeptide sequence, annealing and ligating the oligonucleotides, and then amplifying the ligated oligonucleotides using the polymerase chain reaction.

Alternatively, a polynucleotide encoding a polypeptide sequence of the present invention may be produced from nucleic acid from any suitable source. If no clones containing the nucleic acid encoding a particular antibody are available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from an appropriate source. For example, the source may be a cDNA library of antibodies, or a cDNA library produced from poly A + RNA, or isolated from any tissue or cell capable of expressing an antibody of interest, such as by PCR screening of hybridoma cells capable of expressing an antibody of the invention using synthetic primers capable of hybridizing to the 3 'and 5' ends of a polynucleotide sequence of interest, or by cloning using oligonucleotide probes specific for particular gene sequences to identify, for example, cDNA clones from a cDNA library encoding an antibody, thereby producing nucleic acid molecules for the antibody.

The amplified nucleic acid can be cloned into a replicable cloning vector by any method known in the art. Once the nucleotide and corresponding amino acid sequences of an antibody have been determined, the nucleotide sequence of the antibody can be manipulated by any method known in the art (e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc.) to create amino acid substitutions, deletions, and/or insertions to produce antibodies having different amino acid sequences (see, e.g., Sambrook et al and Ausubel et al editions, new edition, Current Protocols in molecular Biology, John Wiley & Sons, NY).

In particular embodiments, the amino acid sequences of the heavy and/or light chain variable domains can be examined to identify Complementarity Determining Region (CDR) sequences using known methods, such as comparing known amino acid sequences of other heavy and light chain variable regions to determine sequence hypervariable regions. One or both of the CDRs described herein can be inserted into an appropriate framework region, such as a human framework region, using conventional recombinant DNA techniques to reduce immunogenicity. The framework regions may be naturally occurring or common framework regions (or as taught herein), and are preferably human framework regions (for a list of human framework regions, see e.g., Chothia et al 1998 J.mol.biol.278: 457-79). Generally, to identify residues within a human framework that may affect the integrity of the antigen binding site and thus antigen binding, the donor and selected human acceptor sequences may be aligned with several sequence templates derived from an antibody repertoire. Identification of "invariant residues" (Kabat et al 1991) and "Key residues" (Chothia et al 1989) and screening of the sequences against a sequence template to determine the classical-assignment of the donor antigen-binding loops L1-L3, H1 and H2, respectively (Martin)&Thornton, 1996 mol.biol.263: 800-15at http:// www.bioinf.org.uk /). In addition, residues at the VH/VL interface (Chothia et al 1985) and structurally conserved residues at the core position (Chothia et al 1989) were compared to corresponding donor and acceptor residues. Their structure according to known in the Protein database (Protein DataBank) (Berman et al 2000Nucl. acids Res.28 (1): 235-42)Information on his antibody, donor and acceptor framework residues that did not match at these sites were analyzed. The choice of human framework to use as a template for humanization of the foreign V regions will then determine which residues to humanize. Homologous templates are selected from antibodies of known crystal structure, germline or non-germline, or consensus sequences that may optionally be derived from available databases (for a review of this matter, see Routedge et al (Routedge et al 1993in Protein Engineering of Antibody Molecules for Therapeutic and Therapeutic Applications in Man (Clark, M. editor, pages 14-44, Academic notes, Nottingham, UK; and B.Lo, 2004)Antibody Engineering：Methods and ProtocolsHumana Press)). Another strategy for humanizing antibodies is to select the sequence closest to the human germline (Tomlinson et al 1992) as the framework for accepting the donor CDR. This germline approach is based on the same principles as the best-fit strategy, but only searches the database for germline sequences (see, e.g., V BSASE, which is a comprehensive dictionary of all human germline variable region sequences compiled from thousands of published sequences (including sequences currently published by the Genbank and EMBL databases.) the V BASE database serves as an extensive work for sequencing and mapping human antibody genes, and a user-friendly tool for its analysis, which was developed over several years by the MRC protein engineering center (Cambridge, UK.) the germline framework method is very useful because human germline sequences do not exhibit somatic hypermutations with potential immunogenicity. CDRs can also be grafted or embedded in human frameworks using a common human germline strategy with one of the subtypes as the framework (see, e.g., Presta et al, 1993J. Immunol 151: 2623-32; human Coouda et al, 13: 215-9; couto et al 1995Cancer Res. (Suppl.), 55, 5973s-7 s; werther et al 1996j.immunol., 157: 4986-95; o' Connor et al 1998Protein Engng 11: 321-8)

The polynucleotides produced by the framework region and CDR combination preferably encode an antibody (or fragment thereof) that specifically and/or selectively binds TGF β 1 (or epitope thereof). As discussed herein, one or more amino acid substitutions are preferably made in the framework regions to improve the binding of the antibody to its antigen.

In addition, such methods can be used to replace or delete one or more cysteine residues of the variable region involved in the formation of an intrachain disulfide bond, to produce an antibody molecule lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotides are encompassed by the present invention and are within the ability of those of ordinary skill in the art, such as molecular biology.

In addition, these techniques can be adapted for the production of single chain antibodies (see, e.g., U.S. Pat. No.4,946,778; Bird, Science 242: 423-42 (1988); Huston, et al PNAS 85: 5879-. The Fv regions of the heavy and light chain fragments are joined by amino acid bridges to produce a single chain polypeptide, thereby forming a single chain antibody. The technique of assembling functional Fv fragments in E.coli can also be used (Skerra et al (1988) Science 242: 1038-1041).

Polypeptide fragments

The invention also encompasses fragments of the binding composition. A "polypeptide fragment or segment" comprises a portion of a sequence described herein or SEQ ID NO: an amino acid sequence of a portion of a polypeptide sequence. The protein and/or polypeptide fragments or segments may be in free form or they may form part of a larger polypeptide or protein, wherein the fragments or segments form part or region, as shown here in SEQ ID NO: to a single contiguous region on the fusion protein.

A polypeptide segment preferably possesses 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 consecutive amino acids in length. In this context, "about" includes the specified range or value described herein, and also includes values at either or both ends of the fragment that differ by a few amino acid residues (e.g., plus or minus 5, plus or minus 4, plus or minus 3, plus or minus 2, plus or minus 1 amino acid residue) from the recited value. Polynucleotides encoding such polypeptide fragments are also encompassed by the invention.

Furthermore, the invention encompasses proteins or polypeptides comprising a plurality of such amino acid segments or fragments (e.g., non-overlapping segments of a specified length). Typically, the plurality should be at least 2, more typically at least 3, and preferably at least 4, 5,6, 7,8, 9 or more. Although a minimum length for a segment is provided, a maximum length of varying size for any particular plurality of segments is also encompassed, e.g., up to 3 segments may in total contain 1 segment of 7 contiguous amino acids in length and two additional non-overlapping segments, each 12 amino acids in length.

Also preferred are polypeptide fragments or segments (and their corresponding polynucleotide fragments) of structural or functional domains such as fragments or combinations thereof so characterized: for example, it may comprise complementarity determining regions (CDRs such as VL or VH: CDR1, CDR2 or CDR3), variable regions (such as the variable region of a heavy or light chain, VL or VH), framework regions (FH1, 2, 3 or 4), D or J regions, constant regions (such as C1, 2, 3 or 4)_L、C_H1、C_H2 or CH₃) A hinge region, an Fc γ receptor binding region, an alpha helix and alpha helix forming region, an alpha 0 fold and alpha 1 fold forming region, a turn and turn forming region, a coil and coil forming region, a hydrophilic region, a hydrophobic region, an alpha amphipathic region, a beta amphipathic region, a flexible region, a loop region, a hairpin domain, a beta-alpha-beta motif, a helical bundle, an alpha/beta barrel, an upper and lower beta barrel, a jam roll (jelly roll) or Swiss roll (Swiss roll) motif, a transmembrane domain, a surface forming region, a substrate binding region, a transmembrane region, a linker, an immunogenic region, an epitope region and a high antigenic index region. Furthermore, the present invention also encompasses polynucleotides encoding these domains.

Other preferred polypeptide segments are biologically active fragments. A biologically active fragment exhibits similar, but not necessarily identical, activity as the polypeptide (or fragment thereof) of the binding composition, e.g., Fab, Fv, scFv, or F (ab') 2. Polynucleotides encoding these polypeptide fragments are also encompassed by the present invention.

The polynucleotide fragment preferably encodes a polypeptide exhibiting functional activity. Word (word) "Functional Activity"comprises polypeptide segments that can achieve one or more known functional activities. Such functional activities include, but are not limited to, biological activity, antigenicity (an antibody binding composition that binds to or competes with a polypeptide for binding to a polypeptide of the invention), immunogenicity (the ability to generate antibodies that bind to a polypeptide of the invention), the ability to form multimers with a polypeptide of the invention, and the ability to bind to a receptor or ligand of a polypeptide described herein.

The functional activity of polypeptides (including fragments, variants, derivatives and analogs thereof) can be determined in a variety of ways. For example, in determining the ability of an antibody to bind or compete with a full-length polypeptide of the invention for binding to a polypeptide of the invention, a variety of immunoassays known in the art can be used, including, but not limited to, competitive and non-competitive assay systems, using techniques such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., labeling with colloidal gold, enzymes, or radioisotopes), 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.).

In another embodiment, detection of antibody binding is achieved by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the second antibody is labeled. Many methods for detecting binding in an immunoassay are known in the art and are within the scope of the present invention.

In another embodiment, when ligands are identified, or the ability of fragments, variants or derivatives of the polypeptides of the invention to form multimers is assessed, binding can be detected, for example, by reducing or non-reducing gel chromatography, protein affinity chromatography and affinity blotting (see generally Phizicky et al (1995) Microbial. Rev.59: 94-123). In another embodiment, the physiological relationship of the polypeptide to its substrate (e.g., signal transduction) can be determined by conventional techniques. In addition, the assays described herein (see, e.g., the "examples" section of the application) or other assays known in the art can be routinely used to determine the ability of binding compositions (fragments, variants, derivatives, and analogs thereof) to modulate the relevant biological activity of TGF β 1in vitro or in vivo.

Method for producing antibody

The antibody-binding composition may be produced by any method known in the art, in particular by chemical synthesis or preferably by recombinant expression techniques.

Recombinant expression of a binding composition or fragment, derivative or analog thereof (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention) requires construction of an expression vector containing an antibody-encoding polynucleotide. Once the sequences of the polynucleotides encoding the antibody molecules or antibody heavy or light chains or portions thereof (preferably comprising the variable domain regions of the heavy or light chains) of the present invention are obtained, vectors for the production of the antibody molecules may be prepared using known recombinant DNA techniques.

Expression vectors containing the coding sequences in combination with appropriate transcriptional and translational control signals can be constructed by methods known in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the invention encompasses a replicable vector comprising a nucleotide sequence encoding an antibody of the invention (or a fragment thereof), or a heavy or light chain thereof, or a variable domain or CDR of a heavy or light chain, operably linked to a promoter. Such vectors may include, for example, nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., WO 86/05807; WO 89/01036; or U.S. Pat. No.5,122,464), and the variable domains of the antibodies may be cloned into such vectors to express the entire heavy or light chain or any portion thereof.

Generally, the expression vector is transferred to a host cell using conventional techniques, and the transfected cells are then cultured to produce the antibody or portion thereof. Thus, the invention also encompasses a host cell comprising a polynucleotide encoding an antibody of the invention or its heavy or light chain, or a single chain antibody of the invention, operably linked to a heterologous promoter. In a preferred embodiment of expressing a diabody, vectors encoding the heavy and light chains may be co-expressed in a host cell to express an intact immunoglobulin molecule, as described in detail herein or as known in the art.

Various host expression vector systems can be used to express the antibody molecules of the invention. Such host expression systems provide a means for producing and subsequently purifying any coding sequence of interest. However, when transformed or transfected with the appropriate nucleotide coding sequences, the host expression system cell also provides the antibody molecule of the invention in situ. Such cells include, but are not limited to, bacterial microorganisms (e.g., E.coli, Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences, yeast (e.g., Saccharomyces, Pichia), insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences, plant cell systems infected with recombinant viral expression vectors (e.g., Cauliflower mosaic Virus, tobacco mosaic Virus TMV) containing antibody coding sequences or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid), or plant cell systems containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoters, vaccinia virus 7.5k promoter) in a mammalian cell system (e.g., COS, CHO, BHK, 293, 3T3 cells).

Preferably, bacterial cells such as E.coli are used, more preferably eukaryotic cells are used to express the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary Cells (CHO), together with vectors such as the major, i.e., early, Gene promoter from human cytomegalovirus, are efficient expression systems for antibodies (Foecking et al (1986) Gene 45: 101; Cockett et al (1990) Bio/Technology 8: 2).

In bacterial systems, a wide variety of expression vectors may be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when large amounts of protein are to be produced, such as in pharmaceutical compositions for the production of antibody molecules, vectors that direct high levels of expression of the fusion protein product and that are easily purified are required. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al EMBO J.2: 1791(1983)), to which antibody coding sequences can be ligated in-frame with the lac Z coding region, respectively, to generate fusion proteins, the pIN vector (Inouye and Inouye, Nucleic acids sRs.13: 3101 and 3109 (1985); van Heeke and Schuster, j.biol.chem.24: 5503-5509(1989)), and the like. pGEX vectors may also be used to express foreign polypeptides in the form of fusion proteins with glutathione-S-transferase (GST).

Typically, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix, and subsequent elution in the presence of free glutathione. The pGEX vectors were designed to include thrombin or factor Xa protease cleavage sites to allow cleavage of the cloned target gene product from the GST moiety. One insect system used to express foreign genes is the Autographa californica nuclear polyhedrosis virus (AcNPV) system. AcNPV virus is grown in Spodopterus frugiperda (Spodopteru frugiperda) cells. Antibody coding sequences can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcPNV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems are available. In the case of an adenovirus used as an expression vector, the antibody coding sequence of interest can be linked to an adenovirus transcription/translation control complex, such as a late promoter and a triple leader sequence. This chimeric gene is then inserted into the adenovirus genome by in vivo or in vitro recombination. Insertion into non-essential regions of the viral genome (e.g., the E1 or E3 regions) results in a recombinant virus that is capable of surviving and expressing antibody molecules in infected host cells (see, e.g., Logan and Shenk, 1984PNAS 81: 355-9). Specific initiation signals are required for efficient translation of the inserted antibody coding sequence. These signals include, for example, the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in frame with the desired coding sequence to ensure proper translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, including naturally occurring and synthetic.

Expression efficiency can be increased by the incorporation of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bittner et al 1987Methods in enzymol.153: 5 l-4). In addition, host cell strains are selected which are capable of modulating the expression of the inserted sequences or modifying and processing the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products are important to the function of the protein.

Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. The appropriate cell line or host system is selected to ensure proper modification and processing of the foreign protein expressed. For this, eukaryotic host cells are used which possess the cellular machinery for the correct processing of the initial transcript, glycosylation and phosphorylation. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, in particular breast cancer cell lines such as BT483, Hs578T, HTB2, BT20 and T47D and normal breast cell lines such as CRL7030 and Hs578 Bst.

The invention encompasses recombinant fusion or chemical conjugation (including covalent conjugation and non-covalent conjugation) of an antibody to a polypeptide (or portion thereof, preferably containing at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 contiguous amino acids of the polypeptide) to produce a fusion protein. The fusion need not be direct, but can be performed by a linker sequence.

Antibodies fused or conjugated to polypeptides may also be used in vitro immunoassays and purification methods using methods known in the art (see, e.g., Harbor et al supra, and WO 93121232; EP 439,095; Naramura et al (1994) Immunol. Lett.39: 9 l-9; U.S. Pat. No.5,474,981; Gillies, et al 1992PNAS 89: 1428-32; Fell et al 1991J. Immunol.146: 2446-52).

The invention also includes compositions comprising a polypeptide (or fragment thereof) fused or conjugated to an antibody domain other than a variable region. For example, a polypeptide of the invention (or fragment thereof) may be fused or conjugated to an antibody constant region, D or J region, Fc region, or portion thereof. The antibody portion fused to the polypeptide of the invention (or fragment thereof) may comprise any combination of constant region, hinge region, CH1 domain, CH2 domain and/or CH3 domain or complete domain or portions thereof. The polypeptide (or fragment thereof) can also be fused or conjugated to an antibody moiety described herein to form a multimer. For example, an Fc portion fused to a polypeptide of the invention (or fragment thereof) may form a dimer through disulfide bonds between the Fc portions. Higher multimeric forms can be formed by fusing polypeptides to IgA and IgM moieties. Methods for fusing or conjugating the polypeptides of the invention (or fragments thereof) to antibody moieties are known (see, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP307,434; EP 367,166; WO 96/04388; WO9106,570; Ashkenazi et al (1991) PNAS 88: 10535-.

As discussed herein, the polypeptides, polypeptide fragments may be fused or conjugated to antibody moieties described herein or known in the art to increase their half-life in vivo. In addition, the polypeptides, polypeptide fragments may also be fused or conjugated to antibody moieties to facilitate purification. An example is a chimeric protein comprising the first two domains of a human CD4 polypeptide and multiple domains of a mammalian immunoglobulin heavy or light chain constant region (see, e.g., EP 394,827; Traunker et al (1988) Nature 331: 84-86).

In many cases, the Fc portion of the fusion protein is useful in therapy and diagnosis, and thus improves pharmacokinetic properties (see, e.g., EP A232,262). Optionally, the Fc portion may advantageously be deleted after expression, detection and purification of the fusion protein. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may hinder therapy and diagnosis. For high throughput screening assays to identify antagonists of hIL-5, as in drug development, human proteins such as hIL-5 are fused to the Fc portion (see, e.g., Bennett et al (1995) J.molecular Recognition 8: 52-58; Johanson et al (1995) J.biol. chem.270: 9459-9471).

Furthermore, the binding composition (or fragment thereof) may be fused to a marker sequence, such as a polypeptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as a tag provided on the pQE vector (QIAGEN, inc., Chatsworth, CA) as well as on other commercially available vectors. Hexahistidine facilitates purification of the fusion protein (Gentz et al (1989) PNAS 86: 821-824). Other peptide tags that may be used for purification include, for example, the "HA" tag, which corresponds to an epitope derived from the influenza virus hemagglutinin protein (Wilson et al (1984) Cell 37: 767) and the "flag" tag.

