MX2007005650A - Ligands that enhance endogenous compounds. - Google Patents

Abstract

The invention relates to ligands that comprise a moiety (e.g., a dAb) that has a binding site with binding specificity for an endogenous target compound but do not substantially inhibit the activity of said endogenous target compound. Preferably, the ligand does not bind to the active site of an endogenous target compound. The invention relates to the use of such a ligand for the manufacture of a medicament for increasing the half-life, bioavailability, activity or amount of an endogenous target compound to which the ligand binds.

Description

LIGANDS THAT IMPROVE ENDOGENOUS COMPOUNDS Related Requests This application is a continuation in part of the US Patent Application. UU No. 10 / 985,847, filed on November 10, 2004, the total teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION Animals, including mammals, and humans produce a variety of endogenous compounds that have activities that help maintain the normal health and physiology of the animal. Such compounds are included in many physiological processes such as hemostasis, immune function, metabolism, regulation of cell proliferation and differentiation and the like. Many endogenous compounds have been isolated and synthetic or recombinant forms can be made and administered to patients to combat the disease. Current therapeutic approaches often include administering exogenous or exogenously produced forms of endogenous compounds to patients. Such therapeutic approaches may produce undesirable effects due to the high doses that are normally administered, and certain therapeutic agents, such as recombinant forms of human proteins, generally must be produced by expression in mammalian cells which is expensive and produces lower productions than expression in human protein systems. yeast or bacterial. These problems could be overcome by the development of therapeutic agents and methods that can strengthen and exploit the beneficial activities of endogenous compounds to produce therapeutic effects. There is a need for such therapeutic agents and methods.

Brief Description of the Invention The invention relates to the use of a ligand comprising a portion having a binding site for an endogenous target compound wherein said ligand binds said endogenous target compound and does not substantially inhibit the activity of said endogenous target compound, for the manufacture of a medicine. Preferably, the ligand does not bind to the active site of the endogenous target compound. The medicament may be for increasing the amount of an endogenous target compound in a subject, to increase the bioavailability of an endogenous target compound in a subject, to increase the in vivo half-life of an endogenous target compound. The medicament may also be for increasing the activity (e.g., binding activity) of an endogenous target compound. The medication can be for local or systemic supply. In some embodiments, the amount of endogenous target ligand in the subject approximately 4 hours after administration of the medicament is increased by at least 1.5 times, relative to the amount prior to administration of the medicament. In some embodiments, the in vivo half-life of the endogenous target compound increases by at least 1.5 times after drug administration. Generally, the ligand inhibits the activity of said endogenous target compound by no more than about 10%, or has an inhibitory concentration 50 (IC 50) of at least 1 micromolar. The ligand may comprise two or more copies of said portion having a binding site for an endogenous target. For example, the ligand can be a dimer of a portion having a binding site for an endogenous target, such as a dAb dimer. The portion that has a binding site for an endogenous target compound can be an affibrant, an SpA domain, a class A domain of the LDL receptor, an EGF domain, an avimer, antibody or an antibody fragment (e.g., an Fv fragment). , a single chain Fv fragment, a disulfide linked Fv fragment, a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment> a diabody, a single immunoglobulin variable domain (eg, a VH) human, and a human VL, a VHH). If desired, the ligand may further comprise a portion extending the half-life as described herein. For example, the ligand may comprise a portion extending the half-life selected from the group consisting of a portion of polyalkylene glycol, serum albumin or a fragment thereof, transferrin receptor or a transferrin binding portion thereof, or a portion comprising a binding site for a polypeptide that improves the half-life in vivo. Suitably a portion comprising a binding site for a polypeptide that improves half-life in vivo includes an affibody, an SpA domain, an LDL receptor class A domain, an EGF domain, an avimer, an antibody or antibody fragment. , such as a Fv fragment, a single-chain Fv fragment, a disulfide-linked Fv fragment, a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, a diabody, and a unique immunoglobulin variable domain ( for example, human VH, human VL, VHH) - The medicaments and ligands of the invention are substantially non-immunogenic. In some embodiments, the medication is a depot formulation. In some embodiments, the endogenous target compound is a soluble agonist (e.g., cytokines, growth factors, hormones), soluble receptor (e.g., soluble cytokine receptors, such as soluble TNFR1, soluble TNFR2, soluble I L-1 receptor , soluble I receptor L-4, soluble receptor I L-13), endogenous receptor antagonist (eg, receptor antagonist I L-1 (I LI ra)) or an enzyme. In particular embodiments, the endogenous target compound is a soluble cytokine receptor, such as soluble TNFR1. Preferably, the ligand binds the endogenous target compound with high affinity, such as Kd of 1 x 10 ~ 7 M or less. In certain embodiments, the medicaments or ligands of the invention do not include an antibody, fragment, or region thereof described in U.S. patent application publication. published no. 2003/0144484. In certain embodiments, the drugs or ligands of the invention do not inhibit the binding of the A2 antibody or chimeric A2 antibody (cA2) to human TNF-alpha. In particular embodiments, the drugs or ligands of the invention do not bind to an epitope included in amino acids 87-108 or an epitope included in both amino acids 59-80 and 87-108 of human TNF (tumor necrosis factor). These amino acids are: 59-80: Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His-Thr- lle (SEQ ID NO: 26); and 87-108: Tyr-GIn-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Lys-Ser-Pro-Cys-Gln-Arg Glu-Thr-Pro-Glu-Gly (SEQ ID NO: 27). The invention also relates to the use of endogenous target compound for the manufacture of a medicament for increasing the activity of said endogenous target, wherein said ligand binds said endogenous target and does not substantially inhibit the activity of said endogenous target compound. Preferably, the ligand does not bind to the active site of said endogenous target compound. In some embodiments, the binding activity of the endogenous target ligand is increased, for example the binding activity (eg, affinity, avidity) can be increased by a factor of at least 10. The invention also relates to a ligand that binds an endogenous target compound having suitable activity for treating a disease in a subject, wherein said ligand does not bind the active site of said endogenous target compound or substantially inhibits the activity of said endogenous target compound, for use in therapy of a disease suitable for treatment with said endogenous target compound. The invention also relates to a pharmaceutical composition comprising a ligand that binds an endogenous target compound having suitable activity for treating a disease in a subject and a physiologically acceptable carrier, wherein said ligand does not bind the active site of said endogenous or substantially objective target compound. inhibits the activity of said endogenous target compound. The invention also relates to a drug delivery system comprising a pharmaceutical composition of the invention. In some embodiments, that drug delivery device is a parenteral delivery device, intravenous delivery device, intramuscular delivery device, intraperitoneal delivery device, transdermal delivery device, pulmonary delivery device, intraartepal delivery device, intrathecal delivery device. , intra-articular delivery device, subcutaneous delivery device, intranasal delivery device, vaginal delivery device, and rectal delivery device For example, the drug delivery device can be a syringe, a transdermal delivery device, a capsule, a tablet, a nebulizer, an inhaler, an atomizer, an aerosolizer, a vaporizer, a dry powder inhaler, a metered dose inhaler, a metered dose sprayer, a metered dose vaporizer, a metered dose sprayer, a catheter invention refers to a method for increasing the half-life of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said endogenous target compound. refers to a method for increasing the amount of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said target compound Endogenous The invention relates to a method for increasing the bioavailability of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said endogenous target compound. The invention relates to a method for increasing the activity (eg, binding activity) of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a site. of binding with binding specificity for said endogenous target compound. The invention relates to a method for treating a subject having a disease that is suitable for treatment with an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a site. of binding with binding specificity for said endogenous target compound. The invention also relates to new ligands (e.g., dAbs) described therein Brief Description of the Drawings Figure 1 is a graph of the relative concentration of Soluble TNFR1 detected by ELISA in serum over time (concentration time curve) after a single intravenous administration of a PEGylated dAb monomer that binds TNFR 1 (TAR2m-21 -23 / 40K branched PEG, also referred to as PEG anti-TNFR1 dAb). The data points in the diagram are obtained by ELISA using a 1: 5 dilution of serum. The graph clearly shows that the bioavailability of TNFR1 in the systemic circulation is increased by the administration of a PEGylated dAb monomer that binds TNFR1. As shown by the concentration time curve, the level of soluble TNFR1 in serum increased after the PEGylated dAb monomer was administered, reached a maximum concentration, and then reduced over time to basic levels as the PEGylated dAb it clears The results demonstrate that administering a dAb that binds a soluble receptor can increase the bioavailability of the receptor and increase the level or amount of soluble receptor in the systemic circulation, and that the increased level or amount of soluble receptor in the systemic circulation is cleared and returned at basic levels. The results indicate that the level of soluble receptor in the systemic circulation can be controlled by administering a PEGylated dAb monomer that binds the soluble receptor.