The invention also encompasses antibodies or fragments thereof conjugated to diagnostic or therapeutic agents. For example, antibodies can be used to diagnostically monitor the development and progression of tumors as part of a clinical testing procedure to determine the efficacy of a given treatment regimen. Coupling the antibody to a detectable substance facilitates detection. Examples of detectable substances include, for example, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography techniques, and nonradioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated to the antibody (or fragment thereof) either directly or indirectly by established techniques through an intermediate, such as a linker as is known in the art (see, e.g., U.S. Pat. No.4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents of the invention). Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidinAnd biotin; suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include, but are not limited to, luminol; examples of bioluminescent materials include, but are not limited to, luciferase, and aequorin; examples of suitable radioactive materials include I¹²⁵、I¹³¹、I¹¹¹Or Tc⁹⁹。

The binding compositions of the invention may also be attached to a solid support, which is particularly useful in immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Techniques for conjugating therapeutic moieties to Antibodies are known, e.g., Amon et al, "Monoclonal Antibodies against Drugs In Cancer Therapy", In Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.), pp 243-56 (orange r. loss, inc. 1985); hellstrom, et al, "Antibodies For Drug Delivery," inControlled Drug Delivery (2 nd edition), Robinson et al (ed.), pages 623-53 (MarcelDekker, Inc. 1987); thorpe, "Antibody Carriers Of Cytotoxin Agents Incancer Therapy: a Review ", in Monoclonal Antibodies' 84: biological and clinical Applications, Pinchera et al (ed), pp.475-; "Analysis, Results, and Future productive Of The Therapeutic Use Of radioLabeledAntibody in Cancer Therapy", in Monoclonal Antibodies for Cancer Therapy and Therapy, Baldwin et al (ed.), pp.303-16 (Academic Press1985), and "The prediction and cytological Properties Of antibody-Toxin Conjugates" Immunol. Rev.62: 119-58(1982)).

B immunoassay

Specific proteins such as TGF-beta 1 may be assayed by a variety of immunoassay methods, including, for example, but not limited to, competitive and non-competitive assay systems, using techniques including, but not limited toBut are not limited to, western blots, radioimmunoassays, ELISA ("enzyme chain immunoadsorption 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. For a general review of immunological and immunoassay methods, see Stits and Terr (eds) (1991)Basic and Clinical Immunology(7 th edition). Furthermore, the immunoassays of the present invention can be performed in a variety of formats, which are extensively reviewed in the following literature: maggio (editor) (1980)Enzyme Immunoassay CRC Press，Boca Raton，Florida；Gosling J P 2000Immunoassays：A Practical Approach(Practical Approach Series) OxfordUniv Press; diamandis and Christopouus, 1996Immunoassay AcademicPress，San Diego，CA；Tijan(1985)″Practice and Theory of EnzymeImmunoassays，″Laboratory Techniques in Biochemistry and Molecular BiologyElsevier Science Publishers b.v., Amsterdam; wild, D. (eds.), 2001The Immunoassay Handbook(2 nd edition) Nature Pub Group; james t.wu, 2000Quantitative Immunoassay：A Practical Guide for Assay Establishment，Troubleshooting，and Clinical ApplicationAmer Assn for clinical Chemistry, Brousseau and Beaudet (eds.)Manual of Immunological MethodsCRC Press Boca Raton, Florida; and Harlow and LaneAntibodies，A Laboratory ManualAs before. See also Chan (edit) (1987)Immunoassay：A Practical GuideAcademic Press, Orlando, FL; price and Newman (edit) (1991)Principles and Practice of ImmunoassaysStocktonPress, NY; and Ngo (edit) (1988)Non-isotopic Immunoassays Plenum Press，NY。

Immunoassays can be carried out using a variety of methods known in the art. Briefly, immunoassays for the determination of proteins may be competitive or noncompetitive binding assays. In a competitive binding assay, the sample to be analyzed competes with the labeled analyte for binding to specific binding sites on the capture reagent that have been bound to the solid surface. Preferably the capture reagent is an antibody specifically reactive with TGF-beta 1 as described herein. The concentration of labeled analyte bound to the capture reagent is inversely proportional to the amount of free analyte present in the sample.

In a competitive binding immunoassay, a target protein present in a sample competes with a labeled protein for binding to a specific binding composition. For example, binding compositions such as antibodies can specifically and/or selectively react with a target protein. The binding composition may be bound to a solid surface to effect separation of bound tagged proteins from unbound tagged proteins. Alternatively, competitive binding assays can be performed in liquid phase, and a variety of techniques known in the art can be used to separate bound, labeled protein from unbound, labeled protein. After separation, the amount of bound labeled protein is measured. The amount of protein present in the sample is inversely proportional to the amount of bound marker protein.

Alternatively, homogeneous immunoassays can be performed without a separation step. In these immunoassays, the label on the protein is changed by the binding of the protein to its specific binding composition. Such a change in the labeled protein can result in an increase or decrease in the signal emitted by the label, such that the protein can be detected or quantified by detecting the label at the end of the immunoassay.

When the cell is contacted with a labeled binding partner or antibody having a known binding affinity for the protein (e.g., a peptide)¹²⁵I-antibody) and incubated together, and a competition assay is also useful in determining the binding affinity of the test sample to the binding composition. Bound and free labeled binding compositions were then separated to assess the extent of protein binding. The amount of test compound bound is inversely proportional to the amount of label binding partner bound to the known source. The bound protein can be separated from the free protein by any of a number of techniques to assess the extent of protein binding. Such separation steps typically include, for example, adhering to a filter, and thenWashing, adhering to plastic and then washing, or centrifuging cell membranes. Live cells may also be used to screen drugs for their effect on TGF-beta protein-mediated functions (e.g., levels of second messengers, e.g., cell amplification, changes in phosphoinositide pools, transcription using luciferase-based assays, etc.). Some detection methods may eliminate a separation step, such as proximity sensitive detection systems.

Proteins can also be qualitatively or quantitatively analyzed by a variety of non-competitive immunoassays. For example, a two-site solid phase sandwich immunoassay may be used. In this type of assay, a protein binding composition (e.g., an antibody) is attached to a solid support. The second protein binding composition (which may also be an antibody, and binds the protein at a different site) is labeled. After binding has occurred at both sites of the protein, unbound labeled binding composition is removed and the amount of labeled binding composition bound to the solid phase is determined. The amount of labeled binding composition bound is directly proportional to the amount of protein in the sample.

The immunoassay format described above employs labeled assay components. The label may be coupled directly or indirectly to the desired assay component according to methods well known in the art. A variety of markers and methods may be employed. Incorporation is conventionally used³H、¹²⁵I、³⁵S、¹⁴C or³²A radiolabel for P. Non-radioactive labels include proteins that bind antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can specifically bind members of a pair as a labeled protein. The choice of label depends on the sensitivity required, the ease of conjugation with the compound, the stability requirements and the equipment available. For an overview of the various marker or signal generating systems that can be used, see U.S. Pat. No.4,391,904.

The reactivity of the antibody-binding composition with a particular protein can also be determined using a variety of immunoassay methods. For a review of the immunological and immunoassay procedures for antibody assays by immunoassay techniques, see Stits and Terr (eds.)Basic and Clinical Immunology(7 th edition) same as above;maggio (edit)Enzyme ImmunoassayThe same as above; and Harlow and LaneUsing Antibodies，A Laboratory ManualThe same as above.

Briefly, an immunoassay that detects the reaction of serum with a target protein can be a competitive or non-competitive binding assay. In a competitive binding assay, the sample analyte competes with the labeled analyte for binding to specific binding sites of a capture reagent that has bound to a solid surface. The capture reagent is preferably a purified recombinant protein. Other sources of protein, including isolated or partially purified naturally occurring proteins, may also be used. Non-competitive assays include sandwich assays, wherein the sample analyte is bound between two analyte-specific binding reagents. One of the binding compositions is used as a capture reagent to bind to a solid surface. The second binding composition is labeled and used to measure or detect the formed complex either visually or by a device. Various combinations of capture reagents and labeled binding compositions can be used. A wide variety of immunoassay formats, separation techniques and labels similar to those described above can be used to determine the protein.

For example, Western blot analysis can be used to assess the ability of an antibody of interest to immunoprecipitate a particular antigen. Those skilled in the art should have profound knowledge to alter parameters to increase antibody binding to antigen to reduce background, such as pre-washing cell lysates with agarose beads. A discussion of immunoprecipitation Protocols can also be found in Ausubel et al, eds, 1994, Current Protocols in molecular Biology, Vol.1, John Wiley & Sons, Inc., New York.

The ELISA assay involves preparing an antigen, coating a 96-well microtiter plate with the antigen, adding an antibody of interest conjugated to a detectable compound, such as an enzyme substrate (horseradish peroxidase or alkaline phosphatase), to the wells and incubating for a period of time, and detecting the presence of the antigen. In an ELISA, the antibody of interest is not necessarily conjugated to a detectable compound; instead, a second antibody conjugated to a detectable compound (which recognizes the antibody of interest) may be added to the well. In addition, the microwells may also be coated with antibodies rather than antigens. In this case, the second antibody conjugated to the detectable compound may be added after the antigen of interest is added to the coated wells. Those of ordinary skill can determine without undue experimentation which parameters to adjust to increase signal, and which other variations of ELISA to use (see, e.g., Ausubel et al, eds., 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York).

The binding affinity of an antibody to an antigen and the rate of binding and dissociation of antibody-antigen interactions can be determined using, for example, a competitive binding assay. A non-limiting example is a radioimmunoassay that involves the addition of labeled antigen in the presence of a gradual increase of unlabeled antigen (e.g.³H or¹²⁵I) Incubated with the antibody of interest and the amount of antibody bound to the labeled antigen is then detected. The affinity and the association dissociation rate of the antibody of interest for a particular antigen can be determined using data obtained from Scatchard plot analysis. Competition with the second antibody can also be determined using, for example, radioimmunoassay. In this case, the antigen is conjugated with a labeling compound: (³H or¹²⁵I) The antibody of interest of (1) is incubated with gradually increasing amounts of non-labeled secondary antibody.

Physical variants

The invention also encompasses polypeptide sequences having amino acid sequences substantially similar and/or identical to the amino acid sequences described herein. Amino acid sequence similarity or sequence identity can be determined by optimizing residue matching. This situation changes when conservative substitutions are considered for pairing. 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-; sankoff et al (1983)Time Warps，String Edits，and Macromolecules：The Theory and Practice of Sequence ComparisonChapter I, Addison-Wesley, Reading, MA; and from IntelliGenetics, mountain view, CA; and the University of Wisconsin Genetics Computer Group, Madison, WI..

The present invention includes, but is not limited to, polypeptide sequences that are functionally related to the polypeptides encoded by the specific sequence identifiers of the present application. Functionally related polypeptides include any polypeptide that shares functional characteristics with a binding composition, such as the ability to selectively and/or specifically bind TGF β 1 and not TGF β 2 and/or 3. Such functionally related polypeptides include, but are not limited to, the addition or substitution of amino acid residues within the amino acid sequences encoded by the sequences described herein, particularly amino acid additions or substitutions that result in silent changes, thereby resulting in functionally equivalent polypeptides. Amino acid substitutions may be based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the relevant residues.

For example, 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; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Furthermore, non-classical amino acids or chemical amino acid analogs can be substituted or added to the polypeptide sequence. Non-canonical amino acids generally include, but are not limited to, e.g., the D-isomer of a common amino acid; 2, 4-diaminobutyric acid, aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaproic acid, Aid, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cystine (cysteine acid), tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylglycine, b-alanine, fluoroamino acids, designed amino acids such as b-methylamino, Ca-methylamino, Na-methylamino and amino acid analogs in general. Furthermore, the amino acid may be dextrorotatory (D) or levorotatory (L).

The addition of peptide moieties for ease of handling is well known and routine in the art. Furthermore, the binding composition (including any fragment thereof, especially an epitope) may be combined with an immunoglobulin [ e.g., a constant domain portion of IgA, IgE, IgG, IgM, or any combination thereof (CH1, CH2, CH3) and including the entire domain and portions thereof ], to produce a chimeric polypeptide. Such fusion proteins facilitate purification and are typically used to increase the half-life of the protein in vivo. This has been demonstrated, for example, by chimeric proteins comprising the first two domains of the human CD4 polypeptide and various domains of the constant region of a mammalian immunoglobulin heavy or light chain (EP 394,827; Traunker et al 1988Nature 331: 84-6). Fusion proteins of disulfide-linked dimeric structure (due to IgG domains) are also more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone (fountain et al 1995J. biochem. 15270: 3958-64). It has been demonstrated that conjugation of an antigen (e.g. insulin) to an Fc γ R binding partner, such as IgG or Fc fragment, facilitates delivery of the antigen across epithelial barriers to the immune system (see e.g. WO 96/22024 and WO 99/104813).

In addition, the fusion protein may comprise portions of constant regions of immunoglobulin molecules as well as human proteins or portions thereof. In many cases, the Fc portion of the fusion protein is of great benefit in therapy and diagnosis, and thus, for example, may improve pharmacokinetic properties. Alternatively, it may be desirable to delete the Fc portion after expression, detection and purification of the fusion protein. For example, if the fusion protein is used to immunize as an immunogen, the Fc portion may hinder therapy and/or diagnosis. For example, in drug development, human proteins are fused to the Fc portion to identify antagonists by high throughput screening assays.

Moreover, similar functional domains of other proteins can be combined to form new constructs. For example, protein binding or other segments can be "swapped" between different new fusion polypeptides or fragments. Thus, a new chimeric polypeptide exhibiting a new combination specificity will result from the functional linkage of the protein binding specificity to other functional domains. In addition, fusion constructs can be generated by gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling techniques.

"substantially pure"Refers to the isolation of a nucleic acid, protein or polypeptide from its natural environment and from other contaminating proteins, nucleic acids and other biological agents. Purity or "isolation" can be measured by standard methods and is typically at least about 50% pure, more typically at least about 60% pure, typically at least about 80% pure, often at least about 85% pure, more typically at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in the most preferred embodiment at least 99% pure. The same concept applies to binding compositions, such as the antibodies of the invention. For example, it may be desirable to purify the polypeptide from a recombinant cellular protein or polypeptide.

Of proteins or polypeptidesSolubility "This can be reflected in sedimentation measured in Svedberg units, which measure the rate of molecular sedimentation under specific conditions. Determination of sedimentation rate has been traditionally performed in the past with analytical ultracentrifugation, but is now commonly performed with standard ultracentrifuges (see Freifelder 1982)Physical Biochemistry(2 nd edition) w.h.freeman&Co, SanFrancisco, CA; and Cantor and Schimmel (1980)Biophysical ChemistryParts 1-3, w.h.freeman&Co, San Francisco, CA). In a rough assay, the sample containing the putative soluble polypeptide is spun in a standard full size ultracentrifuge at about 50K rpm for about 10 minutes and the soluble molecule will remain in the supernatant. The soluble particle or polypeptide is typically less than about 30S, more typically less than about 15S, typically less than 10S, and more typically less than 6S, in particular embodiments preferably less than about 4S, and more preferably less than about 3S. The solubility of the polypeptide or fragment depends on the environment and the polypeptide. Many parameters affect the solubility of the polypeptide, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and the nature of the solvent. Generally, the temperature range used for the polypeptide is from about 4 ℃ toAbout 65 ℃. Temperatures above about 18 ℃ and more typically above about 22 ℃ are typically used. For diagnostic purposes, the temperature is typically about room temperature or warmer, but below the denaturation temperature of the assay components. For therapeutic purposes, the temperature is typically body temperature, typically about 37 ℃ for humans, although under certain conditions, the temperature may be adjusted up or down, in situ or in vitro. The size and structure of a polypeptide is generally in a substantially stable state, usually not in a denatured state. The polypeptide may be associated with other polypeptides having quaternary structure to impart solubility, or with phospholipids or detergents in a manner that approximates natural phospholipid bilayer interactions.

Solvents are generally a class of biologically compatible buffers used to preserve biological activity, often in proximity to physiological solvents. The solvent is generally at a neutral pH, generally about 5 to 10, preferably about 7.5. In some cases, a detergent, typically a mild non-denaturing detergent, such as CHS (cholesterol hemisuccinate) or CHAPS (3- [ 3-cholamidopropyl ] -dimethylamino) -1-propanesulfonic acid inner salt), is added; or at a concentration low enough to avoid significantly disrupting the structural or physiological properties of the protein. In preferred embodiments, the solubility (pH 5.0-6.0 and 150mM NaCl) of a binding composition of the invention is greater than 10, 15, 20, 25, 30, 35 or 40mg/ml, in more preferred embodiments greater than 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50mg/ml, and in even more preferred embodiments greater than 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60mg/ml, and in even more preferred embodiments greater than 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 or 150mg/ml (or a range between any two such values, which range may or may not include any one or both boundary points).

Variants

The present invention relates to polypeptides comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide sequence of the present invention (or a fragment thereof). Methods known in the art can be used to determine whether a particular sequence exhibits identity to a binding composition sequence.

In such alignments, one sequence is usually used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, followed by assigning coordinates, if necessary, and assigning parameters of the sequence algorithm program. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the parameters of the designated program.

Optimal alignment of sequences for comparison can be achieved by Smith and Waterman (1981)Adv.Appl. Math.2: 482 local homology algorithm, needleman and Wunsch (1970)J.Mol.Biol.48: 443 homology alignment algorithm, Pearson and Lipman (1988)Proc.Nat′l Acad.Sci. USA85: 2444, computer executions of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin genetic software package, Genetics computer group, 575Science Dr., Madison, Wis.), the LASERGENE bioinformatics calculation package produced by DNASTAR corporation (Madison, Wisconsin), sequence alignment methods developed by Notredame et al (e.g., 3 Dcofee or Tcoffe in, e.g., Nucleic Acids Res.2004: 32(Web Server issue): W37-40; Nucleic Acids Res.2003, 7.1.7.31 (13): 3503-6; or Pharmacogenomics.2002.1.3 (1): 131-4) or by visual inspection (see generally Ausubel et al supra). Other methods of comparing nucleotide or amino acid sequences by determining optimal alignments are known (see e.g. Peruski and Peruski,The Internet and the New Biology：Tools for Genumic and Molecular Research(ASM Press, Inc. 1997); wu et al (edit)',Information Superhighway and Computer Databases of Nucleic Acids and Proteins，″in Methods in Gene Biotechnologypage 123-; or a Bishop (edition),Guide to Human Genome Computing2 nd edition (Academic Press, Inc.1998)).

A polypeptide that exhibits or has at least about 95% sequence identity to other amino acid sequences can include up to 5 amino acid changes per 100 amino acid length of the test amino acid chain. In other words, a first amino acid sequence that is at least 95% identical to a second amino acid sequence may differ from the second sequence by up to 5% of the total number of amino acid residues due to insertions, deletions or substitutions of amino acid residues. Changes in amino acid residues of a polypeptide can occur at the amino or carboxy terminal position or anywhere between these two terminal positions, or interspersed among residues of the sequence, respectively, or interspersed among one or more adjacent amino acid residue amino acid intervals, portions, or fragments within the sequence, respectively. Indeed, methods known in the art can be used to determine whether any particular polypeptide sequence exhibits at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to other sequences, such as those shown in table 1 herein.

The variants encompassed by the present invention may contain changes in coding regions, non-coding regions, or both. Furthermore, variants in which 1, 2, 3, 4, 5,6, 7,8, 9 or 10 amino acids are substituted, deleted or added in any combination are also preferred. Non-naturally occurring variants may be generated by mutagenesis techniques or synthesized directly by known methods of protein engineering or recombinant DNA techniques. Production of such variants can enhance or alter the characteristics of the binding composition polypeptide (or fragment thereof). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of a secreted polypeptide of the invention (or fragment thereof) without substantial loss of biological function. For example, Ron et al (1993 J.biol.chem.268: 2984-8) report that variant KGF proteins still have hairpin binding activity even after deletion of 3, 8 or 27 amino-terminal amino acid residues. For example, when a small portion of the residues are removed from the N-terminus or C-terminus of the secreted form, antigenicity and/or immunogenicity (e.g., the ability of the deletion variant to induce and/or bind antibodies that recognize the mature form of the polypeptide) may be retained. Whether a polypeptide lacking an N-or C-terminal residue of a protein retains such activity can be readily determined using routine methods described herein or known in the art. Thus, the invention also encompasses polypeptide variants that exhibit biological activity such as immunogenicity or antigenicity. Such variants include deletions, insertions, rearrangements, repeats and substitutions selected with the general rules known in the art which have little effect on their activity. For example, by Bowie et al (1990) Science 247: 1306-1310 provides teachings on the preparation of phenotypically silent amino acid substitutions.

In addition to using conservative amino acid substitutions, other variants of the invention include, but are not limited to, (i) substitution with one or more non-conservative amino acid residues, wherein the substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code; or (ii) substitution with one or more amino acid residues having a substituent group; or (iii) fusing the mature polypeptide to other compounds, such as compounds that increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); or (iv) fusing the polypeptide with additional amino acids (such as an IgG Fc fusion domain peptide, or a leader or secretory sequence, or a sequence that facilitates purification). All such variants are within the ability of those skilled in the art of molecular biology given the teachings of this application, e.g., specifying unique polynucleotide and polypeptide sequences.

For example, polypeptide variants in which charged amino acids are substituted with other charged or neutral amino acids can result in polypeptides with improved characteristics (e.g., reduced aggregation). Aggregation of pharmaceutical agents both reduces activity and increases clearance due to the immunogenicity of the aggregate (Pinckard et al (1967) Clin. exp. Immunol.2: 331-. In a preferred embodiment, the binding compositions of the invention are formulated in a suitable pH/buffer system (described herein or known in the art) that does not exhibit significant aggregation after incubation for at least 1, 2, 3, 4, 5,6, 7,8, or 9 months at a temperature range of about 1-10 ℃, more preferably 2-8 ℃, even more preferably 3-7 ℃ and more preferably 5-6 ℃.

Another embodiment of the invention also encompasses compositions comprising an amino acid sequence of the invention comprising at least 1 amino acid substitution, but no more than 50 amino acid substitutions, even more preferably no more than 40 amino acid substitutions, yet more preferably no more than 30 amino acid substitutions, yet even more preferably no more than 20 amino acid substitutions, or no more than 15 amino acid substitutions. Of course, in increasing order of preference, peptides or polypeptides comprising an amino acid sequence of the invention containing at least one but not more than 10, 9, 8, 7, 6,5, 4,3, 2 or 1 amino acid substitution are highly preferred. In particular embodiments, the number of additions, substitutions, and/or deletions in a polypeptide sequence of the invention or fragment 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 substitutions of conservative amino acids are preferred over substitutions of non-conservative amino acids.

Therapeutic uses

The invention also provides agents of beneficial therapeutic value. Therapeutic binding compositions of the invention include, but are not limited to, antibody binding compositions of the invention (including fragments, analogs and derivatives thereof described herein) and nucleic acid molecules encoding them (including fragments, analogs and derivatives thereof described herein and anti-idiotypic antibodies). Such antibodies may be used to modulate, treat, inhibit, ameliorate, or prevent a disease, disorder, or condition associated with aberrant expression and/or activity of TGF β 1 (or fragments thereof), including, but not limited to, any one or more of the diseases, disorders, syndromes, or conditions described herein. Treatment, amelioration and/or prevention of diseases, disorders or conditions associated with aberrant expression and/or activity of TGF β 1 includes, but is not limited to, amelioration of symptoms associated with such diseases, disorders or conditions.

For example, diseases or disorders associated with aberrant expression or aberrant signaling of TGF β 1 are targets for TGF β 1 antagonists, such as binding compositions of the invention. The invention encompasses treatment based on a combination composition, which includes administering a combination composition to an animal, preferably a mammal, more preferably a primate (e.g., a human), to modulate, treat, inhibit, affect or ameliorate one or more of the diseases, disorders or conditions disclosed herein. For example, while not being limited by theory, antibodies specific for human TGF have been shown to be effective in animal models of the treatment of TGF receptor overexpression (TGFR) diseases. Antisera to TGF β have been shown to be effective in the treatment of glomerulonephritis (Border et al 1990Nature 346: 371-4), pulmonary fibrosis (Gir et al 1993Thorax 48: 959-66). Thus, the novel binding compositions improved by features of the invention will be effective in ameliorating conditions, states or diseases such as TGF β 1-associated fibrotic diseases and conditions.

Recombinant and/or isolated binding compositions of the invention, such as antibodies, can be purified and administered to a subject for treatment. These agents may be used in combination with other active or inert ingredients, such as conventional pharmaceutically acceptable carriers or diluents, e.g. immunological adjuvants, as well as physiologically harmless stabilizers and excipients. These combination products may be sterile filtered, formulated in dosage form, e.g., lyophilized and stored in a dosage bottle or in a stable aqueous preparation. The invention also contemplates the use of antibodies or binding fragments thereof, including forms that are not complement-binding.

Another therapeutic means encompassed by the present invention includes direct administration of the agent, formulation or composition to a subject using any conventional administration technique, such as, but not limited to, local injection, inhalation or systemic administration. The invention also provides a pharmaceutical pack or kit (i.e., a kit or pack) comprising one or more containers filled with one or more ingredients of the compositions of the invention and instructions, such as processing instructions (typically in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products). Methods of administration include intravenous, intraperitoneal or intramuscular administration, transdermal diffusion, and the like. Pharmaceutically acceptable carriers include water, salts, buffers andMerck Index，Merck&other compounds described by co, Rahway, NJ.

Other dysplastic conditions encompassed herein are known in cell types that have been shown to possess TGF β 1mRNA by Northern blot analysis (see, e.g., Berkow (eds.), The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn et al Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; and Rich (eds.) Clinical Immunology; Principles and Practice, Mosby, St.Louis (New edition) and infra). Developmental or functional abnormalities, such as those of the neurological, immunological, or hematopoietic systems, cause significant medical abnormalities and conditions that may be prevented or treated using the compositions provided herein.

Another therapeutic means encompassed by the present invention includes the direct administration of the agent, formulation or composition to the subject using conventional administration techniques such as, but not limited to, local injection, inhalation or systemic administration. The agents, formulations, or compositions contained may be targeted using any of the methods described herein or known in the art. The actual dosage of an agent, formulation or composition that modulates a disease, disorder, condition, syndrome, etc., depends on many factors, including the size and health of the organism, however, one of ordinary skill in the art can determine the Clinical dosage using the methods and techniques described in the following teachings (see, e.g., Spilker (1984) Guide Clinical Studies and development Protocols, Raven Press Books, Ltd., New York, pages 7-13, pages 54-60; Spilker (1991) Guide to Clinical Trials, Raven Press, Ltd., New York, pages 93-101; Craig and Stitzel (eds. 1986) model Pharmacology, 2 nd edition, Little, Brown and Co., Boston, pages 127-33; Speight (eds. 1987) average's drive: Principles and action of Clinical Trials, pages 3, Williams, 56, Balancies & 855, pages 858; Verlag, pages 52; William & 858; William & 52; William et al; pages 42-5,206,344; spring et al; Verlag, publication No. 18; publication No. 11; Green Press, York, pages 3, William & 858; William & 2000, pages 52; Yirk, Tailie, York, pages). Typically, a final concentration of about 0.5fg/ml to 500ug/ml, inclusive, is administered daily to an adult human in any pharmaceutically acceptable carrier. Furthermore, animal testing provides a reliable guide for the determination of therapeutically effective dosages for humans. Interspecies Scaling of effective doses is carried out according to principles known in The art (see, e.g., Mordenti and Chappell (1989) "The Use of interactions screening in affinity assays," in affinity assays and New drug development; Yacobi et al (eds.) Pergamon Press, NY).

Effective doses may also be extrapolated from dose response curves from in vitro or animal model test systems. For example, for antibodies, the dosage is typically 0.1mg/kg to 100mg/kg of the body weight of the recipient. The dosage is preferably 0.1mg/kg to 20mg/kg of the body weight of the recipient, more preferably 1mg/kg to 10mg/kg of the body weight of the recipient. Typically, a homologously specific antibody has a longer half-life than a heterologously specific antibody (e.g., a human antibody persists longer in a human host than an antibody derived from another species (e.g., a mouse), possibly due to the host's immune response to the foreign composition). Thus, if the antibody is administered to a human subject, it is generally possible to use lower doses of human antibody and lower frequency of administration. Furthermore, by modifying (e.g., lipidizing) to increase uptake and tissue penetration (e.g., in the brain), the dosage and frequency of administration of the antibodies of the invention can be reduced.

The invention also provides a pharmaceutical package or kit containing one or more containers filled with one or more ingredients of the compositions of the invention and instructions, such as instructions for processing (typically, in a manner directed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products).

The amount of agent required for effective treatment depends on many factors, including the mode of administration, the target site, the physiological state of the patient, and the other drugs being administered. Thus, the therapeutic dose should be titrated to optimize safety and efficacy. Generally, the dosages of these agents used in vitro may provide useful guidance for the amount administered in situ. Animal testing of effective dosages for the treatment of particular disorders can also provide predictive guidance for dosages for human use. In a system such as Gilman et al (eds) (1990) Goodman and Gilman's: the Pharmacological Bases of Therapeutics (8 th edition) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17 th edition) Mack publishing Co., Easton, Pa. Methods of administration are discussed therein and below, such as for oral, intravenous, intraperitoneal or intramuscular administration, transdermal diffusion, and the like. Pharmaceutically acceptable carriers include water, salts, buffers and other compounds such as those described in Merck Index, Merck & co, Rahway, NJ. With a suitable carrier, a dosage range of less than 1mM concentration is ordinarily contemplated. Typically less than 10 μ M concentration, often less than 100nM concentration, preferably less than 10pM concentration (picomolar), most preferably less than 1fM concentration (femtomolar). Sustained release formulations or sustained release containers are typically used for continuous administration.

The binding compositions may be administered directly to the host to be treated, or depending on the size of the compounds, it may be desirable to conjugate them to a carrier protein such as ovalbumin or serum albumin prior to administration. The therapeutic formulation may be administered in any conventional dosage form. Although it is possible to administer the active ingredient alone, it is preferably presented as a pharmaceutical formulation. The formulations will generally comprise at least one active ingredient as defined herein and one or more carriers acceptable therefor. Each carrier should be 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 and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the 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 (8 th edition) Pergamon Press; and (1990) Remington's pharmaceutical Sciences (17 th edition) Mack Publishing Co., Easton, Pa; avis et al (eds) (1993) Pharmaceutical Dosage Forms: pareteral medical dekker, NY; lieberman, et al (eds) (1990) Pharmaceutical DosageForms: tablets Dekker, NY; and Lieberman et al (eds.) (1990) Pharmaceutical Dosage Forms: disperse Systems Dekker, NY. The treatment of the present invention may be used in combination or association with other therapeutic agents, such as ACE inhibitors.

The invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a composition of the invention, e.g., in a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means a carrier approved by a regulatory/regulatory agency of the federal or a state government of the united states, or listed in the U.S. pharmacopeia or other pharmacopeia, which is generally recognized by those skilled in the art for use in animals, such as mammals, and particularly in primates, such as human primates.

The term "carrier" as used herein refers to a diluent, adjuvant, excipient, or carrier with which the composition of the invention is administered. Pharmaceutical carriers are typically sterile liquids, such as water or oils (including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Generally, sterile water is preferred as a carrier when the pharmaceutical composition is to be administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions.

Suitable pharmaceutical excipients include, for example, but are not limited to, 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. The compositions of the present invention may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired.

The compositions of the present invention may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like, or may be formulated as suppositories (with conventional binders and/or carriers, such as triglycerides). Other examples of suitable drug carriers are e.w. Martin new editionRemington’s Pharmaceutical SciencesAs described in (1). Such formulations contain a therapeutically effective amount of a composition of the invention, preferably in purified form, and an appropriate amount of carrier to provide for proper administration to the subject. Traditionally, the formulation will be adapted for the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with conventional methods into a pharmaceutical composition suitable for intravenous administration to a human. Typically, the composition for intravenous administration is a sterile isotonic buffered solution. Where desired, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to promote comfort at the injection site. The ingredients are typically provided separately or mixed in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a sealed container (e.g., an ampoule or sachet labeled with the amount of active agent). When the composition is administered by infusion, it can be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline. When administered by injection, sterile water or saline for injection in an ampoule may be provided to mix the various ingredients prior to administration.

The compositions of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include, but are not limited to, anionic salts (e.g., salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like) and cationic salts (e.g., salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylethanolamine, histidine, procaine, and the like).

Fibrotic diseases, disorders or conditions

Binding compositions, such as antibodies, are useful for the treatment of conditions associated with fibrotic diseases. Accumulation of extracellular matrix components or replacement of normal cellular material in various cells, tissues and organs by extracellular matrix components can lead to disease-causing fibrosis. Cumulative fibrosis is fatal and can lead to the eventual failure of many organs, such as the kidneys. Both preclinical and clinical data suggest that TGF β 1 is a major contributor to matrix protein deposition in interstitial fibrosis, and is involved in the initiation and progression of many relevant fibrotic disease states, including renal fibrosis, which is common in various forms of chronic kidney disease (CRD). The degree of renal fibrosis is positively correlated with the development of chronic renal failure (CRF, also known as end-stage renal disease (ESRFD)), which can lead to long-term dialysis or kidney transplantation or death. TGF β 1 is the most experimentally relevant cytokine and has been implicated in the progression of this type of fibrosis in human and animal studies.

The profound effects of TGF β 1 on extracellular matrix, including stimulating the synthesis of extracellular matrix components and inhibiting their degradation, have been the subject of many reviews (see, e.g., Rocco & Ziyadeh 1991in environmental Issues in physiology v.23, "Hormons, autocoids and the kit" eds Jay Stein, Churchill Livingston, New York 391 page 410; Roberts et al 1988Rec. prog. Hornone Res.44: 157-97). TGF-beta 1 can induce extracellular matrix accumulation in a variety of synergistic ways, for example, TGF-beta 1 stimulates the expression of mRNA and protein production of major extracellular matrix components including collagen type I, collagen type IV, fibronectin and laminin (Sharma and Ziyadeh 1997Semin Nephrol 17: 80-92). Meanwhile, TGF-. beta.1 prevents degradation of extracellular matrix by inhibiting the production of proteases that digest matrix (e.g., plasminogen activator, collagenase, elastase, and stromelysin) and by activating inhibitors of these proteases (e.g., metalloprotease and inhibitor 1 of plasminogen activator) (Sharma and Ziyadeh 1995Kidney Int 51: S34-6). TGF-beta 1 also up-regulates cell surface receptors for synproteins and ECM, thereby enhancing the ability of cells to bind specific ECM proteins (Heino et al 1989J Biol Chem 264: 380-8). In addition, TGF β 1 also has strong chemotactic properties, attracting ECM cells such as fibroblasts and phagocytes (Reibman et al 1991PNAS 88: 6805-9). Moreover, TGF β 1 has the specific ability to induce its own expression, potentially augmenting the progression of aberrant fibrosis (Kim et al 1990Mol Cell Biol 10: 1492-7).