Detailed Description of the Invention Within this specification the modalities have been described in a manner that allows a clear and concise specification to be written, but it is proposed and well appreciated that the modalities may be combined in a varied or separate manner without departing from the invention .

As used herein, the term "ligand" refers to a compound that comprises at least one peptide, polypeptide or protein portion having a binding site with binding specificity for a desired endogenous target compound. For example, the portion that has a binding site for a desired endogenous target compound may be a unique immunoglobulin variable domain (eg, VH, V, VHH) that has binding specificity for a desired endogenous target compound (e.g. a soluble receptor protein, endogenous receptor antagonist, cytokine or growth factor). The portion having a binding site for an endogenous target compound may also comprise one or more complementary determination regions (CDRs) of a single immunoglobulin variable domain having binding specificity for a desired endogenous target compound in a suitable format, so that the portion has binding specificity for the endogenous target compound. For example, CDRs can be grafted onto a suitable protein structure or micro-scaffold, such as an affibody, a SpA micro-scaffold, a class A domain of the LDL receptor, or an EGF domain. In addition, the ligand may be monovalent (for example, a dAb monomer), bivalent (homobivalent, heterobivalent) or multivalent (homomultivalent, heteromultivalent) as described herein. In this manner, "ligands" include polypeptides consisting of dAb, include polypeptides consisting essentially of such dAb, polypeptides comprising a dAb (or the CDRs of a dAb) in a suitable format, such as an antibody format (e.g. , similar format to IgG, scFv, Fab, Fab ', F (ab') 2) or a suitable protein structure or micro-scaffold, such as an affibody, a SpA micro-scaffold, a class A domain of the LDL receptor or an EGF domain , dual specific ligands comprising a dAb that binds a first endogenous target compound and a second dAb that binds another endogenous target compound (e.g., serum albumin), and multispecific ligands as described herein. The portion that has a binding site for an endogenous target compound may also be a protein domain comprising a binding site for a desired target, for example, a protein domain is selected from an affibody, an SpA domain, a class A domain of the LDL receptor, an EGF domain, an avimer (see, for example, US Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301). If desired, a "ligand" may further comprise one or more additional portions, which may each independently be a peptide, polypeptide or protein portion or a non-peptide moiety (eg, a polyalkylene glycol, a lipid, a carbohydrate ). For example, the ligand may further comprise a portion extending the half-life as described herein (eg, a portion of polyalkylene glycol, a portion comprising albumin, a fragment of albumin or albumin variant, a portion comprising transferrin, a fragment of transferrin or transferrin variant, a portion that binds albumin, a portion that binds neonatal receptor Fe). As used herein, the term "endogenous target compound" refers to a soluble compound that is produced by a subject (e.g., a mammal, a human) of interest, endogenous target compounds include, for example, soluble agonists (eg, example, cytokines, growth factors, hormones), soluble receptors (eg, soluble cytokine receptors, such as soluble, soluble TNFR1, soluble TNFR2, soluble I receptor L-1, soluble receptor I L-4, receptor I L- 13 soluble), endogenous receptor antagonists (e.g., receptor antagonist I L-1 (I L-1 ra)), enzymes, and the like. As used herein, the term "active site" refers to the site or domain of an endogenous target compound (e.g., protein) that interacts with (e.g., binds) an endogenous binding partner of an endogenous target compound ( for example, site or domain of a receptor protein that makes contact with the ligand of the receptor, site or domain of an agonist protein (eg, a cytokine) that makes contact with the agonist receptor, the catalytic domain of an enzyme). For example, as used herein, the "active site" of a soluble endogenous compound such as a cytokine, growth factor or hormone is the site on the cytokine, growth factor or hormone that makes contact with the endogenous receptor that binds the cytokine, growth factor or hormone. As used herein, "bioavailability" refers to the degree to which an endogenous target compound or endogenous target compound-ligand complex is present or gains access to a desired site of action (e.g., systemic circulation, nervous system central, a local action site (eg, a particular organ, a particular area of tissue (eg, muscle, skin))). Bioavailability of an endogenous target compound can be increased independently of any change in the in vivo half-life of the endogenous target compound, or of any change in the amount of the endogenous target compound in a subject. For example, a ligand can bind an endogenous target compound that has beneficial activity in the systemic circulation but that is rapidly distributed in tissues, and decreases the rate of tissue distribution. Such a ligand would increase the bioavailability of the endogenous target compound in the systemic circulation where the beneficial activity of the endogenous target compound may have therapeutic effect. As used herein, "binding activity" refers to the ability of the binding partners to bond with each other. For example, the ability of a solid receiver or receiver to bind its cognate ligand. The binding activity can be expressed, for example, as affinity (Kd); or avidity Binding activity can be increased, for example, by increasing affinity (for example, increasing the binding rate (Kencendida) or decreasing the dissociation rate (Kapagada)) or by increasing avidity. Avidity can be increased, for example, by increasing the number of sites in which a first attachment partner and a second union partner make contact. As used herein, the phrase "substantially non-immunogenic" means high affinity antibodies that bind the ligand, ligand-endogenous target compound or endogenous target compound do not occur with a ligand that is administered to a subject. High affinity antibodies bind the ligand, endogenous target compound-ligand complex or endogenous target compound with an affinity of 500 nM or less, or 300 nM or less, or 100 nM or less, or 10 nM or less, or 1 nM or less. lower. Antibodies binding the ligand, ligand-endogenous target compound or endogenous target compound complex and the affinity of such antibodies can be identified using any suitable method. Several such methods are well known in the art. For example, a serum sample can be obtained from a human to whom a ligand has been administered (e.g., about 1 week, 2 weeks, 3 weeks, 4 weeks after administration) and serum can be tested for the presence of antibodies which bind ligand, ligand-endogenous target compound or endogenous target compound using, for example, ELISA or other suitable assay. The affinity of antibodies can be determined using any suitable method, such as by equilibrium dialysis or by surface plasmon resonance. The phrase "single variable immunoglobulin domain" refers to a variable region of antibody (VH, VH H, VL) that specifically binds an antigen or epitope independently of other V regions or domains; however, as the term is used herein, a unique immunoglobulin variable domain may be present in one format (eg, homo- or hetero-multimer) with other variable regions or variable domes where the other regions or domains they are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the unique immunoglobulin variable domain binds the antigen independently of the additional variable domains). "Single immunoglobulin variable domain" comprises not only a single variable polypeptide of isolated antibody, but also larger polypeptides comprising one or more monomers of a single variable antibody domain polypeptide sequence. A "domain antibody" or "dAb" is the same as a "single immunoglobulin variable domain" polypeptide as the term is used herein. A unique immunoglobulin variable domain polypeptide, as used herein, refers to a mammalian immunoglobulin unique variable domain polypeptide, preferably human, but also includes rodent VH H dAbs (e.g., as described in WO 00 / 29004, the contents of which are incorporated herein for reference in its entirety) or camelid. camelid dAbs are single variable immunoglobulin domain polypeptides that are derived from species including camel, llama, alpaca, Arabian camel, and guanaco, and comprise heavy chain antibodies naturally devoid of light chain: VH H - VH H molecules are approximately ten times smaller than the IgG molecules, and as unique polypeptides, they are very stable, resisting extreme temperature and pH conditions. "Complementary" Two immunoglobulin domains are "complementary" where they belong to families of structures that form cognate pairs or groups or are