TGF β 1 is implicated in the following diseases, syndromes and/or conditions:

● nephropathy, such as chronic kidney disease (CRD), chronic kidney failure (CRF), End Stage Renal Disease (ESRD), Glomerulonephritis (GN) (including mesangial hyperplasia GN, capillary mesangial GN, membranous GN, focal nodes GN, immune GN and crescent GN), glomerulosclerosis, nephrosclerosis, membranous nephropathy, immunoglobulin A (IgA) nephropathy, interstitial Renal fibrosis, focal-node glomerulosclerosis, Renal fibrosis in cyclosporine-receiving transplant patients, chronic kidney transplant rejection, HIV-related nephropathy, Renal cell hypersensitivity, tubulointerstitial fibrosis with chronic kidney disease, including Renal failure derived from urinary obstruction or analgesic or secondary to a segment of acute kidney failure (such as acute kidney failure derived from Renal ischemia) (Spurgel et al am. J Physiol Rephysiol 288: 568-F577, 2005); renal thrombotic microangiopathy associated with, e.g., glomerular endothelial cell injury or other microvascular endothelial cell injury, e.g., renal thrombotic microangiopathy associated with pre-eclampsia, endotoxemia, radiation exposure; renovascularis, focal necrotic glomerulonephritis, diabetic nephropathy [ TGF β 1 is associated with CRF by a complex and diverse event affecting most kidney cells (Bottinger, 2002, j.am. soc. nephrol.13, 2600). These events ultimately lead to tubulointerstitial fibrosis and glomerulosclerosis, leading to loss of nephron function, and ultimately to chronic renal failure. Among the three isoforms of TGF β, TGF β 1 is dominant in mediating the development of chronic kidney disease not only because TGF β 1 is the most preferentially expressed isoform, but also because TGF β 2 and TGF β 3 mediate their effects by up-regulating the expression of TGF β 1 (Yu, 2003kid. int.64: 844). Both in vivo and in vitro studies suggest a role for TGF β 1in the pathogenesis of diabetic nephropathy, including complications associated with type I and type II diabetes, such as glomerulosclerosis and tubulointerstitial fibrosis (Ziyadeh 1998Cur. practice. Med. 1: 87-9). Antisera to TGF β are effective in the treatment of glomerulosclerosis (Border et al 1990Nature 346: 371-4) ]; fibrotic nephropathy [ TGF β 1 mediates renal cell hypertrophy, another feature of diabetic nephropathy caused by interference with normal regulation of the cell cycle by the induction of cyclin-dependent kinase inhibitors such as p27Kip1 and p21Cip2 (Wolf and Ziyadeh 1999Kidney Int 56: 393-405). These inhibitors are also increased by high sugar and diabetic conditions (Wolf et al 2001Am J Pathol; 158: 1091-1100; Wolf et al 1999 diabetes 42: 1425-32; Wolf et al 1997Am J Physiol 273: F348-56). They inhibit the activity of cyclin-dependent kinases, mainly cyclin-dependent kinase 2/cyclin E kinase (Liu and Preisig 1999Am J Physiol 277: F186-94), thus inhibiting the phosphorylation of ocular oncoproteins and repressing the cells at late G1. The cell enters a stage where there is protein synthesis without DNA replication, and undergoes cellular hypertrophy. Thus, TGF β 1 causes alterations in the pathophysiological features that translate into diabetic nephropathy at the cellular level ]; experimental diabetic nephropathy; it is not surprising that TGF β 1 has profound effects on the kidney, since the structural and filtering properties of glomeruli are largely determined by the extracellular matrix composition of the vascular mesangium and the glomerular membrane. TGF β 1 mediates pathological changes in diabetic nephropathy (Ziyadeh 1998Cur Prac Med 1: 87-89). The accumulation of mesangial matrix is a clear and major pathological feature of proliferative glomerulonephritis (Border et al 1990Kidney int.37: 689-. Increased levels of TGF β 1in human diabetic glomerulosclerosis (end stage renal disease) (Yamamoto et al 1993Proc. Natl. Acad. Sci.90: 1814-one 1818). TGF β 1 has been found to be an important mediator of renal fibrosis development in a number of animal models (Phan et al 1990Kidney Int.37: 426; Okuda et al 1990 J.Clin.invest.86: 453). Inhibition of experimentally induced glomerulonephritis has been demonstrated in rats using antisera to TGF β (Border et al 1990Nature 346: 371) and extracellular matrix proteins, i.e.decorin (decorin) which binds TGF β 1 (Border et al 1992Nature 360: 361-363; see also e.g.Border and Noble 1994 N.Engl.J.Med.331: 1286-92; Border et al 1989Semin. Nephrol.9: 307-17; Han et al 2000Am J Physiol Renal Physiol 278: F628-34; and Ziyadeh, et al 2000PNAS 97: 8015-20). Accordingly, the binding compositions of the present invention are useful for treating, ameliorating, modulating, diagnosing and/or inhibiting the above-mentioned diseases, disorders, syndromes and/or conditions.

● liver diseases-such as cirrhosis, liver fibrosis [ liver fibrosis is a common response to necrosis or injury of liver cells and can be induced by a wide variety of agents, such as any process that disrupts hepatic homeostasis (especially inflammation, toxin injury or altered liver blood flow) and liver infections (viruses, bacteria, fungi and parasites). Numerous storage disorders caused by congenital metabolic errors are often associated with fibrosis, including lipid abnormalities (Gaucher disease), glycogen storage diseases (especially types III, IV, VI, IX and X), 1-insulin resistance deficiency, storage of foreign substances, as seen in iron overload syndrome (hemochromatosis) and copper storage disease (Wilson disease); accumulation of toxic metabolites (such as in tyrosinemia, fructosamia and galactosemia) and peroxisome disorders (Zellweger syndrome). Many chemicals and drugs can cause fibrosis, particularly ethanol, methotrexate, isoniazid, phentermine, methyldopa, chlorpromazine, tolbutamide, and amiodarone. Fibrosis can be caused by disorders of the liver circulation (e.g., chronic heart failure, buddy-Chiari syndrome, venous occlusive disease, portal thrombosis) and chronic obstructive bile flow. Finally, congenital liver fibrosis is an autosomal invisible malformation.

Most people worldwide have chronic viral hepatitis or steatohepatitis associated with ethanol or obesity, but other fibrosis-inducing injuries include, for example, parasitic diseases (e.g., schistosomiasis), autoimmune attacks of hepatocytes or biliary epithelia, neonatal liver disease, metabolic disorders including Wilson hemochromatosis and various storage diseases, chronic inflammatory conditions (e.g., sarcoidosis), drug toxicity (e.g., methotrexate or vitamin a hyperactivity), and congenital or acquired vascular disorders.

Hepatic astrocytes (HSCs) are an important cell source of the ECM in liver fibrosis (Li and Friedman 1999J. gastroenterol. hepatol.14: 618-33). HSCs are located in the perisinus space of disia, which separates hepatocytes from sinus-like endothelium. As described, liver injury usually activates quiescent HSCs, leading to their subsequent proliferation and activation. Activated HSCs then undergo phenotypic transdifferentiation into contractile Myofibroblasts (MFBs) that express smooth muscle actin and an excess of ECM molecules seen in liver fibrosis. The transdifferentiation of HSCs into MFBs is due to the overexpression of TGF β 1, a major regulator of hepatic fibrosis in response to stress and injury (Gressner et al 2002Front biosci.7: d 793-807). TGF β 1 is the strongest fibrogenesis-inducing factor known in the effector cells of liver fibrosis (Schuppan et al 2003Cell Death Differ.10Supl 1: S59-67). Thus, the level of active TGF-beta 1 is elevated in the tissues and serum of liver fibrosis, and overexpression of TGF-beta 1in transgenic mice and use of exogenous TGF-beta 1 has been shown to induce organ fibrosis (Kanzler et al 1999am. J. physiol.276: G1059-68; Sanderson et al 1995PNAS 2572-76). Furthermore, experimental fibrosis can be inhibited by anti-TGF-beta 1 therapy, such as with neutralizing antibodies or soluble TGF receptors (George et al 1999PNAS 12719-24; Qi et al 1999PNAS 2345-49). The expression of TGF β 1 seen in activated HSC/MFB, the efficacy of upregulation of ECM expression by TGF β 1, and expression of TGF receptors on HSCs have been widely accepted models in which the continuous autocrine/paracrine stimulation of HSC/MFB activated by TGF β 1 is the primary fibrogenic response that promotes organ fibrosis. Thus, a reduction in TGF β 1 is predicted to reduce the incidence of liver fibrosis (Wu and Zern 2000J. gastroenterol.35: 665-72).

● pulmonary diseases-such as pulmonary fibrosis (Giri et al Thorax 48, 959-966, 1993); idiopathic pulmonary fibrosis; adult respiratory distress syndrome; cryptogenic histopneumonia, Bronchiolitis Obliterans (BO), transplantation-related bronchiolitis obliterans (aiba); drug-induced lung disease; radiation-induced lung disease; local fibrosis around the respiratory tract in asthma and emphysema; hypersensitivity pneumonitis; cryptogenic tissue pneumonia (COP); chronic Obstructive Pulmonary Disease (COPD); acute Interstitial Pneumonia (AIP); asbestos stasis; interstitial pulmonary fibrosis associated with autoimmune disorders, such as systemic lupus erythematosus and scleroderma, chemical exposure or anaphylaxis; asthma; chronic neonatal lung disease (CLD) [ uncontrolled expression of TGF β 1 plays an important role in the pathology of the abnormal development of the airways of many chronic lung diseases including asthma, pulmonary fibrosis and chronic neonatal lung disease (CLD) (Holgate et al 2001Int Arch Allergy Immunol 1241-3: 253-8; Khalil et al 2001Thorax 5612: 907-15; Lecart et al 2000Biol Neonate: 217-23; Pulley et al 2001Hum Genet 1096: 623-7; Sagara et al 2002J Allergy Clin Immunol 1102: 249-54; Shepard, 2001Chest 1201 Suppl: 49S-53S; Strieter, 2001Chest 1201 p: 77S-85S; Toti et al Peditar pulmonary disease 241-8: 22-8). Overexpression of active TGF-. beta.1 on the mouse alveolar surface leads to a potent fibrotic response, whereas inhibition of TGF-. beta.1 with antibodies or decoys abolishes bleomycin-induced fibrosis (Kelly et al 2003Cur. pharm. Des.9: 3949). Furthermore, microarray analysis of all lung mRNA chips showed that most of the known TGF-beta 1-induced genes were up-regulated during experimentally induced fibrosis (Kaminski et al 2000PNAS 97: 1778-83) ].

● cardiovascular disease-atherosclerosis [ TGF β is associated with atherosclerosis, see e.g. t.a. mccaffrey: 2000 "TGF-. beta.s and TGF-. beta.receptors in Atherosclerosis" cytokines and Growth Factor Reviews 11: 103-114] ]; hypertrophic cardiac growth (Schultz et al 2002J Clin invest.109 (6): 787-96; Brand and Schneider 1995J.mol.cell Cardiol.27: 518); progressive systemic sclerosis [ parallels the increase in TGF β 1in aged vasculature is a significant increase in fibronectin levels (Li et al 1999). These changes undoubtedly affect the transition from a flexible pipe to a fiberized/stiff pipe ]; mechanically induced microvascular fibrosis; sarcoidosis; ventricular fibrosis; radiation-induced ischemic heart disease [ it has been reported that patients treated with radiation for Hodgkin's disease and breast cancer have a significantly increased risk of death due to ischemic heart disease. Certain cytokines and growth factors, such as TGF β 1, can stimulate radiation-induced endothelial proliferation, fibroblast proliferation, collagen deposition and fibrosis leading to exacerbated atherosclerotic lesions ]; arterial injury (Wolf et al 1994j. clin. invest.93, 1172-8); chronic Venous Insufficiency (CVI); restenosis following percutaneous transluminal coronary angioplasty or stenting (PTCA); Hermansky-Pudlak syndrome; polymyositis; scleroderma; dermatomyositis; eosinophilic fasciitis; hard spot disease, or hard spot disease associated with the occurrence of Raynaud's syndrome; perivascular fibrosis of the internal coronary vasculature of non-infarcted myocardium; injury-induced hyperplasia, such as restenosis; atherosclerosis fibrosis associated with collagen vascular disease TGF β 1 is a factor in the progressive thickening of arterial walls due to smooth muscle cell proliferation and extracellular matrix deposition in arteries following balloon angioplasty. Because of this thickening, the diameter of the restenosis artery can be reduced by 90%, since most of the reduction in diameter is due to extracellular matrix rather than smooth muscle cell bodies, and it is possible to enlarge the vessel by only 50% by reducing the deposition of large amounts of extracellular matrix. In intact porcine arteries transfected with the TGF β 1 gene in vivo, expression of the TGF β 1 gene is associated with both extracellular matrix synthesis and proliferation (Nabel et al 1993PNAS 90: 10759-63); histiocytosis (eosinophilic granuloma);

● Soft tissue fibrosis (Border and Ruoslahti 1992J Clin Inv 90: 1-7; Border and Noble 1994N Engl J Med 331: 1286-91); erectile dysfunction (Moreland, R.1998int J Impot Res 10: 113-20); rheumatoid arthritis (Wahl et al 1993J.Exp. medicine 177, 225-30).

● wound healing-skin scarring; burns; hypertrophy and scarring; keloid formation; conjunctival scarring (Cordeiro MF, 2003Clin Sci (Lond) 104 (2): 181-7) [ treatment of fetal wounds with different concentrations of TGF β 1 resulted in significant scarring of these wounds, suggesting that TGF β is directly involved in skin scarring (Sullivan et al 1995J Peditar Surg 30: 198-) -203). It has been shown that injection of neutralizing TGF β 1 antibody into the margins of a rat healing wound can inhibit scarring without affecting the rate of wound healing or the tensile strength of the wound. At the same time, angiogenesis is reduced, the number of macrophages and monocytes in the wound is reduced, and the amount of unorganized collagen fiber deposits in the scar tissue is reduced (Shah et al 1992Lancet 339: 213-4; Shah et al 1994J. Cell Science 107: 1137-57; Shah et al 1995J Cell science.108: 985-. Scar reduction response commonly occurs in mice wounds treated with antisense TGF β 1 (Choi et al 1996 ImmunoCelbiol 74: 144-50). Topical application of synthetic TGF-beta 1 antagonists reduces scarring of swine burn and excision surgical wounds and rabbit skin excision wounds (Huang, et al 2002FASEB J16: 1269-70; Werner and Grose 2003Physiol Rev 83 (3): 835-70).

● inflammatory diseases-Inflammatory Bowel Disease (IBD), such as Crohn's Disease (CD) and Ulcerative Colitis (UC) [ inflammatory bowel disease depends on the balance between inflammatory cytokines, in particular IFN-. gamma.and TGF-. beta.1 activity (Strober et al 1997Immunol.today 18: 61-4; Neurath et al 1996J.Exp.Med.183, 2605-16; Ludviksson et al 1997 J.Immunol.159; Boirivant et al 1998J.Exp.Med.188, 1929-39; Powre et al 1996J.Exp.Med.183, 2669-74). Immunologically mediated tissue damage in the gut is associated with increased production of inflammatory cytokines, which activate the transcription factor NF κ -B in a variety of different cell types; rheumatoid Arthritis (RA); synovial hyperplasia [ elevated levels of TGF β 1in the synovial fluid of human rheumatoid arthritis (Lotz et al 1990J. Immunol.144: 4189-94). Excess TGF β 1 forms a painful osteoarticular byproduct called osteophyte, synovial hyperplasia and inflammation in RA (Hamilton et al 1991PNAS 88: 7180-4). Inhibition of endogenous TGF-beta 1in a murine arthritis model can prevent osteophyte formation and impair cartilage repair, suggesting a role for TGF-beta 1in these pathological events (Scharstuhl et al 2002 immunol.169: 507-14) ];

● Cell proliferative diseases-numerous data demonstrate that TGF β 1 not only has transforming potential, but is also capable of driving malignant development, invasion and metastasis in vivo and in vitro (Derynck et al 2001nat. Genet29: 117-29; Cui et al 1996Cell 86: 531-42). Examples of hyperproliferative states, diseases, disorders, syndromes, and/or conditions include, but are not limited to, neoplasms of the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine system (e.g., adrenal, parathyroid, pituitary, testicle, ovary, thymus, or thyroid), eye, head, neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, spleen, chest, and urogenital system. Likewise, other hyperproliferative conditions include, but are not limited to, hyper-gammaglobulinemia, lymphoproliferative conditions, pathoproteinemia, purpura, sarcoidosis, hamartomas, Sezary syndrome, Waldenstron macroglobulinemia, Gaucher's syndrome, histiocytosis and other hyperproliferative states.

Despite TGF β 1 activity, tumor cells often exhibit this increase in growth factor production (Derynck et al 1987Cancer Res.47: 707-12; Dickson et al 1987PNAS 84: 837-41), and considerable evidence suggests that it may contribute to tumor promotion by affecting tumor cell invasion and changes in the tumor microenvironment. It is now clear that TGF β 1 can act as both an inhibitor of tumor and an important stimulator of tumor development, invasion and metastasis. TGF β can act directly on cancer cells to inhibit tumor growth early in tumorigenesis, such as when the tumor is still benign. However, as the tumor progresses, genetic and/or biochemical changes allow TGF β 1 to stimulate tumor development through its pleiotropic activity on the cancer cell itself and the non-malignant stromal cell type of the tumor. Stimulation of invasion and metastasis by TGF-beta 1 may be a more important clinical outcome than its tumor-inhibiting effect, since most human tumors retain a functional TGF-beta signaling pathway (Akhurst and Derynck 2001Trends Cell biol.11: S44-51).

There is ample evidence that overproduction of TGF β 1in cell proliferative diseases is associated with poor prognosis (Tssushima et al 1996Gastroenterology 110: 375-82; Adler et al 1999 J.Urol.161: 182-7). There are several types of cancer in which TGF β 1 produced by the tumor may be detrimental. MATLyLu rat prostate cancer cells (Steiner and Barrack 1992mol. Endocrinol 6: 15-25) and MCF-7 human breast cancer cells (Aretaga et al 1993CellGrowth and Differ.4: 193-201) became more tumorigenic and metastatic after transfection of a vector expressing mouse TGF β 1. TGF β 1 is associated with angiogenesis, metastasis and poor prognosis in human Prostate and advanced gastric cancers (Wikstrom, P. et al (1998) Prostate 37: 19-29; Saito, H et al (1999) Cancer 86: 1455-62). In breast Cancer, poor prognosis is associated with increased levels of TGF β 1 (Dickson et al 1987PNAS 84: 837-41; Kasid et al 1987; Cancer Res.47: 5733-8; Daly et al 1990J. cell biochem.43: 199-211; Barrett-Lee et al 1990Br. J Cancer 61: 612-7; King et al 1989J. Steroid biochem.34: 133-8; Welch et al 1990PNAS 87: 7678-82; Walker et al 1992Eur. J. Cancer 238: 641-4), and tamoxifen treatment (Butta et al 1992Cancer Res.52: 4261-4) induced TGF β 1 is associated with failure to treat breast Cancer with tamoxifen (Thompson et al 1991Br. J. Cancer 63: 609-14). anti-TGF β 1 antibody inhibits the growth of human breast cancer cells MDA-231 in athymic mice (artemiga et al 1993J. Clin. invest.92: 2569-76), a treatment associated with increased splenic natural killer cell activity. CHO cells transfected with latent TGF-. beta.1 also showed decreased NK activity and increased tumor growth in nude mice (Wallick et al 1990 J.exp.Med.172: 1777-84). Thus, TGF β 1 secreted by breast tumors may cause endocrine immunosuppression. High plasma concentrations of TGF β 1 indicate a poor prognosis for patients with advanced breast cancer (Anscher et al 1993 N.Engl.J.Med.328: 1592-8). Patients with high TGF β 1 circulation prior to high dose chemotherapy and autologous bone marrow transplantation are at high risk for liver vascular obstructive disease (15-50% of all patients have a high mortality rate of up to 50%) and congenital interstitial pneumonia (40-60% of all patients). These findings indicate that an increase in TGF β 1 plasma levels can identify patients at risk, and that lowering TGF β 1 can reduce morbidity in breast cancer patients and mortality from these treatments.