derived from such families and retain this feature. For example, a VH domain and a VL domain of an antibody are complementary; Two VH domains are not complementary, and two VL domains are not complementary. The complementary domains can be found in other members of the immunoglobulin superfamily, such as the Va and Vp (or? And d) domains of the T cell receptor. Domains that are artificial, such as domains based on protein microannads that do not bind epitopes unless they are planned to do that, they are not complementary. Similarly, two domains based on (for example) an immunoglobulin domain and a fibronectin domain are not complementary. "Immunoglobulin family" This refers to a family of polypeptides that retain the immunoglobulin fold characteristic of antibody molecules, which contains two β-sheets and, usually, a conserved disulfide bond. Members of the immunoglobulin superfamily are included in many aspects of cellular and non-cellular interactions in vivo, including diffusion roles in the immune system (eg, antibodies, T cell receptor molecules and the like), inclusion in cell adhesion (e.g., ICAM molecules) and intracellular signaling (e.g., receptor molecules, such as the PDGF receptor). The present invention is applicable to all immunoglobulin superfamily molecules that possess binding domains. Preferably, the present invention relates to antibodies. "Domain" A domain is a bent protein structure that retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases they can be added, removed or transferred to other proteins without loss of function of the rest of the protein and / or the domain. "Single antibody variable domain" means a bent polypeptide domain comprising characteristic sequences of antibody variable domains. Thus it includes variable domains of whole antibody and modified variable domains, for example, in which one or more cycles have been replaced by sequences that are not characteristic of antibody variable domains, or variable antibody domains that have been truncated or comprise extensions of terminal N or C, as well as bent fragments of variable domains that retain at least in part the binding activity and specificity of the full-length domain. "Repertory" A collection of various variants, for example, polypeptide variants that differ in their primary sequence. A library used in the present invention will comprise a repertoire of polypeptides comprising at least 1000 members. "Library" The term "library" refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, each of which has a unique nucleic acid or polypeptide sequence. For this degree, library is synonymous with repertoire. The sequence differences between members of the library are responsible for the diversity present in the library. The library can take the form of a simple mixture of polypeptides or nucleic acids, or it can be in the form of organisms or cells, for example bacteria, viruses, plant or animal cells and the like, transformed with a nucleic acid library.

Preferably, each individual organism or cell contains only one or a limited number of library members. Advantageously, the nucleic acids are incorporated into expression vectors, to allow the expression of the polypeptides encoded by the nucleic acids. In a preferred aspect, therefore, a library can take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in the form of nucleic acid that can be expressed for produce its corresponding polypeptide member. In this way, the population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants. "Antibody" An antibody (eg, IgG, IgM, IgA, IgD or IgE) or fragment (such as a Fab, F (ab ') 2, Fv, disulfide linked Fv, scFv, closed conformation multispecific antibody, bound scFv a disulfide, diabody) either derived from any species that naturally produces an antibody, or created by recombinant DNA technology, either isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria).

"Dual specific ligand" A ligand comprising a single immunoglobulin unique variable domain and a second immunoglobulin unique variable domain as defined herein, wherein the variable regions are capable of binding to two different antigens or two epitopes on the same antigen which are not normally bound by a monospecific immunoglobulin. For example, the two epitopes may be in the same hapten, but not the same epitope or sufficiently adjacent to bind by a monospecific ligand. The specific dual ligands according to the invention are composed of variable domains that have different specificities, and do not contain mutually complementary variable domain pairs that have the same specificity. Specific dual ligands and methods suitable for preparing dual specific ligands are described in WO 2004/058821, WO 2004/003019, and WO 03/002609, the full teachings of each of these published international applications are incorporated herein by reference. "Antigen" A molecule that is bound by a ligand according to the present invention. Typically, antigens are bound by antibody ligands and are capable of obtaining an antibody response in vivo. It can be a polypeptide, protein, nucleic acid or other molecule. Generally, the dual specific ligands according to the invention are selected for objective specificity against a particular antigen. In the case of conventional antibodies and fragments thereof, the antibody-binding site defined by the variable cycles (L1, L2, L3 and H1, H2, H3) is capable of binding to the antigen.

"Epitope" A structure unit conventionally linked by an immunoglobulin VH / V pair. Epitopes define the minimum binding site for an antibody, and thus represent the specificity target of an antibody. In the case of a single domain antibody, an epitope represents the unit structure bound by a variable domain in isolation. "Universal structure" A single antibody structure sequence corresponding to the regions of an antibody conserved in sequence as defined by Kabat ("Sequences of Proteins of Immunological Interest", US Department of Health and Human Services) or corresponding to the structure or repertoire of human germline immunoglobulin as defined by Chothia and Lesk, (1987) J. Mol. Biol. 196: 910-917. The invention provides the use of a unique structure, or set of such structures, that has been found to allow derivation of virtually any binding specificity through variation in hypervariable regions alone. "Half-life" The time taken for the serum concentration of the ligand to be reduced by 50%, in vivo, for example due to degradation of the ligand and / or clearance or sequestration of the ligand by natural mechanisms. The ligands of the invention are stabilized in vivo and their half-life is increased by binding to molecules that resist degradation and / or clearance or sequestration. Typically, such molecules are naturally occurring proteins that themselves have a long half-life in vivo. The half-life of a ligand is increased if its functional activity persists, in vivo, for a longer period than a similar ligand that is not specific for the molecule by increasing the half-life. In this way, a specific ligand for HSA and a target molecule is compared to the same ligand where the specificity for HSA is not present, which does not bind HSA but binds another molecule. For example, it can be a second epitope in the target molecule. Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50% or more. I ncrements in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x or more of the half-life are possible. Alternatively, or in addition, increments in the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x of the half-life are possible. "Substantially identical (or" substantially homologous ") A first nucleotide or amino acid sequence that contains a sufficient number of nucleotides or amino acid residues identical or equivalent (eg, with a similar side chain, eg, conserved amino acid substitutions) to a second sequence of nucleotides or amino acids so that the nucleotide or amino acid sequences, first and second, have similar activities In the case of antibody, the second antibody has the same binding specificity and has at least 50% of the affinity of As referred to herein, the term "about" is preferably interpreted to mean plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, even more preferably plus or minus 2 %, more preferably plus or minus 1% As used herein, the terms "low severity", "medium severity", "high" "severity", or "very high severity conditions" describe conditions for nucleic acid hybridization and rinsing. Gupia to perform hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & amp; amp;; Sons, N.Y. (1989), 6.3.1 -6.3.6, which is incorporated herein by reference in its entirety. Aqueous and non-aqueous methods are described in that reference and any can be used. The specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by two rinses in 0.2X SSC, 0.1 % SDS at least at 50 ° C (the temperature of the rinses can be increased to 55 ° C for conditions of low severity, (2) conditions of hybridization of medium severity in 6X SSC at about 45 ° C, followed by one or more rinses in 0.2X SSC, 0.1% SDS at 60 ° C, (3) high severity hybridization conditions in 6X SSC at approximately 45 ° C, followed by one or more 0.2X SSC rinses, 0.1% SDS at 65 ° C, and preferably (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65 ° C, followed by one or more rinses at 0.2X SSC, 1% S DS at 65 ° C. severity (4) are the preferred conditions and those that should be used unless specified otherwise. As referred to herein, the term "competes" means that the binding of a first epitope to its cognate epitope binding domain is inhibited when a second epitope is attached to its cognate epitope binding domain. For example, the linkage can be sterically inhibited, for example by physically blocking a binding domain or by altering the structure or environment of a binding domain so that its affinity or avidity for an epitope is reduced. Similar or homologous sequences (eg, at least about 70% sequence identity) to the sequences described herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level may be approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher At the level of nucleic acid, the sequence identity may be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very low stringency hybridization conditions) to the filament complement. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

The calculations of "homology" or "sequence identity" or "similarity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (eg, spaces can be introduced into one or both of a first and a second nucleic acid or amino acid sequence for optimal alignment and non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of an aligned reference sequence for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The nucleotides or amino acid residues at corresponding nucleotide positions or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid residue as the corresponding position in the second sequence, then the molecules are identical in that position (as used in the present "homology" of nucleic acid or amino acid is equivalent to "identity" of nucleic acid or amino acid). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of spaces, and the length of each space, that need to be introduced for optimal alignment of the two sequences. The alignments of the nucleotide and amino acid sequence and homology, similarity or identity, as defined herein, are preferably prepared and determined using the BLAST 2 algorithm sequences, using failure parameters (Tatusova, TA et al., FEMS Microbiol Lett , 174: 187-188 (1999)). Alternatively, advantageously, the BLAST algorithm (version 2.0) is used for sequence alignment, with parameters set to fault values. The BLAST algorithm is described in detail on the website ("www") of the National Center for Biotechnology Information (".ncbi") of the National Institutes of Health ("NIH") of the US Government. UU (".gov"), in the "/ Blast /" directory, in the "blast_help. html" file. The search parameters are defined as follows, and are advantageously set to the defined failure parameters. BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm used by the blastp, blastn, blastx, tblastn, and tblastx programs; these programs ascribe meaning to their discoveries using the statistical methods of Karlin and Altschul, 1990, Proc. Nati Acad. Sci. USA 87 (6): 2264-8 (see the file "blast_help. Html", as described above) with a few improvements. BLAST programs are suitable for sequence similarity search, for example, to identify homologues with a query sequence. Programs are usually not useful for reason-style search. For a discussion of the basic fields in search of similarity of sequence databases, see Altschul et al. (1994). The five BLAST programs available on the website of the National Center for Biotechnology Information carry out the following tasks: "blastp" compares an amino acid query sequence against a database of protein sequences; "blastn" compares a nucleotide query sequence against a database of nucleotide sequences; "blastx" compares the conceptual translation products of six structures of a nucleotide query sequence (both strands) against a database of protein sequences; "tblastn" compares a protein query sequence against a database of dynamically translated nucleotide sequences in all six reading structures (both strands). "tblastx" compares the translations of six structures of a nucleotide query sequence against the translations of six structures of a nucleotide sequence database. BLAST uses the following search parameters: HISTOGRAM Displays a histogram of scores for each search; Failure is yes. (See parameter H in the BLAST Manual). DESCRI PTIONS Restricts the number of short descriptions of coupling sequences reported for the specified number; The failure limit is 100 descriptions. (See parameter V on the manual page). See also WAIT and CUT. ALI NEATIONS Restricts database sequences to the specified number for which high-scoring segment pairs (HSPs) are reported; the limit of failure is 50. If more sequences of databases than this pass to satisfy the threshold of statistical significance to report (see WAIT and CUT below), only the couplings that ascribe the greatest statistical significance are reported. (See parameter B in the BLAST Manual). WAITING The threshold of statistical significance for reporting links against database sequences; the value of failure is 10, so that 10 couplings are expected to be found purely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a coupling is greater than the WAIT threshold, the coupling will not be reported. The low WAIT thresholds are more severe, leading to fewer couplings by luck reporting. The fraction values are acceptable. (See parameter E in the BLAST Manual). CUT Cut score to report high score segment pairs. The fault value is calculated from the ESPE RA value (see above). HSPs are reported for a database sequence only if the statistical significance attached to them is at least as high as it would be ascribed to HSP only having a score equal to the CUT value. High CUT values are more severe, leading to fewer couplings by luck reporting. (See parameter S in the BLAST Manual). Typically, meaning thresholds can be handled more intuitively using WAIT. MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The fault matrix is BLOSUM62 (Henikoff &Henikoff, 1992, Proc. Nati, Acad. Sci. USA 89 (22): 10915-9). Valid alternative choices include: PAM40, PAM 120, PAM250 and I DENTITY. No alternate scoring matrix is available for BLASTN; specifying the directive MATRIX in BLASTN requests returns an error response. FI LAM ENTO Restrict a TBLASTN search to only the upper or lower filament of the database sequences; or restrict a BLASTN, BLASTX, or TBLASTX search to only the reading structures in the upper or lower thread of the query sequence. FI LTRO Hide segments of the query sequence that have low composition complexity, as determined by the Wootton &SEG program. Federhen (1993) Computers and Chemistry 17: 149-163, or segments consisting of internal repetitions of short periodicity, as determined by the XNU program of Claverie & States, 1993, Computers and Chemistry 17: 191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see the NCBI website). Filtering can eliminate statistically significant but biologically uninteresting reports of blast output (for example, hits against common acidic, basic or proline-rich regions), leading to more biologically interesting regions of the query sequence available for specific coupling against sequences of databases. Low complexity sequence found by a filter program is substituted using the letter "N" in nucleotide sequence (for example, "N" repeated 13 times) and the letter "X" in protein sequences (for example, "X" repeated 9 times). Filtering only applies to the query sequence (or its translation products), no sequence of databases. The fault filtering is DUST for BLASTN, SEG for other programs. It is not unusual for all to be hidden by SEG, XNU, or both, when applied to SWISS-PROT sequences, so filtering should not be expected to always produce an effect. In addition, in some cases, the sequences are hidden in their entirety, indicating that the statistical significance of any reported coupling against the unfiltered query sequence should be expected. Causes NCBI-gi NCBI gi identifiers are displayed in the output, in addition to the access and / or location number. More preferably, the sequence comparisons are conducted using the simple BLAST search algorithm provided on the NCBI website described above, in the "/ BLAST" directory. Unless defined otherwise, all the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, in molecular genetic cell culture, nucleic acid chemistry). , techniques of hybridization and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al, Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley &Sons, Inc. which is incorporated herein by reference) and chemical methods. The invention relates to ligands comprising a binding portion having binding specificity