Many malignant cells secrete TGF β 1, suggesting that the production of TGF β 1 can provide a mechanism for tumor cells to evade host immune surveillance. The establishment of a subpopulation of leukocytes that disrupt TGF β 1 signaling in a tumor-bearing host provides a potential means for immunotherapy of cancer. Transgenic animal models with disruption of TGF β 1 signaling in T cells can eradicate the commonly lethal TGF β 1-overexpressing lymphoid tumor EL4(Gorelik and Flavell, 2001Nature Medicine 7 (10): 1118-22). Downregulation of TGF β 1 secretion in tumour cells leads to restoration of immunogenicity in the host, while T cells insensitive to TGF β 1 lead to accelerated differentiation and autoimmunity, which elements may be necessary to tolerate the host against self-antigen expressing tumours. The immunosuppressive effects of TGF-beta 1 are also associated with a lower than expected immune response in a subset of HIV patients based on CD4/CD8T cell counts (Garba et al 2002J. immunology 168: 2247-54). TGF β 1 neutralizing antibodies can reverse this effect in culture, implying that other TGF β 1 antibody inhibitors have the immunosuppressive effect of reversing the appearance in this HIV patient subpopulation.

The dual tumor-inhibiting/tumor-promoting effects of TGF β 1 are clearly demonstrated in keratinocyte transgenic systems overexpressing TGF β 1. Although transgenic systems are more resistant to the development of benign skin lesions, the rate of metastatic transformation is greatly increased in transgenic systems (Cui et al 1996Cell86 (4): 531-42). TGF β 1 production by malignant cells in primary tumors increases as the tumor progresses to an advanced stage. Studies with many major epithelial cancers suggest that increased production of TGF β 1 by human cancers is a relatively advanced event in tumor development. Furthermore, tumor-associated TGF β 1 also provides a selective advantage for tumor cells and promotes tumor development. The effect of TGF β 1 on cell/cell and cell/matrix interactions leads to greater propensity for invasion and metastasis. Tumor-associated TGF β 1 may allow tumor cells to evade immune surveillance, since TGF β 1 is a potent inhibitor of activated lymphocyte clonal proliferation as seen in patients with aggressive brain or breast tumors (artemiga et al 1993j. clin. invest.92: 2569-76). TGF β 1 inhibits the production of angiostatin. Forms of cancer therapy, such as radiation therapy and chemotherapy, induce the production of activated TGF β 1in tumors, thereby selecting the growth of malignant cells that are resistant to the growth inhibitory effects of TGF β 1. Thus, these anti-cancer treatments increase the risk and accelerate the development of tumor progression. Agents that prevent TGF β 1 mediated signal transduction may be very effective therapeutic strategies in such situations. Resistance of tumor cells to TGF β 1 can counteract most of the cytotoxic effects of radiation therapy and chemotherapy, and treatment-dependent activation of TGF β 1in the matrix is even detrimental as it can make the microenvironment more conducive to tumor development and contribute to tissue damage leading to fibrosis. Prostate cancer cells express high levels of TGF β 1, which appears to enhance prostate cancer growth and metastasis by stimulating angiogenesis and inhibiting immune responses against tumor cells. Prostate cancer cells typically lose their TGF β receptor and acquire resistance to the antiproliferative and pro-apoptotic effects of TGF β 1. Thus, high expression of TGF β 1 and loss of TGF β receptor expression are associated with a particularly unfavorable prognosis for human prostate cancer patients (Wikstrom et al 2000Scand J Urol Nephrol.34 (2): 85-94). In hepatocellular carcinoma (HCC), the induction of TGF-beta 1 expression is accompanied by a loss of TGF-beta 1 receptor expression in liver foci and nodules, altering hepatocytes to escape TGF-beta 1-induced apoptosis, thereby conferring growth advantage on the altered hepatocytes during hepatogenesis (Lim 2003Mech Ageing Dev.124 (5): 697-708). In squamous cell carcinoma of the head and neck, early TGF β 1 overexpression provides a favorable microenvironment for tumors (Lu et al 2004Cancer Res 64 (13): 4405-10); the binding compositions can modulate, ameliorate, treat, prevent and/or diagnose a hyperproliferative disease, condition, disorder or syndrome (such as a neoplasm) by interacting directly or indirectly. For example, the hyperproliferative state is modulated by preventing inhibition of cell proliferation (e.g., TGF β 1 inhibits proliferation and functional differentiation of T lymphocytes, lymphokine-activated killer cells, NK cells, neutrophils, macrophages, and B cells (Letterio and Roberts 1998 "Regulation of the immunophenocarpy by TGF Beta" an ren Immunol 16: 137-61)), or by increasing an immune response (e.g., by increasing the immunogenicity of a protein involved in the hyperproliferative condition), or by causing proliferation, differentiation, or mobilization of a particular cell type (e.g., T cells) Such as, but not limited to, transfection, electroporation, cell microinjection, or with a vector (e.g., liposome, lipofection, or naked polynucleotide). "cellular proliferative condition" refers to any human or animal disease, syndrome, disorder, condition or state, affecting any cell, tissue, any site, or any combination of organs, tissues or body part, characterized by one or more local abnormal proliferations of cells, cell groups or tissues, whether benign or malignant ].

● neurological diseases-neurological scars (Logan et al, 1994Eur. J. Neurosci.6: 355-63), cerebrovascular abnormalities, Alzheimer's disease (Masliah et al, 2001neurochemistry International 39: 393 400) [ positive correlation between the mRNA level of TGF β 1in the mid-forebrain gyrus and the relative degree of cerebrovascular amyloid deposits in this brain region, suggesting a possible role for TGF β 1in human cerebrovascular abnormalities. Transgenic mice overexpressing TGF β 1in astrocytes develop Alzheimer' S disease-like cerebrovascular abnormalities including perivascular astrocytosis, thickening of microvascular basement membranes, and accumulation of thioflavin S-positive amyloid peptide without substantial degeneration. Long-term overexpression of TGF β 1in the brain leads to structural and functional impairment, reminiscent of the situation in AD cases of amyloid angiopathy (Buckwalter et al 2002Ann N Y Acad Sci.977: 87-95) ]; proliferative retinopathy (Chaturvedi et al 2002Diabetes Care 25: 2320-7); other ocular diseases associated with conditions of fibroproliferative associated with TGF β 1 overproduction include retinal reattachment surgery, cataract extraction, intraocular lens transplantation, and proliferative vitreoretinopathy associated with glaucoma exclusion surgery.

● hematopoietic state/condition- [ inhibitory effects in the naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis usually predominate (see e.g. Rastinejad et al Cell 56565632355 (1989)). When new angiogenesis occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development and the female reproductive process, angiogenesis is tightly regulated and spatially and temporally delimited. In pathological angiogenesis, such as during solid tumor formation, these regulations fail because of the continued progression of neoplastic and non-neoplastic diseases, and deregulated angiogenesis can become pathologic. Many serious diseases are dominated by aberrant neovascularization, including, for example, solid tumor growth and metastasis, arthritis, some types of eye disease, and psoriasis, as described by Moses et al 1991 Biotech.9630-634; folkman et al 1995n.engl.j.med., 333: 1757 to 63; auerbach et al 1985J. Microvasc. Res.29: 401-11; folkman, "Advances in cancer Research," Klein, Weinhouse, editors, Academic Press, New York, pp.175-203 (1985); patz, 1982am.j. opthalmol.94: 715-43; and Folkman et al 1983Science 221: 719-25. Tumor angiogenesis is critical to tumor growth and invasion, as blood vessels transport nutrients and oxygen to tumor cells and allow tumor cells to invade the blood system, resulting in metastasis. TGF-beta 1 acts as an effective inducer of angiogenesis in several assays (Roberts et al 1986PNAS 83: 4167-71; Madri et al 1988J.cell biol.106: 1375-84; Yang & Moses 1990J.cell biol.111: 731-74; Gajduek et al 1993J.cell. physiol.157: 133-44; Choi and Ballermann 1995 J.biol.chem.270: 21144-50), whereas TGF-signaling component deficient mouse models indicate the importance of TGF-beta 1in normal vascular development. Moreover, TGF β 1 and its receptors ALK-5 and ALK-1 may be involved in the vascular maturation phase of angiogenesis (see, e.g., Bull Acad Natl Med.2000; 184 (3): 537-44). Under a number of pathological conditions, angiogenesis contributes to the disease state, and important data, such as accumulation, suggest that the formation of solid tumors is dependent on angiogenesis (see, e.g., Folkman and Klagsbrun1987Science 235: 442-7). In another embodiment of the invention, administration of the combination composition is provided to treat, ameliorate, regulate, diagnose and/or inhibit a disease, disorder, syndrome and/or condition associated with cardiovascular production or hematopoietic cell expansion. Malignant and metastatic conditions that can be affected in the intended manner using the conjugate compositions include, but are not limited to, malignant tumors, solid tumors, and other cancers as described herein or known in the art (for reviews of such disorders, syndromes, see, e.g., fisherman et al, Medicine, new edition, j.b. lippincott co., philiadelphia 2002). For example, cancers that can be affected using the compositions of the invention include, but are not limited to, solid tumors (including prostate, lung, breast, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer), primary and metastatic tumors, melanoma, glioblastoma, Kaposi sarcoma, leiomyosarcoma, non-small cell lung cancer, colorectal cancer, advanced malignancies, and hematologic tumors such as leukemia.

● infection-sepsis [ TGF. beta.1 dramatically alters the course of mice infected with Leishmania. Genetically resistant mice become susceptible to leishmania infection after administration of TGF β 1. TGF-1 can exacerbate the disease, while TGF-1 antibodies can stop the disease progression (Barral-Netto et al 1992Science 257: 545-7) ].

● alopecia-alopecia; alopecia [ neutralizing anti-TGF β 1 antibodies can reverse androgen-induced growth inhibition of keratinocytes in a dose-dependent manner, suggesting that androgen-induced TGF β 1 from bald skin papilla cells mediates hair growth inhibition in androgenic alopecia and thus plays a role in human model alopecia (Inui et al 2002FASEB j.16 (14): 1967-9) ]; accordingly, the present invention provides a method for ameliorating, modulating, treating, preventing and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to a subject in need thereof an effective amount of a binding composition.

The broad scope of the invention can best be understood by reference to the following examples. These examples are not intended to limit the invention to the specific embodiments.

Examples

General procedure

Many of the standard procedures described herein are described, for example, in Sambrook et al (2001)Molecular Cloning，A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; and its associated website (www.MolecularCloning.com); ausubel et alBiologyGreene Publishing Associates, Brooklyn, NY; or Ausubel et al (1987 and supplements)Current Protocols in Molecular BiologyWiley/Greene，NY；Bartlett & Stirling(2003) PCR Protocols：Methods in Molecular Biology Vol.226Humana Press, NJ, is described or referenced. Protein purification methods include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and the like; see, e.g., Ausubel et al (1987 and periodical supplementations); deutscher (1990) "Guide to protein purificationMethods in EnzymologyVolume 182, and other volumes of this series: coligan et al (1995 and supplement)Current Protocols in Protein Science JohnWiley&Sons, New York, NY; matsudaira (eds) (1993)A Practical Guide to Protein and Peptide Purification for MicrosequencingAcademic Press, San Diego, CA; and the manufacturer's literature on the use of protein purification products, e.g. Pharmacia, Piscataway, NJ, or Bio-Rad, Richmond, CA. In combination with recombinant technology allows for fusion to a suitable segment (epitope tag), such as the FLAG sequence or a fusible equivalent, such as a sequence cleavable by a protease. See, e.g., Hochuli (1989)Chemische Industrie12: 69-70 parts of; hochuli (1990) "Purification of Recombinant Proteins with Metal chemical Absorbent" in Setlow (eds.)Genetic Engineering，Principle and Methods12: 87-98, Plenum Press, NY; and Crowe et al (1992)QIAexpress：The High Level Expression and Protein Purification SystemQUIAGEN，Inc.，Chatsworth，CA。

In Hertzenberg et al (eds.) 5 th edition 1996Weir′s Handbook of Experimental ImmunologyVolumes 1-4, Blackwell Science; bierer et al eds. (2004)Current Protocols in ImmunologyWiley/Greene, NY; andMethods in Enzymologyand (3) rolling: standard immunological techniques are described in 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162 and 163Elsevier, usa.

Example 1: construction and screening of Fab fragments Using CDR and human frameworks

A library of CDRs with single mutations in the variable regions of the antibody was characterized and synthesized using standard methods (see, e.g., Wu et al 1998PNAS 95: 6037-42). Libraries containing antibody light and heavy chain genes composed of human constant and variable region framework sequences as described herein together with the CDR sequences of the invention were constructed using the phage M13 expression vector. In some cases, the target CDR is deleted prior to nucleotide annealing. Oligonucleotide synthesis was performed using codon-based mutagenesis to generate the CDR sequences of the invention.

The library was initially screened by capture screening (lift) to identify the highest affinity variants. The capture screening method (Watkins, 2002Methods mol. biol. 178: 187-93) is known in the art and described in WO/0164751 and US 2002/0098189. Subsequently, the desired clones were titrated on immobilized antigen in ELISA and the cell proliferative capacity assay described herein for further characterization.After such screening, the dissociation constant (K) of the objective clone was determined_d) Binding Rate (K)_on) And dissociation Rate (K)_off)。

To identify potential antibody binding compositions comprising the present embedded donor CDRs, synthetic CDR libraries are inserted onto the deletion template as described herein or known in the art. The library of mutagenic oligonucleotides was used to replace specific CDRs using standard mutagenesis techniques (Kunkel, 1985PNAS 82: 488-92). Typically, the CDRs are placed in frame using the system defined by Kabat, except for CDRH1, where CDRH1 is the sum of Kabat and Chothia definitions. The mutagenic oligonucleotides were annealed to uridylated phage templates lacking the corresponding CDRs.

Annealing is carried out by incubating the reaction at 85 ℃ for 5 minutes and then slowly cooling to 20 ℃ for 45 minutes. The annealed samples were placed on ice, T4DNA polymerase and T4DNA ligase were added to produce double-stranded DNA, and the reaction was incubated at 4 ℃ for 5 minutes and then at 37 ℃ for 90 minutes. The reaction was extracted with phenol, ethanol precipitated, and the resulting DNA was electroporated into DH10B cells. XLOLR cells were added to the reaction to allow phage amplification, followed by plating with the library. Phage stocks were prepared by adding 8ml of growth medium to the plate followed by incubation at 4 ℃ for a minimum of 4 hours. The phage-containing medium was harvested, clarified by centrifugation, and added with sodium azide (0.02%) as preservative.

Used in Watkins et al 1998Anal Biochem 256: 169 to 77; and Watkins, 2002Methods mol. biol., 178: plaque screening the library was initially screened as described by 187-93. Subsequently, the filters containing the fabs expressed by the different plaques bound to the immobilized anti-human kappa antibody were incubated with biotinylated TGF β 1 (biotin-TGF β 1), Neutravidin-alkaline phosphatase (NA-AP), during which they were briefly washed. Clones of interest were sequenced and further characterized by ELISA. ELISA was typically coated with 0.4ug/ml TGF β 1, TGF β 2 or TGF β 3 overnight at 4 ℃ in Coster 3366 microplates. The plate was then washed twice before 100ul of blocking solution (10mg/ml BSA wash) was added to each well. Dilutions of Fab were incubated in coated wells for 1.5 hours at 22 ℃. After washing, anti-human kappa-alkaline phosphatase conjugate was added and incubated at 22 ℃ for 1 hour. After extensive washing, a colorimetric substrate is added and the absorbance at a560 is measured to identify positive clones.

Detection of Fab by ELISA

In one non-limiting example, an ELISA was used to coat Coster 3366 microplates overnight at 4 ℃ with 0.4ug/mL of TGF β 1, TGF β 2 or TGF β 3 (TGF β 1(R & D Systems, Cat #240-B/CF, 239ug/mL), TGF β 2(RDI, Cat # RDI-1035, 50ug/mL) and TGF β 3(RDI, Cat # RDI-1036/CF, 50ug/mL) diluted with coating buffer to 0.4 ug/mL). The plates were then washed twice before 100ul of blocking solution (10mg/ml BSA wash) was added to each well. The Fab dilutions of the invention were incubated in coated wells for 1.5 hours at 22 ℃. After washing, anti-human kappa-alkaline phosphatase conjugate was added and incubated at 22 ℃ for 1 hour. After washing well, a colorimetric substrate is added and the absorption at a560 is determined.

In another example, the binding compositions of the invention are tested in a competitive ELISA assay. Typically, a liquid phase assay is performed in which a compound that competes with an antigen for binding to a binding composition (e.g., an antibody) first binds to the antibody in the liquid phase, and the extent to which the antibody binds to the antigen is subsequently determined.