for a desired endogenous target compound, to such ligands for use in therapy and to the use of such ligands for the manufacture of a medicament and to therapeutic methods comprising administering such ligands. It has been found that agents that bind soluble endogenous target compounds but do not substantially alter the activity of such compounds, such as antibodies and antibody fragments (eg, Fab fragment, Fab 'fragment, F (ab') 2 fragment, Fv fragment. (eg, scFv, disulfide linked Fv), dAb, diabody), can be used to alter certain properties of the endogenous target compound, and thus make the endogenous target compound more effective in treating, suppressing or mitigating a disease, or for purposes of diagnosis. Accordingly, the invention relates to ligands, as described herein, that bind a soluble endogenous target compound but do not substantially inhibit the activity of the endogenous target compound (eg, they do not bind the active site of the endogenous target compound). ), but which are useful for altering certain properties of the endogenous target compounds. For example, a ligand comprising a binding portion having binding specificity for a desired endogenous target compound can bind the endogenous target compound, thereby increasing the hydrodynamic size of the endogenous target compound and increasing the serum half-life of the endogenous target compound. Beneficially, the endogenous target compound retains its biological activity when bound by the ligand (e.g., it is active in a complex consisting of ligand and endogenous target compound), and may produce beneficial effects for therapeutic and / or diagnostic purposes. The ligand, as described herein, can increase the serum half-life of an endogenous target compound by, for example, reducing the clearance rate of the endogenous target compound. In such a situation, the amount of endogenous target compound in a subject (e.g., in the systemic circulation) may increase to an amount that is greater than the amount normally present in a subject, and the increased amount of endogenous target ligand it can produce beneficial therapeutic effects that are not produced by lower or normal amounts of endogenous target compound. A ligand comprising a binding portion having binding specificity for a desired endogenous target compound can bind the endogenous target compound and increase the bioavailability of the endogenous target compound independent of any change in the in vivo half-life of the endogenous target compound, or of any change in the amount of the endogenous target compound in a subject. For example, a ligand can bind an endogenous target compound that has beneficial activity in the systemic circulation but is rapidly distributed in the tissues or clears (eg, a spheroid or peptide hormone), and decreases the rate of distribution. bución or clearance. Such a ligand may increase the biodi spongibility of the endogenous target compound in the systemic circulation where the activity of the endogenous target compound may have a beneficial (eg, therapeutic) effect. A ligand comprising a binding portion having binding specificity for a desired endogenous target compound can bind the endogenous target compound and increase the activity (e.g., binding activity) of the endogenous target compound. For example, a ligand can bind an endogenous target compound such as an endogenous agonist (eg, a cytokine) or a soluble cytokine receptor and improve the binding activity of the endogenous target compound by increasing the affinity and / or avidity of the target compound endogenous to its endogenous binding partner. For example, a ligand comprising two or more portions having binding sites with binding specificity for a desired cytokine can bind two or more individual cytokine molecules thereby producing a cytokine, trimer, oligomer or multimer dimer. Such a dimer, trimer, oligomer or multimer will have improved binding activity towards, for example, a cell expressing the receptor for the cytokine due to higher avidity. Similarly, the binding activity of soluble receptors (eg, soluble TNFR1) can be increased by a ligand comprising two or more binding sites for soluble TNFR1. Such a ligand can, for example, bind two soluble TNFR1 chains to form a soluble TNFR1 dimer that will bind trimeric TNF with higher avidity than a single TNFR1 chain. The ligands described herein can be administered systemically, for example, to bind endogenous target ligands in the systemic circulation and produce beneficial effects in the systemic circulation. Additionally, the ligands can be administered locally (eg, by subcutaneous injection, intramuscular injection, intraarticular injection, intradermal injection, inhalation, and the like) and thus improve the local bioavailability of an endogenous target compound or to increase the amount of endogenous target compound to the site where the ligand is administered locally. For example, a depot formulation of a ligand can be administered locally by subcutaneous injection, and the administered ligand can recruit endogenous target ligand to the reservoir site and bind the endogenous target ligand to improve local bioavailability or to produce a locally high amount of target ligand. endogenous. Such an approach is advantageous, for example, to promote healing of the wound, or to inhibit local inflammatory reactions (for example, in the skin, in the lung, in a joint). The invention provides several advantages over conventional therapeutic agents and approaches. For example, the ligands described herein are generally small polypeptides that can be produced economically and in large amounts using yeast or bacterial expression systems, rather than more expensive mammalian expression systems that produce lower yields. In addition, the ligands described herein are administered to exploit and exploit the beneficial activities of the compounds (e.g., proteins) that are endogenous to the patient, and generally the ligand is composed of protein domains (e.g., dAb) that are of the same origin as the patient (e.g., human origin) and therefore are substantially non-immunogenic. Advantageously, this significantly reduces or eliminates the risk that the patient will produce neutralizing antibodies of high affinity against the ligand., endogenous target compound-ligand complex or endogenous target compound. This is a distinct advantage over therapeutic approaches comprising administering exogenous compounds, or even recombinantly produced endogenous proteins, which often induce the production of high affinity antibodies in the patient that can restrict or eliminate the efficacy of the therapeutic agent. Ligands Binding an Endogenous Target Compound The invention relates to ligands comprising a portion (e.g., dAb) that has a binding site with binding specificity for an endogenous target compound but does not substantially inhibit the activity of said endogenous target compound. Preferably, the ligand does not bind to the active site of an endogenous target compound. The ligands described herein do not substantially inhibit the activity of the endogenous target compounds to which they bind, and the endogenous target compound remains active when bound by the ligand. For example, a soluble receptor is bound by a ligand of the invention, and the receptor soluble in the resulting receptor-ligand complex can bind the endogenous ligand of the soluble receptor. In another example, an enzyme is bound by a ligand of the invention, and the enzyme in the resulting enzyme-ligand complex can bind substrate and catalyze the enzymatic reaction. Generally, the ligand inhibits the activity of the endogenous target compound to which it binds by no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6% , no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or substantially no inhibitory effect on the activity of the endogenous target compound. In some examples, a ligand that does not substantially inhibit the activity of an endogenous target compound to which it binds, inhibits that activity of said endogenous target compound with a high inhibitory concentration (I C50), such as an IC50 of at least 1 nanomolar. , at least 10 nanomolar, at least 100 nanomolar, at least 1 micromolar, or at least 10 micromolar. The ability of a ligand to inhibit the activity of the endogenous target compound can be assessed using any in vitro or in vivo assay. Many suitable assays are well known in the art and used routinely to assess the inhibitory activity of the compounds. For example, the ability of ligands comprising a moiety that has a binding site for TNFR1 to inhibit TNFR1 activity or can be assessed using in vivo endotoxin shock models or skin necrosis induced by TNF (see, Sheehan et al., J. Exp. Med., 181: 607-61 1 (1995)), and / or the receptor binding assay in vitro, cytoxicity assay L929, assay I L-8 HeLa, or release assay I L-8 M RC-5 described herein. Other suitable assays to assess whether a ligand inhibits the activity of a desired endogenous target compound, such as those described herein, are well known in the art. The ligands per se generally have no substantial therapeutic activity but are used to exploit and exploit the activity of endogenous target compounds for diagnostic and therapeutic purposes. In some embodiments, the ligands do not have substantial activity in an in vitro assay suitable for the activity of an endogenous target compound. For example, a ligand that binds an endogenous target compound and increases the half-life of the endogenous target compound will generally not alter the activity of an endogenous target compound in the assay as compared to the activity of the endogenous target compound in the same assay but without a ligand. Ligands (e.g., dAbs) that bind a desired endogenous target compound, such as a receptor protein, a soluble agonist (e.g., a cytokine), or an enzyme, but do not bind the active site of the endogenous target compound can be identified using any suitable method. For example, individual ligands, or portions having a binding site for an endogenous target compound or collections of such portions (e.g., a library or repertoire) may be selected in an appropriate competition assay in which the binding of a partner Endogenous binding (eg, natural ligand, substrate, cofactor) to the endogenous target compound is assessed in the presence of the ligand or portion (or collection of ligands or portions) being selected. No substantial reduction (e.g., inhibition) in binding of the endogenous binding partner to the endogenous target compound relative to a suitable control indicates that the ligand or portion being selected does not bind the active site of the endogenous target compound. Many assays suitable for selecting ligands and portions comprising a binding site for an endogenous target compound, such as enzymes, receptors (e.g., cytokine receptors, growth factor receptors), and soluble agonists (e.g., cytokines, chemokines, growth factors, hormones) are well known in the art. Additionally, competition assays are used routinely to select collections of compounds containing 106 to 109 or more members. In this way, such selection activity is within the ordinary experience in the matter and is not considered problematic or undue by those in the field. For example, the variable domains described in WO 03002609, WO 2004101790, WO 2005035572, WO 2004061026, WO 2004003019, WO 2004058821, WO 9404678, WO 9749805, WO 9923221, WO 9937681, WO 0024884, WO 0043507, WO 0065067, WO 0140310, WO 03035694, WO 03053531, WO 03054015, WO 0305527, WO 2004015425, WO 2004041862, WO 2004041863, WO 2004041865, WO 2004062551, WO 2005044858 and EP1 134231 can be selected by the ability to bind a desired endogenous target compound without binding to the active site in an adequate competition trial. The description of the variable domains, their sequences and methods of production and selection in the above documents are explicitly incorporated herein by reference to provide expert guidance with examples of portions having a binding site for an endogenous target compound for use in the invention. Ligands can increase the half-life of the targets to which they bind, increase the amount of endogenous targets to which they bind in a subject (for example, the systemic amount, the amount in a desired location or location), and / or increase the bioavailability of the endogenous objectives to which they are linked. A ligand can increase the half-life of an endogenous target compound to which it binds, for example, by binding the endogenous target compound and thereby increase the hydrodynamic size of the endogenous target compound, by binding and stabilizing the endogenous target compound, or by binding and decrease the rate of clearance or metabolism of the endogenous target compound. Generally, the ligand will increase the half-life of the endogenous target compound to which it binds by a factor of at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, relative to the half-life of the endogenous target compound in the absence of ligand. For example, an endogenous target compound may have a half-life of about 1 hour, and after administration of a ligand comprising a portion with a binding site that binds said endogenous target compound, the half-life of the endogenous target compound may be increased. to approximately 1.5 hours, to approximately 10 hours, or more time. The level or amount of endogenous target compound may increase after administration of a ligand, as described herein, due to the effect that increases the half-life of the ligands. For example, the level or amount of endogenous target ligand (e.g., in serum) 1 hour, or 2 hours, or 3 hours, or 4 hours, or 5 hours, or 6 hours, or 7 hours, or 8 hours, or 9 hours, or 10 hours, or 11 hours, or 12 hours, or 1 day after administration of a ligand of the invention can be increased by a factor of at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, less about 50,000, or at least about 100,000. The increase of the levels or amounts of an endogenous target compound can be easily detected using any suitable method. For example, a suitable sample (e.g., serum) can be obtained prior to administration of the ligand and at one or more time points after administration, and the level or amount of endogenous target compound in the samples can be determined using any method suitable. The sample can be, for example, a sample of blood, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage, enema or a biopsy. In some embodiments, the amount or level of ligand in such a sample is increased, relative to the amount or level before the ligand is administered. Ligands can increase the bioavailability of an endogenous target compound. The bioavailability can be increased systemically, or at a desired site of action (eg, at a site where the ligand is administered locally). For example, a ligand can bind an endogenous target compound that has beneficial activity in the systemic circulation but is rapidly distributed in the tissues or clears (e.g., a steroid or peptide hormone), and decreases the rate of distribution or clearance. Such a ligand may increase bioavailability of the endogenous target compound in the systemic circulation where the activity of the endogenous target compound may have a beneficial (e.g., therapeutic) effect. A ligand can increase the bioavailability of an endogenous target compound at a desired site by recruiting the endogenous target compound to that site. For example, a ligand comprising a binding site for a desired endogenous target compound can be administered locally to a patient (e.g., as a depot formulation). The locally administered ligand can bind the desired endogenous target compound, and recruit additional endogenous target compound to the site. In this way, an increased level or amount of endogenous target compound may be present at the site to produce beneficial effects. For example, in one embodiment, the ligand comprises a portion with a binding site for TGF beta 3 isoform. Such a ligand can be administered locally to bind isoform of TGF beta 3 and recruit the TGF beta 3 isoform to the local administration site , and in this way promote the healing of the wound.

Similar ligands comprising a portion with a binding site for a desired cell surface target can be prepared using the methods described herein and used, for example, to recruit a desired cell type to a desired site (eg, a site). of injury, site of inflammation). Generally, the ligand will increase the bioavailability (e.g., in the systemic circulation, at a desired site of action) of the endogenous target compound to which it binds by a factor of at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, relative to the bioavailability of the endogenous target compound in the absence of ligand. For example, when the ligand increases the bioavailability in the systemic circulation, a concentration time curve for the endogenous ligand starting at the time of administration of the ligand can be prepared, and compared to the normal systemic levels of the endogenous target ligand to determine the degree of increase in systemic bioavailability. Ligands can increase the activity (eg, binding activity) of the endogenous targets to which they bind. For example, a ligand can bind an endogenous target compound such as an endogenous agonist (eg, a cytokine) or a soluble cytokine receptor and enhance the binding activity of the endogenous target compound by increasing the affinity and / or avidity of the compound endogenous target for its endogenous binding partner. Ligands comprising two or more portions having binding sites with binding specificity for a desired endogenous target compound can bind two or more individual endogenous target compound molecules thereby producing a dimer, trimer, oligomer or multimer of endogenous target compound. Such a dimer, trimer, oligomer or multimer will have enhanced binding activity towards, for example, a cell expressing the natural binding partner for the endogenous target compound due to higher avidity. Generally, such a ligand