Materials:

the carbonate coating buffer comprised 50mM sodium carbonate pH 9.6. The antigen is TGF beta 1 (R) diluted to 0.4ug/ml with coating buffer&D Systems, Cat #240-B/CF, 239ug/ml), TGF- β 2(RDI, Cat # RDI-1035, 50ug/ml) and TGF- β 3(RDI, Cat # RDI-1036/CF, 50 ug/ml). The washing buffer was composed of 0.02M Tris pH7.4, 0.15M NaCl, 0.1% Tween 20 and 10mg/ml BSA (Sigma A-4503) in the washing buffer, i.e., blocking solution. The protein used as a positive control was mouse anti-human TGF-. beta.1, 2 or 3 (R)&D Systems, cat #1D11), mouse anti-human TGF-. beta.2 (R)&D Systems, cat # BAF302) and mouse anti-human TGF-. beta.3 (R)&D Systems, cat # BAF243) diluted to 1ug/ml with blocking solution. Detection antibodies usedThe conjugate was an anti-mouse kappa-peroxidase conjugate (Southern Biotech, cat #1050-05) at working concentrations diluted 1: 2000 with blocking solution. The substrate used for the color reaction was an o-phenylenediamine (OPD) plate (Sigma cat # P-6912), which was dissolved in a substrate buffer (0.1 MNa)₂HPO₄pH adjusted to 5.0 with 0.05M citric acid). Working solutions of OPD substrate (i.e.volume for each 196. mu.plate) were freshly prepared before development of color on each plate by dissolving 1X 5mg OPD plates in 12.5ml substrate buffer followed by addition of 5ul of 30% H₂O₂。

The scheme is as follows:

individual 96-well plates were coated with antigen (0.4ug/ml TGF-. beta.1, 2 or 3, 50ul per well), sealed with tape, and stored at 4 ℃ for 16-20 hours. The plate was then washed 2 times with the wash buffer described previously and then 100ul of blocking solution (10mg/ml BSA wash buffer) was added to each well. After incubation at 22 ℃ for approximately 1 hour, the plates were washed 2 times with wash buffer. Then, 100ul of the buffer diluted sample or PBS diluted control was added to each well and incubated at 22 ℃ for 1.5 hours. After incubation, the plates were washed 6 times with wash buffer, and then 100ul of anti-mouse kappa-peroxidase conjugate (diluted 1: 2000 with blocking solution) or SA-HRP (diluted 1: 10,000 with blocking solution) was added to each well. The test samples were incubated at 22 ℃ for 1 hour, and then 100ul of OPD substrate was added to each well. After development (approximately 10 minutes), the absorbance at 490nm was measured in 96-well plates.

The successful result of the Fab embodiment in this case is that TGF β 1 produces an absorption of greater than 1.6 units at 490nm, whereas TGF β 2 and TGF β 3 exhibit significantly lower absorption values, indicating specific and/or selective binding to TGF β 1.

Detection of mAbs in cell-based assays

To test the ability of the binding compositions of the present invention to neutralize TGF-beta bioactivity and to neutralize specific TGF-beta isozymes, a HT-2 cell proliferation assay by Tsang et al (1995Cytokine 7: 389-97) was used. The potency of the Fab and mAb compositions in vitro was determined using an HT-2 cell proliferation assay. Briefly, HT-2 cells proliferate in the presence of IL-4 but undergo apoptosis in the presence of TGF β. TGF β -induced cell death can be prevented by addition of TGF β 1 neutralizing fabs or mabs. The human cell line HT-2 proliferates in response to IL-4, but IL-4-induced proliferation is inhibited by TGF β 1, TGF β 2 or TGF β 3. Thus, a binding composition that is specific and/or selective for TGF-beta 1 is neutralizing if it prevents the normal inhibitory effects of TGF-beta 1 on IL-4 induced HT-2 cells. Thus, if sufficient TGF-beta 1-specific binding composition is added to a mixture of HT-2 cells containing TGF-beta 1, IL-4-induced cell proliferation should proceed indefinitely. Thus, the HT-2 assay is used to assess the dose response neutralizing capacity of the binding compositions of the invention in the presence of specific TGF-beta isoforms and IL-4 proliferation signals. The extent of cell proliferation is assessed using a commercial colorimetric cell proliferation assay (e.g., the cell proliferation assay in solution as described by CellTiter 96 ® Aqueous from Promega).

With 100U/ml and 100ug/ml penicillin/streptomycin supplemented with 10% FBS, 50uM beta-mercaptoethanol and 10ng/ml human IL-2 (R)&D Systems) RPMI 1640 medium maintained HT-2 cells. Cells were centrifuged at 1000RPM with a jouran CR422 centrifuge and resuspended in PBS. After washing 2 times with PBS, the assay medium (containing phenol red-free RPMI 1640 supplemented with 2% FBS, penicillin/streptomycin 100U/ml and 100ug/ml, respectively, and 50 uM. beta. mercaptoethanol) was finally used at 0.15X 10⁶Cells/ml resuspended cells. 50ul of cells in assay medium was added to each well of a 96-well plate. Different concentrations of mAb were pre-incubated with TGF-beta 1, TGF-beta 2 or TGF-beta 3in 300pg/ml assay medium prior to addition of test mAb to the cell bioassay. After 30 min incubation, 50ul of TGF-. beta./Fab mixture was added to the cells, followed immediately by 50ul of assay medium containing 45ng/ml murine IL-4 (final concentration 15 ng/ml). After 20-48 hours of incubation, 35ul of CellTiter 96 aqueous solution (Promega Corp) was added. Moisture at 37 deg.C and 5% CO₂After further incubation for 2-3 hours under air conditions, the cells were incubated withA colorimetric assay of CellTiter 96 ® (the amount of formazan product produced-measured as absorbance at 490 nm-is directly and positively correlated with the number of viable cells in culture) was performed on an ELISA plate reader at 490 nm. Table 2 below shows the model data.

TABLE 2HT-2In vitro cell bioassay：

The potency of the Fab and mAb binding compositions of the invention is determined in vitro using an HT-2 cell proliferation assay. HT-2 cells proliferate in the presence of IL-4, but undergo apoptosis in the presence of TGF β. TGF-induced cell death is prevented by the addition of TGF-neutralizing compositions (such as fabs or mabs of the invention). The binding compositions of the invention exhibit at least a 100-fold or greater neutralization potency against TGF-induced cell death as compared to murine IgG1FAB # 2471. The IC50 of the mAb composition ranged from approximately 0.1-1.0ng/ml (IC 50 for mAb2471 was approximately 0.1 ng/ml).

Determination of kinetic constants of Fab

The binding constants were determined with a KinExA 3000 apparatus (Sapidyne inst. inc.). Briefly, an antigen is covalently coupled to an alzactone bead and the binding of the free Fab binding composition of the present invention to the bead is detected using the apparatus. To determine Kd, each tube containing 20pM Fab (for mAb, at a concentration of 200pM) and decreasing serial dilutions of antigen (0-250nM) was incubated in PBS containing 1% BSA, 0.02% azide and 0.01% Tween-20 at 25 ℃ for 1-6 days. After incubation, free Fab in each of the equilibrated samples was determined on a KinExA 3000 according to the manufacturer's instructions. K_dValues were determined using KinExA 3000 software.

To measure K_on2nM of each Fab was mixed with 0-240nM antigen by injection according to the manufacturer's instructions and unbound Fab was detected. The obtained data is used forK was calculated in KinExA 3000 software_on。K_offBy using the formula K_d＝K_off/K_onAnd then calculated.

Determination of kinetic constants of Mab

Alternative methods for determining kinetic constants are known, for example, the BIAcore ® 2000 instrument is used to determine the affinity of binding compositions for human TGF-. beta.1 (R & D Systems, Cat #240-B/CF), TGF-. beta.2 (RDI, Cat # RDI-1035), and TGF-. beta.3 (RDI, Cat # RDI-1036/CF). BIAcore ® 2000 used the optical properties of surface plasmon resonance to detect changes in the protein concentration of interacting molecules in dextran biosensor matrices. All reagents and materials were purchased from BIAcore ® AB (Upsala, Sweden), unless otherwise specified. All assays were performed at room temperature. The sample was dissolved in HBS-EP buffer (150mM sodium chloride, 3mM EDTA, 0.01% (w/v) surfactant P-20, and 10mM HEPES, pH 7.4). Recombinant protein A was immobilized in all four flow cells of the CM4 sensor chip using an amine coupling kit at a level of 400-450 Reaction Units (RU).

Binding was assessed using multiple rounds of analysis. Each round was performed at a flow rate of 50. mu.L/min, and included the following steps: mu.l of 0.5. mu.g/ml antibody binding composition was injected, 250. mu.l TGF-beta 1 (initially 5nM, 2-fold serial dilutions to 0.13nM each round, 2 injections per concentration) was injected, followed by short (5min) or long (120min) delayed dissociation and regeneration with glycine hydrochloride at pH1.5, 50. mu.l, 10 nM. Each round of binding and dissociation rate determination was performed by ClamXP (Center for Biomolecular Interaction Analysis, Univ. of Utah) using a simple binding model to fit biosensor data pairs to extract K_onAnd K_offA rate constant; by K_d＝K_on/K_offThe equilibrium binding constant is calculated. Table 3 below shows model data for compositions of the present invention.

Table 3 Fab and mAb binding affinity and kinetic assays of the invention.

Equilibrium (K) was determined with Kinexa_d) And kinetics (K)_on) Binding parameter (K)_offFrom K_dAnd K_onCalculated in (c). The equilibrium and kinetic binding parameters of the mAb were determined using Biacore. Equilibrium binding constant K_dFrom measured K_onAnd K_offAnd (4) calculating. Comparison was made with murine IgG1Fab # 2471. Due to the very low dissociation, Koff of 21D1 and Dm7 exceeded detectable upper limits, which may be very slow, and thus, the calculated K_dThe value also exceeds the upper limit. The values are the average of repeated measurements (n-3-4).

Determination of Mab specificity

BIAcore methods were used to determine the ability of mAb compositions of the invention to bind other entities, particularly latent forms of TGF-. beta.1 or TGF-. beta.3. All measurements were performed at room temperature. The sample was dissolved in HBS-EP buffer (150mM sodium chloride, 3mM EDTA, 0.01% (w/v) surfactant P-20, and 10mM HEPES, pH 7.4). Recombinant protein A was immobilized on all four flow cells of the CM4 sensor chip using an amine coupling kit at a level of 400-500 Reaction Units (RU).

Binding was assessed using multiple rounds of analysis. Each round is carried out at the flow rate of 100ul/min, and the method comprises the following steps: mu.l of 1. mu.g/ml antibody binding composition was injected, 250. mu.l of 5nM TGF-beta 1 or 5nM latent TGF-beta 1, or 5nM TGF-beta 3 was injected, followed by short (5min) delayed dissociation and 2 injections of 50. mu.l of 10mM glycine hydrochloride, pH1.5, for regeneration. The amount of signal after capture of the first Mab, and then the amount of signal of the ligand, was determined using the instrument control software. Since the signal is proportional to the mass of captured protein, the stoichiometry of the captured ligand can be easily calculated (Table 4).

TABLE 4 binding of TGF-beta 1, latent TGF-beta 1 and TGF-beta 3 to Mab (tested concentration of ligand 5nM)

Specificity of TGF-beta 1

The affinity of the binding composition mAb to TGF □ 3 was determined using the BIAcore method. All measurements were performed at room temperature. The sample was dissolved in HBS-EP buffer (150mM sodium chloride, 3mM EDTA, 0.01% (w/v) surfactant P-20, and 10mM HEPES, pH 7.4). Recombinant protein A was immobilized on all four flow cells of the CM4 sensor chip using an amine coupling kit at a level of 400-500 Reaction Units (RU). Binding was assessed using multiple rounds of analysis. Each round was carried out at a flow rate of 100. mu.l/min, comprising the following steps: mu.l of 0.5. mu.g/ml antibody binding composition was injected, 250. mu.l of TGF □ 3 (2-fold serial dilutions to 0.13nM each round starting at 5nM, 2 injections per concentration) was injected, followed by a short (5min) delayed dissociation and regeneration with 2 injections of 50. mu.l of 10mM glycine hydrochloride, pH 1.5.

The affinity was determined by measuring the average signal during the last 10 seconds of TGF □ 3 injection, based on the equilibrium signal achieved at different concentrations of TGF □ 3, and then the signals obtained at all TGF □ 3 concentrations were fitted to a simple binding equilibrium model in the SCRUBBER (Center for biomolecular interaction Analysis, univ. K of the tested mAb compositions of the present inventors_dAnd specific model data-calculated by dividing the Kd for TGF □ 3 binding by the Kd for TGF □ 1 binding (here) — are shown in table 5 below.

Table 5 binding compositions mabs tested for affinity and relative specificity for TGF β 3 binding.

The error shown represents the standard deviation of multiple repeated measurements (n-3).

Mab	Kd(TGF-□3)，nM	Specificity (K)d，□□3/Kd，□□1)
			21D1	4.90	9800
DM4	0.53±0.01	621
			DM7	1.16±0.18	2320
C27	2.20	3670
			23A3	0.66±0.04	1050

Example 2: liver fibrosis bile duct connector inner model

The in vivo efficacy of anti-TGF β 1 treatment was assessed using the bile duct junction model in a manner similar to that reported by Arias et al (BMC Gastroenterology 3(29), 2003). Briefly, for this purpose, male Sprange Dawley rats (250-300g) were paralyzed by inhalation of isoflurane (2-3%). The abdomen was scraped and scrubbed with povidone iodine (betadine) and 70% ethanol. Under sterile conditions, at midline incision (-4 cm), the common bile duct was isolated and ligated with a 6-0 surgical thread at two locations, approximately 1 cm apart, and then transected between the ligatures. The abdominal wall was closed with 4-0 sutures and the skin stapled. anti-TGF β 1 composition and isotype mouse control mab (igg) were administered every 7 days after the day of surgery. At 4 and 12 days post-surgery, anesthetized rats were subjected to serum liver enzyme, total blood count, and liver histology studies (three-color and H & E staining) to determine the effect of treatment.

Example 3: pulmonary fibrosis in vivo model

There are many models available to evaluate the in vivo efficacy of anti-TGF β compositions for pulmonary fibrosis. For example, JCI 107, 1537-1544, (2001) was performed in a bleomycin model in the manner reported by Pittt et al to assess improvement in anti-TGF β methods. Another model is the respiratory enterovirus 1/L model (see, e.g., Bellum et al am.J. Pathol, 150, 2243; or London et al Clin.Immunol.103, 284; and London et al exp.mol. Pathol, 72, 24-36). Briefly, mice were applied 1X 10 nostril first day with the mice⁷pfu (total 30ul) reovirus 1/L, followed by different concentrations of the anti-TGF-beta binding composition of the invention or isotype control mAbs described herein or known in the art on days 3, 7, and 12. Animals were monitored for signs of respiratory distress, weight loss, and mortality during the treatment period. On day 14 of initial treatment, animals were euthanized and lung samples were prepared for histopathological examination to assess the development and/or progression of lung disease (analysis of hydroxyproline content to determine fibrosis).

Example 4: in vivo model against thy1.1 glomerulonephritis

The rat anti-Thy1.1 model is a well established model of mesangial proliferative glomerulonephritis (see, e.g., Morita et al 1998Am J Kidney Dis 31: 559-73; Bagchus et al 1986Lab. invest.55: 680-7 and Yamamoto and Wilson 1987Kidney int.32: 514-25), into which antibodies directed against Thy antigens localized on the surface of mesangial cells can induce mesangial lysis, with the subsequent stage of mesangial cell overcompensating proliferation resulting in increased levels of urinary protein (urokinase protein). The anti-Thy1.1 nephritis model resembles human IgA nephritis or Henoch-Schonlein purpura in many ways (O' Donoghue et al, 1991J Clin Invest 88: 1522-30) and has been used to test potential treatment for renal disease by determining the ability of test compositions to affect dose-related reductions in urinary protein (see, e.g., Burg et al 1997Lab Invest 76: 505-16; Johnson et al 1995Kidney Int 47: 62-9).

To test the binding compositions of the invention using such a model, separately labeled male Aprague Dawley rats (200-. Precursor urine protein was measured on day 5 of pretreatment. Rats were individually identified by marking the tail with a color marker and marking the ear before blood was drawn from the posterior orbit, and then randomly divided into 5 groups based on the weight of the first day.

Treatment groups were studied blindly, with non-blinding on the last day of the study. Each group was injected initially with 1.25mg thy1.1mab or PBS as a control via the penile blood vessels. The conjugate composition is prepared and purified using standard conditions or conditions described herein. Control mouse IgG1mAb (11513) was protein A purified material and resuspended IN PBS pH 7.2 and purchased from Harlan Bioproducts for Science, Indianapolis, IN 46229-0176.

Mouse □ -Thy1.1 was generated using 2X 10L mouse hybridoma cell culture. Conditioned media were mixed, concentrated to 18 ×, and then purified. Approximately 746mL of the harvested concentrated supernatant was mixed with 1.5M glycine/3.0M sodium chloride pH 8.9 and applied to a clean 137mL protein A Sepharose column pre-equilibrated with 1.5M glycine/3.0M sodium chloride pH 8.9. The protein A column was then washed with 1.5M glycine/3.0M sodium chloride pH 8.9. The column was eluted with citric acid pH 3.0100 mM. The selected fractions corresponding to IgG were pooled, adjusted to pH7.4 with 1M NaOH, and applied to 318ml of a pharmacia Superdex 200 column equilibrated with PBS/sodium chloride pH 7.4. Peaks corresponding to the size of IgG were pooled and stored at-20 ℃ separately.

Animals were dosed subcutaneously with either the same type or the anti-TGF β 1 antibody compositions of the invention 1 hour after administration of anti-thy 1.1mab. Antibody was administered again on day 7. Animals were tested in the following four treatment groups:

1) false processing; injection of PBS

2) anti-Thy1.1 and isotype control antibodies were injected at approximately 12.5mg/kg or 2.5 mg/dose

3) anti-Thy1.1 and DM4 antibodies were injected at approximately 5mg/kg or 1 mg/dose

4) anti-Thy1.1 and DM7 antibodies were injected at approximately 12.5mg/kg or 2.5 mg/dose

5) anti-Thy1.1 and C27 antibodies were injected at approximately 12.5mg/kg or 2.5 mg/dose

Rats were placed in metabolic cages for 24 hours on days-5 and 13. On day 14, rats were sacrificed with carbon dioxide and blood was taken by cardiac puncture for analysis. The left kidney was fixed with 4% paraformaldehyde in PBS and stored in 70% ethanol for later histological analysis. If any rat is moribund, it is killed and treated with carbon dioxide and the concentration of urine protein and blood urea nitrogen is determined. Urine protein and Blood Urea Nitrogen (BUN) concentrations were analyzed on a HITACHI911 automatic analyzer according to the manufacturer's instructions, controls from Biorad.

The model data in table 6 below show that the binding compositions of the invention have the significant ability to attenuate kidney injury in vivo and reduce the increase in proteinuria associated with anti-thy 1.1mab-induced kidney injury

Table 6 rats were injected i.v. with 2.5mg/kg of α -thy1.1mab and studies of 21D1 and DM4 were performed 30 minutes later with 1mg of Herceptin (Herceptin). A second dose of anti-TGF β 1 and control mAb was administered on day 7 and the animals were euthanized on day 14. Both 21D1 and DM4mAb binding compositions reduced urinary protein levels in a dose-dependent manner.

Example 5: epitope mapping of TGF- □ 1 binding compositions

The epitope for TGF □ 1 binding compositions (e.g., antibodies) of the invention is mapped using a combination of H/Dex and a chemical label. Since both H/D exchange and chemical modification rely on solvent accessibility of amino acid residues, changes in solvent accessibility can be used to identify residues involved in antibody binding after the binding composition TGF β 1 complex is formed. Following H/D exchange or chemical modification, the previously bound antigen is proteolytically digested into peptide cleavage fragments, allowing comparison of molecular weights between fragments with LC/MS to determine which amino acid residues are blocked in the H/D exchange or chemical modification following binding complex formation.

Chemical labelling of protein surfaces

15 □ g of a 1mg/ml aliquot of TGF □ 1in 4mM hydrochloric acid solution was transferred to a plastic vial and 180 □ g of a control or TGF □ 1 antibody composition of the invention was added (molar ratio TGF- □ 1/antibody 1/2). Phosphate Buffered Saline (PBS) was added to each vial to a final volume of 150 □ L, and allowed to incubate at room temperature for at least 10 minutes to form binding complexes, followed by protein surface labeling. For chemical labeling, 7.5 □ L of 5mg/mL hydroxysuccinimide Acetate (AHSE) solution was added to each complex vial, and the mixture was incubated at room temperature (AHSE/antibody 200/1 molar ratio). At various times (e.g., 10, 20, and 60 minutes), 50 □ L of the mixed solution was quenched by mixing 50 □ L of 1mg/mL K in 0.1M tris buffer, pH 8.0. The solution was analyzed directly by LC/MS as described herein. The remaining solution of each sample was treated with 3-5 □ L of 50mg/mL DTT solution at 37 ℃ for 10-15 minutes to induce the formation of the mature TGF □ 1 disulfide bond.

The reduced protein solution was then treated with 3 □ L of 0.1mg/mL chymotrypsin solution at 37 ℃ for 2-3 hours, followed by 1 □ L of 0.25mg/mL Glu-C solution at 37 ℃ for an additional 2-3 hours. The reaction was quenched by the addition of 0.5 □ L of glacial acetic acid and then analyzed by LC/MS using a Waters 2795HPLC and Micromass LTC Premier Mass spectrometer. HPLC proteins and peptides were eluted with a gradient of 0.15% formic acid containing acetonitrile at rt using Zorbax, SBC18, 2.1 × 50 nm. The intact protein requires a 14 minute run time and the protease digest 75 minutes.

For lysine residues (K) on the surface or within or structurally near the epitope of TGF □ 1, acetylation of lysine amino groups is partially or completely blocked after the test composition binds TGF □ 1. By comparing the degree of acetylation of the peptides in a complexed sample (TGF- □ 1+ antibody that binds TGF- □ 1) or a non-complexed sample (TGF- □ 1+ control antibody that does not bind TGF- □ 1), the amino acid residues that are blocked from acetylation by the formation of a binding complex can be identified. Table 7 below shows a model of acetylation data obtained in such LC/MS analysis.

TABLE 7 moles of acetylated amino groups per mole of peptide obtained by LC/MS

^＊And # indicates that two different lots of DM4 antibody were characterized separately.

Given such model data, for several TGF □ 1 peptide fragments, particularly the short time acetylation shown (e.g. 10 minutes), TGF □ 1: the difference between the complex of the antibody and the control is discernible. Fragments containing residues 31-39, 33-43, 53-62, and 91-112 demonstrate such discernable differences. Fragments 31-39 and 33-43 all contained residues K37. Fragments 22-32 containing K26 and K31 showed no difference from the control, and thus the difference in acetylation of fragments 31-39 could be attributed to blocking acetylation of K37 after formation of the antigen-antibody complex.

For each antibody tested, fragments 53-62 and 91-112 displayed a difference in persistence over the range tested. Fragment 53-62 showed a decreasing difference in the upper range of acetylation, but with low AHSE/antibody ratios, such difference remained unchanged throughout the range. Without being bound by theory, one explanation for such data is that K60 is not directly involved in the antigen: the antibody binds, but it is sufficiently close to the binding complex to block AHSE accessibility to the K60 residue, thereby blocking acetylation. Alternatively, however, K60 may constitute the epitope defined by the test antibody.

Fragments 91-112 showed acetylation differences throughout the range tested, suggesting that at least one of the three lysine residues (K95, K97, K110) on this fragment is involved in the formation of the binding composition: TGF-beta 1 complex. To identify the relevant lysine residues, the chymotrypsin digest was also treated with Glu-C, generating two additional fragments: 91-99 and 100-112. The latter (fragment 100-112) contains K110, but it does not show a significant acetylation difference, suggesting that it is inaccessible to both solvent or chemical modification.

The former (fragment 91-99) contained K95 and K97, further tested with AHSE treated uncomplexed TGF □ 1, analyzed by MS/MS to determine the elution time of the monoacylated molecule, and to quantify the degree of acetylation of K95 and K97 (model data under such conditions are given in Table 8 below). Such data indicate that acetylation of K97 remains unchanged, regardless of the presence or absence of complex formation. The presence of the antibody composition of the invention significantly affected acetylation of K95, indicating that K95 is directly involved in the formation of the binding complex.

Table 8 acetylation of K95 and K97

^＊And # indicates that two different lots of DM4 antibody were characterized separately.

H/D switching

In epitope mapping, deuterium hydrogen (H/D) exchange technology is similar to protein surface labeling/MS, however, H/D exchange is not residue specific and thus, any amino acid residue change can be detected. In a combination composition: in comparison of TGF-beta 1 complex with non-complexed mature TGF- □ 1 protein, the molecular weight of the complexed protein is about 20Da lower (or at 100% D) than that of non-complexed TGF- □ 1₂30Da at O), and thus by calculating D for both complexed and uncomplexed TGF- □ 1_□O weight differences can indicate that about 30 amino acid residues in the mature TGF- □ 1 dimer may be involved in forming the binding complex of the invention.

120 □ g aliquots (. about.140 or 280 □ L) of antibody solution were exchanged for PBS buffer by serial concentration and dilution using a Microcon (30kD) ultrafiltration protein concentrator (Millipore). After 2 successive concentration and dilution with PBS, the antibody solution was concentrated and taken out, and adjusted to a final volume of 70 □ L with PBS. Then, 4mM HCl solution containing 10 □ L1 mg/mL TGF- □ 1 and 2 □ L1M tris buffer pH 8.0 were added to each antibody vial to form TGF- □ 1: an antibody complex. A control sample of TGF- □ 1 was prepared by mixing 70 □ L1 XPBS, 20 □ L of TGF- □ 1in 4mM HCl and 2 □ L of 1M tris buffer pH 8.0. Subsequently, 9 □ L of TGF- □ 1 or TGF- □ 1 antibody complex was transferred to a mini plastic vial, followed by the addition of 21 □ L of 100% D₂O forms 70% D₂A solution of O. The solution was incubated at room temperature for 10 minutes and then at 0 ℃ for 1 minute.

After incubation, the H/D exchange was quenched, 15 □ L of 1% formic acid solution (0 ℃) and 4 □ L of 2mg/mL pepsin solution (0 ℃) were added, followed by incubation at 0 ℃ for 5 minutes to digest the protein. The digest was immediately injected manually onto the column for LC/MS analysis (as described above except that the tubing and HPLC column were immersed in an ice-water bath).

Mature TGF- □ 1 is resistant to pepsin digestion at low pH (about 2.5) and low temperatures due to disulfide bond formation. As a result, although the digestion time was long, the enzyme: protein concentrations were higher but produced smaller amounts of cleaved peptide, while most of TGF- □ 1 was still intact. Identifiable TGF- □ 1 proteolytic fragments are typically produced in the C-terminal and middle regions of the protein (e.g., fragments 58-64 or 61-64). Model data for the mass change (delta mass) of such fragments after D/H exchange are shown in Table 9 below. The delta mass of fragments 61-64 is close to zero, while the delta mass of fragments 58-64 is about 1Da, suggesting that the region protected from deuterium exchange-after complex formation with the binding composition of the present invention-contains amino acid residues 58-61.

TABLE 9 Delta Mass of Pepsin hydrolysis peptides identifiable after D/H exchange of TGF-. beta.1

TGF-beta 1 mutagenesis

To further elaborate the epitopes of the binding compositions of the invention, conventional mutagenesis techniques are used to identify TGF-beta 1 residues that are critical for forming binding complexes with the compositions of the invention. The important TGF-beta 1 protein mutagenesis site was specified using the crystal structure of the TGF-beta 3/TGF-beta RII complex (2002Nat Struct biol.3: 203-8) as a model: R25K, K26R, V33I, P87T, V89L and K95T (as shown above).

The ability to bind TGF-beta 1 muteins was tested using binding compositions specific for the TGF-beta isoform and commercially available mAbs (1D11 and 240; R & DSystems) that prevent TGF-beta 1 from binding to its cognate receptor (TGF-beta RII). Testing was performed by laser-induced dissociation/ionization time-of-flight mass spectrometry. Test mabs [ e.g., mabs3821 and 2471 (disclosed in PCT/US 2004/018921; US 60/485,820) that specifically bind TGF β 1] and controls [ e.g., mAb1D11 that binds all three TGF β isoforms ] were mixed (providing the opportunity to form complexes with (mutant or wild-type) TGF β 1 protein), immobilized on a detection chip, lasered, and subsequently analyzed with standardized software under production conditions (cipheregen Diagnostics).

Model results indicate that a unique subset of amino acid residues at the TGF-beta 1/TGF-beta RII binding interface are distinct from other TGF-beta isoforms (TGF-beta 2 and TGF-beta 3). Test mAb #2471 (having specific binding affinity for TGF β 1) binds wild-type TGF β 1 at a 5-fold higher rate than TGF β 1 mutein, while mAbs3821 and 1D11 bind TGF β 1 mutein at a 2.5-fold lower rate than wild-type TGF β 1.

Sequence listing

SEQ ID NO: 1 is the primate mature TGF β 1 amino acid sequence.

SEQ ID NO: 2 is a primate VHCDR1 amino acid sequence.

SEQ ID NO: 3 is a primate VHCDR2 amino acid sequence.

SEQ ID NO: 4 is a primate VHCDR3 amino acid sequence.

SEQ ID NO: 5 is the primate VLCDR1 amino acid sequence.

SEQ ID NO: 6 is the primate VLCDR2 amino acid sequence.

SEQ ID NO: 7 is the primate VLCDR3 amino acid sequence.

SEQ ID NO: and 8 is a primate HCVR FR1 framework amino acid sequence.

SEQ ID NO: and 9 is a primate HCVR FR2 framework amino acid sequence.

SEQ ID NO: 10 is the primate HCVR FR3 framework amino acid sequence.

SEQ ID NO: 11 is the primate HCVR FR4 framework amino acid sequence.

SEQ ID NO: 12 is the primate LCVR FR1 framework amino acid sequence.

SEQ ID NO: 13-36 are primate LCVR FR2 framework amino acid sequences.

SEQ ID NO: 37 is the primate LCVR FR3 framework amino acid sequence.

SEQ ID NO: 38 is the primate LCVR FR4 framework amino acid sequence.

SEQ ID NO: 39 is the amino acid sequence of the heavy chain constant region of primate IgG 1.

SEQ ID NO: 40 is the amino acid sequence of the heavy chain constant region of primate IgG 4.

SEQ ID NO: 41 is the primate IgG4 light chain constant region amino acid sequence.

SEQ ID NO: 42-86 is the amino acid sequence of the primate light chain variable region

SEQ ID NO: 87-125 are primate HCVR heavy chain amino acid sequences.

Sequence listing

<110> Elley Co

<120> TGF-beta 1-specific antibodies

<130>X-16598

<160>135

<170>PatentIn version 3.3

<210>1

<211>112

<212>PRT

<213> Primates

<400>1

Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys

1 5 10 15

Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp

20 25 30

Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys

35 40 45

Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu

50 55 60

Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro

65 70 75 80

Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro

85 90 95

Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser

100 105 110

<210>2

<211>10

<212>PRT

<213> Primates

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa at position 3 ═ Thr or Asp

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Xaa at position 5 ═ Thr, Glu or Phe

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223> Xaa at position 9 ═ Met, Ile, Leu or Val

<220>

<221>MISC_FEATURE

<222>(10)..(10)

<223> Xaa at position 10 ═ His, Val or Ala

<400>2

Gly Tyr Xaa Phe Xaa Asp Tyr Asn Xaa Xaa

1 5 10

<210>3

<211>17

<212>PRT

<213> Primates

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa at position 1 ═ Tyr, Gln, or Ser

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa in position 2 ═ Ile or Val

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223> Xaa at position 8 ═ Asp or Glu

<220>

<221>MISC_FEATURE

<222>(11)..(11)

<223> Xaa at position 11 ═ Tyr, Thr, His, or Leu

<220>

<221>MISC_FEATURE

<222>(13)..(13)

<223> Xaa at position 13 ═ Gln, Lys, Pro or Ser

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Xaa at position 15 ═ Phe or Tyr

<400>3

Xaa Xaa Tyr Pro Tyr Asp Gly Xaa Thr Gly Xaa Asn Xaa Lys Xaa Lys

1 5 10 15

Ser

<210>4

<211>7

<212>PRT

<213> Primates

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa at position 4 ═ Trp or Ala

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Xaa at position 5 ═ Phe or Leu

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223> Xaa at position 6 ═ Ala, Glu or Tyr

<400>4

Gly Tyr Arg Xaa Xaa Xaa Tyr

1 5

<210>5

<211>10

<212>PRT

<213> Primates

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa at position 1 ═ Arg, Tyr, Glu, or Gln

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa at position 3 ═ Ser or Thr

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa at position 4 ═ Ser, Val or Ala

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Xaa at position 5 ═ Ser or Leu

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa at position 7 ═ Ser, Pro, Leu or Tyr

<400>5

Xaa Ala Xaa Xaa Xaa Val Xaa Tyr Met His

1 5 10

<210>6

<211>7

<212>PRT

<213> Primates

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Xaa at position 5 ═ Leu, Asn, or Pro

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa at position 7 ═ Ser, Lys, Tyr, Leu, Met, Phe, Glu, Gln, Arg, or His

<400>6

Ala Thr Ser Asn Xaa Ala Xaa

1 5

<210>7

<211>9

<212>PRT

<213> Primates

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa at position 1 ═ Gln or Ser

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Xaa at position 5 ═ Leu, Asp or Pro

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223> Xaa at position 6 ═ Asn or Arg

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa at position 7 ═ Pro, Phe, Tyr or Arg

<400>7

Xaa Gln Trp Asp Xaa Xaa Xaa Pro Ala

1 5

<210>8

<211>25

<212>PRT

<213> Primates

<400>8

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210>9

<211>14

<212>PRT

<213> Primates

<400>9

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210>10

<211>30

<212>PRT

<213> Primates

<400>10

Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg

1 5 10 15

Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>11

<211>11

<212>PRT

<213> Primates

<400>11

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210>12

<211>23

<212>PRT

<213> Primates

<400>12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210>13

<211>15

<212>PRT

<213> Primates

<400>13

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

1 5 10 15

<210>14

<211>15

<212>PRT

<213> Primates

<400>14

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>15

<211>15

<212>PRT

<213> Primates

<400>15

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr

1 5 10 15

<210>16

<211>15

<212>PRT

<213> Primates

<400>16

Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Pro Leu Ile Tyr

1 5 10 15

<210>17

<211>15

<212>PRT

<213> Primates

<400>17

Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>18

<211>15

<212>PRT

<213> Primates

<400>18

Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

1 5 10 15

<210>19

<211>15

<212>PRT

<213> Primates

<400>19

Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

1 5 10 15

<210>20

<211>15

<212>PRT

<213> Primates

<400>20

Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>21

<211>15

<212>PRT

<213> Primates

<400>21

Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr

1 5 10 15

<210>22

<211>15

<212>PRT

<213> Primates

<400>22

Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Pro Leu Ile Tyr

1 5 10 15

<210>23

<211>15

<212>PRT

<213> Primates

<400>23

Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>24

<211>15

<212>PRT

<213> Primates

<400>24

Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

1 5 10 15

<210>25

<211>15

<212>PRT

<213> Primates

<400>25

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr

1 5 10 15

<210>26

<211>15

<212>PRT

<213> Primates

<400>26

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

1 5 10 15

<210>27

<211>15

<212>PRT

<213> Primates

<400>27

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Trp Ile Tyr

1 5 10 15

<210>28

<211>15

<212>PRT

<213> Primates

<400>28

Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Pro Trp Ile Tyr

1 5 10 15

<210>29

<211>15

<212>PRT

<213> Primates

<400>29

Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Leu Trp Ile Tyr

1 5 10 15

<210>30

<211>15

<212>PRT

<213> Primates

<400>30

Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Trp Ile Tyr

1 5 10 15

<210>31

<211>15

<212>PRT

<213> Primates

<400>31

Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr

1 5 10 15

<210>32

<211>15

<212>PRT

<213> Primates

<400>32

Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

1 5 10 15

<210>33

<211>15

<212>PRT

<213> Primates

<400>33

Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Trp Ile Tyr

1 5 10 15

<210>34

<211>15

<212>PRT

<213> Primates

<400>34

Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Pro Trp Ile Tyr

1 5 10 15

<210>35

<211>15

<212>PRT

<213> Primates

<400>35

Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Leu Trp Ile Tyr

1 5 10 15

<210>36

<211>15

<212>PRT

<213> Primates

<400>36

Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Trp Ile Tyr

1 5 10 15

<210>37

<211>32

<212>PRT

<213> Primates

<400>37

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210>38

<211>10

<212>PRT

<213> Primates

<400>38

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210>39

<211>328

<212>PRT

<213> Primates

<400>39

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

1 5 10 15

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

20 25 30

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

35 40 45

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

50 55 60

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

65 70 75 80

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

85 90 95

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

100 105 110

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly

325

<210>40

<211>326

<212>PRT

<213> Primates

<400>40

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210>41

<211>107

<212>PRT

<213> Primates

<400>41

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>42

<211>106

<212>PRT

<213> Primates

<400>42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>43

<211>106

<212>PRT

<213> Primates

<400>43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>44

<211>106

<212>PRT

<213> Primates

<400>44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala SerSer Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Leu Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>45