can improve the binding activity of the endogenous target compound. For example, the binding intensity (e.g., affinity or avidity) of a dimer, trimer, oligomer or multimer of target compound endogenous to its binding partner can be at least about 10, at least about 100, at least about 1000 or at least about 10,000 times stronger than the binding intensity of the endogenous target compound as a monomer (ie, not in a complex with a ligand of the invention). In one example, the ligand comprises two or more portions that have binding sites with binding specificity for a desired endogenous agonist (eg, a cytokine, a growth factor) and binds two or more endogenous agonist molecules thereby producing a dimer, trimer, oligomer or multimer of endogenous agonist. Such a dimer, trimer, oligomer or multimer will have enhanced binding activity towards, for example, a cell expressing the receptor for the endogenous agonist due to higher avidity, and may have improved therapeutic efficacy. Similarly, the binding activity of a soluble receptor (eg, soluble TNFR1) can be increased by a ligand comprising two or more binding sites for the solid receptor. Such a ligand can, for example, bind two soluble receptor chains to form a soluble receptor dimer that will bind the endogenous receptor ligand with higher avidity than a single receptor chain. In a specific embodiment, the ligand comprises two or more portions (dAbs) that have binding specificity for soluble TNFR 1 but does not bind the active site (the active site of soluble TNFR1 is contained within Domains 2 and 3). Such a ligand can bind two or more chains of soluble TNFR1 to form a dimertrimer, oligomer or multimer of soluble TNFR1, which has enhanced binding activity towards its trimeric ligand, TNF, due to increased avidity. It should also be appreciated that the half-life of such a soluble TNFR1 dimer, trimer, oligomer or multimer, which has a hydrodynamic size larger than a single soluble TNFR1 chain, can also be extended relative to a single TNFR1. As a result, such a ligand can improve the TNF binding activity of soluble TNFR1 and improve its therapeutic efficacy. For example, such a ligand can improve the efficacy of soluble TNFR1 by a factor of at least about 10, at least about 100, at least about 1,000, or at least about 10,000, In one example of this embodiment, a ligand which is a dimer of a dAb that binds Mouse TNFR1 Domain 1 is used to demonstrate that ligands that induce soluble TNFR1 dimerization improve the efficacy of soluble TNFR 1. For example, such a ligand can be added to an assay containing M cells RC5 from human, TNF from human and TNFR1 from soluble mouse. Soluble mouse TNFR1 will compete with human TNFR1 in M RC5 cells for binding to human TNF. The ligand that is a dimer of a dAb that binds mouse TNFR1 domain 1 binds two chains of soluble mouse TNFR1 to form a mouse TNFR1 dimer that will be more effective in inhibiting the effects of TNF in the assay. For example, the IC 50 for mouse TNFR1 can be reduced from the nanomolar range to the picomolar range (approximately 10 or approximately 100 or approximately 1000 fold reduction) by the addition of the ligand that induces the dimerization of soluble TNFR1. Ligand administration that induces dimerization (or timerization or oligomerization) of soluble receptor chains to patients can cause clustering and activation of transmembrane forms of certain receptors that are expressed on the cell surface. Generally, any undesired effects of such activation (eg, activation of immune cells) will be minimal compared to the enhanced binding function and efficacy of the dimerized soluble receptor. However, if desired, the interaction of a dimerization, trimerization or oligomerization ligand with the transmembrane form of a receptor can be further reduced by forming the ligand to increase its hydrodynamic size as described herein. For example, the ligand can be formatted to include a PEG portion or other portion extending the half-life, such as a portion that has a binding site for a polypeptide that improves half-life in vivo (eg, a dAb that binds albumin serum). As shown herein using dAbs that bind TNFR1, PEGylation of dAbs dramatically reduced the ability of dAb to inhibit TNFR1 activity in a cell-based assay, but had almost no effect on the ability of dAbs to bind TNFR1 in a receptor binding assay that approximates the conditions for soluble TNFR1 binding in vivo. These results indicate that PEGylation dAbs and ligands comprising two or more dAbs improve the selectivity of dAbs for soluble receptor over the transmembrane form of the receptor. Some endogenous target compounds naturally bind together to form active dimers, trimers or multimers. In this manner, the portion having a binding site for an endogenous target compound can be the endogenous target compound itself of a fragment or portion of the endogenous target compound. In some embodiments, such as when the portion having a binding site for an endogenous target compound is the endogenous target compound, the ligand may have the activity of the endogenous target compound. Accordingly, the administration of such a ligand can, as described herein, increase the half-life, increase the bioavailability, increase the amount and / or increase the activity of the endogenous target compound, and will also provide additional activity of the endogenous target compound due to the presence of a biologically active form of the endogenous target compound in the ligand. In other embodiments, the ligand may comprise a portion that has a binding site for an endogenous target compound that is a portion of the endogenous target compound. For example, the portion of the endogenous target compound may be a portion that binds to the endogenous target compound in vivo, but does not have the activity (e.g., ligand binding activity, receptor binding activity, catalytic activity) of the target compound endogenous. Examples of endogenous target compounds that naturally form dimers, trimers or multimers include, for example, growth factors (eg, PDGF which forms homodimers and heterodimers of A and B chains), cytokines (e.g., cytokines of the TNF superfamily). example, RANK-L) that forms trimers), and receptors (eg, receptor of the TNF receptor superfamily that forms trimers). Suitable portions of such endogenous target compounds can be easily prepared on the basis of knowledge of the structure of the dimer, trimer or multimer forming domains of the endogenous target compounds, or by preparing peptide or polypeptide fragments of the endogenous target compound and analyzing the peptides or polypeptides for the ability to bind the endogenous target compound (e.g., using surface plasmon resonance or any other suitable method). Illustrative of the portions of endogenous target compounds that bind the corresponding endogenous target compound in vivo are the so-called pre-ligand assembly domains (PLAD) of members of the TNF receptor superfamily. (See, for example, WO 01/58953; US Patent Application Publication UU No. 2003/0108992 Al; Deng et al, Nature Medici ne, doi: 10.1038 / nml304 (2005)). PLAD domains of a particular receptor bind to each other in vivo. For example, the PLAD domain of TNFR1 will bind to another PLAD domain of TNFR 1 in vivo. The TNF receptor superfamily is a group of proteins recognized in the art that includes TNFR1 (p55, CD120a, p60, member of the TNF 1 A receptor superfamily, TNFRSF 1 A), TNFR2 (p75, p80, CD120b, member of the superfamily of receptor TNF 1B, TNFRSF1B), CD (TNFRSF3, LTßR, TNFR2-RP, TNFR-RP, TNFCR, TNF-R-III), OX40 (TNFRSF4, ACT35, TXGPIL), CD40 (TNFRSF5, p50, Bp50), Fas (CD95, TNFRSF6, APO-I, APTI), DcR3 (TNFRSF6B), CD27 (TNFRSF7, Tp55, S152), CD30 (TNFRSF8, Ki-1, D1S166E), CD137 (TNFRSF9, 4-1BB, ILA), TRAILR -1 (TNFRSF10A, DR4, Ap? 2), TRAIL-R2 (TNFRSF10B, DR5, KILLER, TRICK2A, TRICKB), TRAILR3 (TNFRSF10C, DcRI, LIT, TRID), TRAILR4 (TNFRSF10D, DcR2, TRUNDD), RANK (TNFRSF11A ), OPG (TNFRSF11B, OCIF, TRI), DR3 (TNFRSF12, TRAMP, WSL-1, LARD, WSL-LR, DDR3, TR3, APO-3), DR3L (TNFRSF12L), TACI (TNFRSF13B), BAFFR (TNFRSF13C) , HVEM (TNFRSF14, ATAR, TR2, LIGHTR, HVEA), NGFR (TNFRSF16), BCMA (TNFRSF17, BCM), AITR (TNFRSF18, GITR), TNFRSF19, FLJ14993 (TNFRSF19L, RELT), DR6 (TNFRSF21), SOBa (TNFRSF22, Tnfrh2, 2810028K06Rik), mSOB (THFRSF23, Tnfrhl). Several PLAD domains are known in the art and other PLAD domains and variants of PLAD domains can be easily isolated and prepared using any suitable method, such as the methods described in WO 01/58953; U.S. Patent Application Publication No. 2003/0108992 A1; Deng et al, Nature Medicine, doi: 10.1038 / nml304 (2005). Many methods suitable for preparing polypeptides, protein fragments, and peptide variants, as well as also suitable binding assays, such as the TNFR1 receptor binding assay described herein are well known and are conventional in the art. Exemplary PLAD domains are presented in Table 1. Table 1 In some embodiments, the ligand comprises a portion that has a binding site with binding specificity for an endogenous