<211>106

<212>PRT

<213> Primates

<400>45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Leu Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>46

<211>106

<212>PRT

<213> Primates

<400>46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Tyr Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>47

<211>106

<212>PRT

<213> Primates

<400>47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Glu Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>48

<211>106

<212>PRT

<213> Primates

<400>48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>49

<211>106

<212>PRT

<213> Primates

<400>49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Asn Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>50

<211>106

<212>PRT

<213> Primates

<400>50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Met Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>51

<211>106

<212>PRT

<213> Primates

<400>51

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>52

<211>106

<212>PRT

<213> Primates

<400>52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Phe Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>53

<211>106

<212>PRT

<213> Primates

<400>53

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Asp Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>54

<211>106

<212>PRT

<213> Primates

<400>54

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Arg Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>55

<211>106

<212>PRT

<213> Primates

<400>55

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>56

<211>106

<212>PRT

<213> Primates

<400>56

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>57

<211>106

<212>PRT

<213> Primates

<400>57

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Pro Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>58

<211>106

<212>PRT

<213> Primates

<400>58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>59

<211>106

<212>PRT

<213> Primates

<400>59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>60

<211>106

<212>PRT

<213> Primates

<400>60

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>61

<211>106

<212>PRT

<213> Primates

<400>61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Phe Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>62

<211>106

<212>PRT

<213> Primates

<400>62

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Val Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>63

<211>106

<212>PRT

<213> Primates

<400>63

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Leu Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>64

<211>106

<212>PRT

<213> Primates

<400>64

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Thr Ser Ser Val Leu Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>65

<211>106

<212>PRT

<213> Primates

<400>65

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Tyr Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>66

<211>106

<212>PRT

<213> Primates

<400>66

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>67

<211>106

<212>PRT

<213> Primates

<400>67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Gln Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>68

<211>106

<212>PRT

<213> Primates

<400>68

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Tyr Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>69

<211>106

<212>PRT

<213> Primates

<400>69

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>70

<211>106

<212>PRT

<213> Primates

<400>70

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>71

<211>106

<212>PRT

<213> Primates

<400>71

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Leu Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>72

<211>106

<212>PRT

<213> Primates

<400>72

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>73

<211>106

<212>PRT

<213> Primates

<400>73

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Phe Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>74

<211>106

<212>PRT

<213> Primates

<400>74

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ala Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>75

<211>106

<212>PRT

<213> Primates

<400>75

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ala Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>76

<211>106

<212>PRT

<213> Primates

<400>76

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Arg Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>77

<211>106

<212>PRT

<213> Primates

<400>77

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Arg Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>78

<211>106

<212>PRT

<213> Primates

<400>78

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Arg Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>79

<211>106

<212>PRT

<213> Primates

<400>79

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Pro Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>80

<211>106

<212>PRT

<213> Primates

<400>80

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>81

<211>106

<212>PRT

<213> Primates

<400>81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Phe Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>82

<211>106

<212>PRT

<213> Primates

<400>82

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>83

<211>106

<212>PRT

<213> Primates

<400>83

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Leu Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>84

<211>106

<212>PRT

<213> Primates

<400>84

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala His Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>85

<211>106

<212>PRT

<213> Primates

<400>85

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>86

<211>106

<212>PRT

<213> Primates

<400>86

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Pro Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>87

<211>116

<212>PRT

<213> Primates

<400>87

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>88

<211>116

<212>PRT

<213> Primates

<400>88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>89

<211>115

<212>PRT

<213> Primates

<400>89

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr Asn

20 25 30

Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

35 40 45

Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe Lys

50 55 60

Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210>90

<211>116

<212>PRT

<213> Primates

<400>90

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>91

<211>116

<212>PRT

<213> Primates

<400>91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Glu Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>92

<211>116

<212>PRT

<213> Primates

<400>92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Tyr Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>93

<211>116

<212>PRT

<213> Primates

<400>93

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>94

<211>116

<212>PRT

<213> Primates

<400>94

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>95

<211>116

<212>PRT

<213> Primates

<400>95

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>96

<211>116

<212>PRT

<213> Primates

<400>96

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>97

<211>116

<212>PRT

<213> Primates

<400>97

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Val Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>98

<211>116

<212>PRT

<213> Primates

<400>98

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>99

<211>116

<212>PRT

<213> Primates

<400>99

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Phe Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>100

<211>116

<212>PRT

<213> Primates

<400>100

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Thr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>101

<211>116

<212>PRT

<213> Primates

<400>101

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly His Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>102

<211>116

<212>PRT

<213> Primates

<400>102

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Leu Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>103

<211>116

<212>PRT

<213> Primates

<400>103

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Lys Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>104

<211>116

<212>PRT

<213> Primates

<400>104

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Ile Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>105

<211>116

<212>PRT

<213> Primates

<400>105

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Met Val Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>106

<211>116

<212>PRT

<213> Primates

<400>106

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Met Val Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Pro Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>107

<211>116

<212>PRT

<213> Primates

<400>107

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Met Val Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ser Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>108

<211>116

<212>PRT

<213> Primates

<400>108

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Glu Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>109

<211>116

<212>PRT

<213> Primates

<400>109

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>110

<211>116

<212>PRT

<213> Primates

<400>110

GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>111

<211>116

<212>PRT

<213> Primates

<400>111

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Glu Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Pro Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>112

<211>116

<212>PRT

<213> Primates

<400>112

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>113

<211>116

<212>PRT

<213> Primates

<400>113

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>114

<211>116

<212>PRT

<213> Primates

<400>114

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>115

<211>116

<212>PRT

<213> Primates

<400>115

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Val Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Ala Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>116

<211>116

<212>PRT

<213> Primates

<400>116

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>117

<211>116

<212>PRT

<213> Primates

<400>117

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>118

<211>116

<212>PRT

<213> Primates

<400>118

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>119

<211>116

<212>PRT

<213> Primates

<400>119

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>120

<211>116

<212>PRT

<213> Primates

<400>120

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Ser Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>121

<211>116

<212>PRT

<213> Primates

<400>121

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Tyr

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>122

<211>116

<212>PRT

<213> Primates

<400>122

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Ser Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>123

<211>116

<212>PRT

<213> Primates

<400>123

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Glu Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>124

<211>116

<212>PRT

<213> Primates

<400>124

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>125

<211>116

<212>PRT

<213> Primates

<400>125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Pro Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>126

<211>9

<212>PRT

<213> Primates

<400>126

Gln Gln Trp Asn Gly Asn Pro Pro Ala

1 5

<210>127

<211>9

<212>PRT

<213> Primates

<400>127

Gln Gln Trp Asp Ser Ash Pro Pro Ala

1 5

<210>128

<211>17

<212>PRT

<213> Primates

<400>128

Tyr Ile Tyr Pro Tyr Asn Gly Asp Thr Gly Tyr Asn Gln Lys Phe Lys

1 5 10 15

Ser

<210>129

<211>10

<212>PRT

<213> Primates

<400>129

Gly Tyr Thr Phe Thr Asp Tyr Thr Met His

1 5 10

<210>130

<211>213

<212>PRT

<213> Primates

<400>130

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Glu Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>131

<211>442

<212>PRT

<213> Primates

<400>131

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210>132

<211>213

<212>PRT

<213> Primates

<400>132

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Pro Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210>133

<211>442

<212>PRT

<213> Primates

<400>133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210>134

<211>442

<212>PRT

<213> Primates

<400>134

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asp Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asp Gly Glu Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210>135

<211>213

<212>PRT

<213> Primates

<400>135

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Leu Asn Pro Pro Ala

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

Claims (16)

1. An isolated binding composition that specifically binds to mature TGF-beta 1 relative to mature TGF-beta 2 and mature TGF-beta 3, the composition comprising a first part having a binding site, the first part comprising GYRX₁X₂X₃Y (SEQ ID NO: 4); wherein: x₁Is W or A; x₂Is F or L; and X₃Is A, E or Y.

2. The composition of claim 1, wherein the first portion further comprises at least one of the following sequences:

i.GYX₁FX₂DYNX₃X₄(SEQ ID NO: 2); wherein: x₁Is T or D; x₂Is T, E or F; x₃Is M, I, L or V; and X₄Is H, V or A; or

ii.X₁X₂YPYDGX₃TGX₄NX₅KX₆KS (SEQ ID NO: 3); wherein: x₁Is Y, Q or S; x₂Is I or V; x₃Is D or E; x₄Is Y, T, H or L; x₅Is Q, K, P or S; and X₆Is F or Y.

3. The composition of claim 2, further comprising a second moiety comprising binding site X₁QWDX₂X₃X₄PA (SEQ ID NO: 7); wherein: x₁Is Q or S; x₂Is L, D or P; x₃Is N or R; and X₄Is P, F, Y or R.

4. The composition of claim 3, wherein the second portion further comprises at least one of the following sequences:

i.X₁AX₂X₃X₄VX₅YMH (SEQ ID NO: 5); wherein: x₁Is R, Y, E or Q; x₂Is S or T; x₃Is S, V or A; x₄Is S or L; x₅Is S, P, L or Y; or

ii.ATSNX₁AX₂(SEQ ID NO: 6); wherein: x₁Is L, N or P; and X₂Is S, K, Y, L, M, F, E, Q, R or H.

5. The recombinant binding composition of claim 1, wherein:

a) the first part:

i) immunoreactive with mature human TGF beta 1;

ii) immunoreactive with mature primate TGF β 1;

iii) is produced against purified or recombinantly produced human TGF-beta 1 protein or fragment thereof;

iv) is a monoclonal antibody, Fab, Fv, scFv, F (ab)2 or part of an antibody variable domain;

v) has at least one, two, three or four conservative substitutions;

vi) is embedded within a human or humanized antibody framework; or

vii) a portion comprising the variable region of an antibody heavy chain, Fab, Fv, scFv, or F (ab) 2.

6. The binding composition of claim 5, which is a monoclonal antibody comprising:

a) a Heavy Chain Variable Region (HCVR) having:

I. selected from the group consisting of GYX₁FX₂DYNX₃X₄[SEQ ID NO：2]CDR1 of the sequence; wherein: x₁Is T or D; x₂Is T, E or F; x₃Is M, I, L or V; and X₄Is H, V or A; and

selected from the group consisting of X₁X₂YPYDGX₃TGX₄NX₅KX₆KS[SEQ ID NO：3]CDR2 of the sequence; wherein: x₁Is Y, Q or S; x₂Is I or V; x₃Is D or E; x₄Is Y, T, H or L; x₅Is Q, K, P or S; and X₆Is F or Y; and

selected from the group consisting of GYRX₁X₂X₃Y[SEQ ID NO：4]CDR3 of the sequence; wherein: x₁Is W or A; x₂Is F or L; and X₃Is A or E; and

b) a Light Chain Variable Region (LCVR) having:

I. selected from the group consisting of X₁AX₂X₃X₄VX₅YMH[SEQ ID NO：5]CDR1 of the sequence; wherein: x₁Is R, Y, E or Q; x₂Is S or T; x₃Is S, V or A; x₄Is S or L; x₅Is S, P, L or Y; and

selected from the group consisting of ATSNX₁AX₂[SEQ ID NO：6]CDR2 of the sequence; wherein: x₁Is L, N or P; and X₂S, K, Y, L,M, F, E, Q, R or H; and

selected from the group consisting of X₁QWDX₂X₃X₄PA[SEQ ID NO：7]CDR3 of the sequence; wherein: x₁Is Q or S; x₂Is L, D or P; x₃Is N or R; and X₄Is P, F, Y or R.

7. The monoclonal antibody of claim 6, wherein:

a) the HCVR has:

I. comprises the amino acid sequence of SEQ ID NO: FR1 framework of 8;

a polypeptide comprising SEQ ID NO: FR2 framework of 9;

a polypeptide comprising SEQ ID NO: FR3 framework of 10; and

comprising SEQ ID NO: FR4 framework of 11; and is

b) The LCVR has:

I. comprises the amino acid sequence of SEQ ID NO: FR1 framework of 12;

comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: the FR2 framework of the sequence of 13-36;

a polypeptide comprising SEQ ID NO: FR3 framework of 37; and

comprising SEQ ID NO: 38 FR4 framework.

8. The monoclonal antibody of claim 7 which:

a) also included are human or humanized constant regions;

b) also included are IgG4 constant regions comprising:

I. comprises the amino acid sequence of SEQ ID NO: 40; and

a polypeptide comprising SEQ ID NO: 41;

c) is a Fab fragment;

d) is an Fv fragment;

e) is a scFv fragment;

f) is a F (ab)2 fragment;

g) fusion to a human constant region;

h) is detectably labeled;

i) is lyophilized;

j) encoded by an isolated nucleic acid molecule;

k) encoded by an isolated nucleic acid molecule operably linked to an expression vector;

l) is encoded by an isolated nucleic acid molecule operably linked to an expression vector which is integrated into a host cell;

m) is a chimeric antibody;

n) conjugation to other chemical moieties;

o) is sterile; or

p) is a pharmaceutical composition.

9. The monoclonal antibody of claim 8, comprising:

a) has a sequence selected from SEQ ID NO: an LCVR of the sequence 42-86; and

b) has a sequence selected from SEQ ID NO: 87-125 in a sequence of HCVR.

10. The monoclonal antibody of claim 9 which is:

a) humanization;

b) fusion to a human constant region;

c) a Fab fragment;

d) (iv) an Fv fragment;

e) a scFv fragment;

f) f (ab) a2 fragment;

g) comprises a constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD;

h) is detectably labeled;

i) freeze-drying;

j) a chimeric antibody;

k) for the preparation of a medicament for administration to a primate for treating a condition that is:

1. renal disease;

2. chronic kidney disease;

3. end stage renal disease;

4. a fibrotic disease, disorder or condition; or

5. Renal fibrosis;

l) conjugation to other chemical moieties;

m) sterile; or

n) comprises a pharmaceutical composition.

11. The monoclonal antibody of claim 10, comprising:

1. comprises the amino acid sequence of SEQ ID NO: 42 and an LCVR comprising SEQ ID NO: 87. 88, 89, 90, 91, 92;

2. comprises the amino acid sequence of SEQ ID NO: 43 and an LCVR comprising SEQ ID NO: 90. a HCVR of 95;

3. comprises the amino acid sequence of SEQ ID NO: 44 and an LCVR comprising SEQ ID NO: 93 or 94;

4. comprises the amino acid sequence of SEQ ID NO: 45. 46, 47, 48, 49, 50, 52, 53, 54, or 55 and an LCVR comprising SEQ ID NO: an HCVR of 96;

5. comprises the amino acid sequence of SEQ ID NO: 51 and an LCVR comprising SEQ ID NO: 97. 98, 99, 100, 101, 102, 103, 104.

12. The monoclonal antibody of claim 8, comprising an LCVR and HCVR as follows:

1. comprises the amino acid sequence of SEQ ID NO: 56 and an LCVR comprising SEQ ID NO: 105. HCVR of 106, 107, 109, 110;

2. comprises the amino acid sequence of SEQ ID NO: 57 and an LCVR comprising SEQ ID NO: 107 HCVR;

3. comprises the amino acid sequence of SEQ ID NO: 58 and an LCVR comprising SEQ ID NO: 106 HCVR;

4. comprises the amino acid sequence of SEQ ID NO: 60 and an LCVR comprising SEQ ID NO: 108 HCVR;

5. comprises the amino acid sequence of SEQ ID NO: 61 and an LCVR comprising SEQ ID NO: 110. 111 HCVR;

6. comprises the amino acid sequence of SEQ ID NO: 62. 63, 64, 66, 67, 68, 69 and a LCVR comprising seq id NO: 112 HCVR;

7. comprises the amino acid sequence of SEQ ID NO: 69 and an LCVR comprising SEQ ID NO: 112. 113, 114, 115 HCVR; or

8. Comprises the amino acid sequence of SEQ ID NO: 70. 71, 72 and a LCVR comprising SEQ ID NO: 116 HCVR.

13. The monoclonal antibody of claim 8, comprising an LCVR and HCVR as follows:

1. comprises the amino acid sequence of SEQ ID NO: 72. 73 and an LCVR comprising SEQ ID NO: 117 of an HCVR;

2. comprises the amino acid sequence of SEQ ID NO: 74 and an LCVR comprising SEQ ID NO: 118. 119 HCVR;

3. comprises the amino acid sequence of SEQ ID NO: 73 and an LCVR comprising SEQ ID NO: 119 HCVR;

4. comprises the amino acid sequence of SEQ ID NO: 75 and an LCVR comprising SEQ ID NO: 96. 117 of an HCVR;

5. comprises the amino acid sequence of SEQ ID NO: 76 and an LCVR comprising SEQ ID NO: 117 of an HCVR;

6. comprises the amino acid sequence of SEQ ID NO: 77. 78, 79 and a LCVR comprising SEQ ID NO: an HCVR of 96;

7. comprises the amino acid sequence of SEQ ID NO: 80 and an LCVR comprising SEQ ID NO: 120. HCVR of 121, 122;

8. comprises the amino acid sequence of SEQ ID NO: 81 and an LCVR comprising SEQ ID NO: 117. HCVR of 120, 121, 122, 123;

9. comprises the amino acid sequence of SEQ ID NO: 82. 84 and an LCVR comprising SEQ ID NO: 117 of an HCVR;

10. comprises the amino acid sequence of SEQ ID NO: 83 and an LCVR comprising SEQ ID NO: 117. 124, HCVR;

11. comprises the amino acid sequence of SEQ ID NO: 84 and an LCVR comprising SEQ ID NO: 117 of an HCVR;

12. comprises the amino acid sequence of SEQ ID NO: 85 and an LCVR comprising SEQ ID NO: 91 HCVR;

13. comprises the amino acid sequence of SEQ ID NO: 86 and an LCVR comprising SEQ ID NO: 117. 125 HCVR;

14. or comprises SEQ ID NO: 82 and a LCVR comprising SEQ ID NO: 89 HCVR.

14. A method of using the binding composition of claim 6, comprising contacting the binding composition with a biological sample comprising an antigen, thereby forming a TGF β 1 binding composition: an antigen complex.

15. The method of claim 14, wherein the biological sample is from a human, and wherein the binding composition is a monoclonal antibody.

16. A test kit comprising the binding composition of claim 6, and:

a) (ii) instructional material for using said binding composition for said detecting; or

b) Compartments separating the binding composition.