target compound that is PLAD, such as PLAD of TNFR1, TNFR2, FAS, LTβR, CD40, CD30, CD27, HVEM, OX40 , DR4 or another member of the TNF receptor superfamily, or a variant of a PLAD Domain. The variant of a PLAD domain can, for example, be a PLAD domain of TNFR1, TNFR2, FAS, LTβR, CD40, CD30, CD27, HVEM, OX40, or DR4, wherein one or more amino acids have been deleted, inserted or substituted, but retains the ability to bind PLAD of TNFR1, TNFR2, FAS, LT ßR, CD40, CD30, CD27, HVEM, OX40, or corresponding DR4. the amino acid sequence of a variant PLAD domain may comprise a region of at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous non-acidic amino acids, at least about 35 contiguous amino acids, or at least about 40 contiguous amino acids that are the same as the amino acids in the corresponding PLAD amino acid sequence (eg, PLAD of TNFR1, TNFR2, FAS, LTβR, CD40, CD30, CD27, HVEM, OX40, DR4). In addition, or alternatively, the amino acid sequence of a variant PLAD domain can be at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93 %, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding PLAD amino acid sequence (e.g. , PLAD of TNFR1, TNFR2, FAS, LT ßR, CD40, CD30, CD27, HVEM, OX40, or DR4). Ligands comprising a binding portion having a binding site for an endogenous target compound that is PLAD or PLAD variant, can be formatted as described herein. For example, the ligand may be a dimer, trimer or multimer of PLAD or variant of PLAD, or a fusion protein comprising PLAD or variant of PLAD and a portion extending the half-life (eg, a portion having a binding site for serum albumin or neonatal Fe receptor). Ligands comprising two or more PLADs or PLAD variants (eg, dimers, trimers, PLAD multimers) can bind two or more soluble receptor chains to form soluble receptor dimers, trimers or multimers which, as described herein , they may have improved binding function in comparison with the soluble receptor monomers. In some circumstances, such a ligand can bind soluble receptor and also bind the transmembrane form of the receptor expressed on the cell surface. If desired, the selectivity for soluble binding receptor can be improved by formatting the ligand to have a larger hydrodynamic size, for example using a PEG portion, or other portion extending the half-life as described herein. In particular embodiments, the ligand comprises PLAD (eg, PLAD of TNFR1, TNFR2, FAS, LT ßR, CD40, CD30, CD27, HVEM, OX40, or DR4) or PLAD variant and a portion extending the half-life, such as a PEG portion or a dAb that binds serum albumin or neonatal Fe receptor. Preferably, a ligand that induces dimerization (or trimerization or oligomerization) of soluble receptor chains does not agonize substantially the cell surface or transmembrane forms of the receptor in a standard cell assay (i.e., when present at a concentration of 1 nM, nM, 100 nM, 1 μM, 10 μM, 100 μM, 1000 μM or 5,000 μM, results in no more than about 5% of the receptor-mediated activity mediated by the receptor's natural ligand in the assay). It is preferred that the portion having a binding site with binding specificity for an endogenous target compound binds the endogenous target compound with high affinity, such as Kd from 300 nM to 5 pM (ie, 3 x 10 ~ 7 to 5 x 10"12M), preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM and more preferably 1 nM to 100 pM, for example 1 x 10 ~ 7 M or less, preferably 1 x 10" 8 M or less, more preferably 1 x 10"9 M or less, advantageously 1 x 10 ~ 10 M or less and more preferably 1 x 10" 11 M or less; and / or a Kapagada velocity constant of 5 x 10 ~ 1 s "1 to 1 x 10 ~ 7 s" \ preferably 1 x 10"2 s" 1 to 1 x 10"6 s' more preferably 5 x 103 s" at 1 x 10"5 s" 1, for example 5 x 10"1 s" 1 or less, preferably 1 x 10'2 s "1 or less, advantageously 1 x 10" 3 s "1 or less, more preferably 1 x 10 ~ 4 s "1 or less, even more preferably 1 x 10" 5 s "1 or less, and more preferably 1 x 10" 6 s "1 or less as determined by surface plasmon resonance. A portion that has a binding site with binding specificity for an endogenous target compound and binds the endogenous target compound with high affinity can be formatted in any suitable ligand format as described herein. In addition to the characteristics of the ligands described herein, it is preferred that the ligand binds the endogenous target compound with high affinity, such as Kd from 300 nM to 5 pM (i.e., 3 x 10"7 to 5 x 10" 1 M), preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM and more preferably 1 nM to 100 pM, for example 1 x 10"7 M or less, preferably 1 x 10" 8 M or less, more preferably 1 x 10"9 M or less, advantageously 1 x 10" 10 M or less and more preferably 1 x 10 ~ 11 M or less; and / or a Kapagada rate constant of 5 x 10"1 s" 1 to 1 x 10"7 s" 1, preferably 1 x 10"2 s" 1 to 1 x 10"6 s" 1, more preferably 5 x 103 s "1 to 1 x 10" 5 s "1, for example 5 X 10" 1 s "1 or less, preferably 1 x 10" 2 s "1 or less, advantageously 1 x 10" 3 s "1 or less , more preferably 1 x 10"4 s" 1 or less, even more preferably 1 x 10"5 s" 1 or less, and more preferably 1 x 10"6 s" 1 or less as determined by surface plasmon resonance In certain embodiments, the ligand comprises a dAb that binds an endogenous target compound.The endogenous target compound may be, for example, a soluble receptor (eg, soluble cytokine receptor, soluble growth factor receptor, hormone receptor). soluble), an endogenous receptor antagonist (e.g., I L-1 ra), an enzyme, an endogenous agonist (e.g., a cytokine, a growth factor, a hormone.) In some embodiments, the ligand is a dimer , trimer, oligomer or multimer of dAb. The ligands may comprise a portion having a binding site for an endogenous target compound, wherein said portion having a binding site for an endogenous target compound is selected from the group consisting of an affinity, an SpA domain, a class domain A LDL receptor, an EGF domain, and an avimer. The ligand may also comprise a portion having a binding site for an endogenous target compound wherein the portion is an antibody or antibody fragment, such as an Fv fragment, a single chain Fv fragment, a disulfide attached Fv fragment, a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, a diabody, a single immunoglobulin variable domain (eg, human VH, human V, VHHH) If desired, the ligand may further comprise a portion extending the average life as described in the present. For example, the ligand may comprise a portion extending the half-life selected from the group consisting of a portion of polyalkylene glycol, serum albumin or a fragment thereof, transferrin receptor or a transferrin binding portion thereof, or a portion comprising a binding site for a polypeptide that improves half-life in vivo. Suitable portions comprising a binding site for a polypeptide that improves half-life in vivo include an affibrant, an SpA domain, an LDL receptor class A domain, an EGF domain, an avimer, an antibody or antibody fragment, such as a Fv fragment, a single-chain Fv fragment, a disulfide-linked Fv fragment, a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, a diabody, and a unique immunoglobulin variable domain (e.g. Human VH, human VL, VHH) - As described in this, the ligands of the invention do not substantially inhibit the activity of the endogenous target compound to which they bind. However, where an objective has more than one activity (e.g., binding activity and signaling activity), the ligand can inhibit activity that is not associated with the proposed therapeutic activity or benefit. For example, a dAb ligand or monomer can bind a soluble receptor and also bind the transmembrane form of the receptor. In such a situation, the transmembrane form of the receptor can have ligand binding activity and also signaling activity. Preferably, the ligand of the invention will not inhibit the binding activity of the ligand (of the soluble and transmembrane forms of the receptor) which acts to inhibit the ligand-induced processes, but may inhibit the signaling activity of the transmembrane form of the receptor. . To further illustrate the invention, embodiments of exemplary ligands that bind soluble TNFR1 are described below. Modalities of ligands that binds soluble TNFR1 are illustrative of ligands of the invention that bind other receptors. TNFR1 is a transmembrane receptor containing an extracellular region that binds the ligand and an intracellular domain that lacks intrinsic signal transduction activity but can be associated with signal transduction molecules. The TNFR1 complex with bound TNF contains three TNFR 1 chains and three TNF chains. (Banner et al, Cell, 75 (3) 431-445 (1993)). The TNF ligand is present as a trimer, which is linked by three chains TNFR1. (Id.) The three TNFR1 chains are clustered closely together in the receptor-ligand complex, and this clustering is a prerequisite to signal transduction mediated by TNFR1. in fact, multivalent agents that bind TNFR1, such as anti-TNFR1 antibodies, can induce clustering of TNFR1 and signal transduction in the absence of TNF and are commonly used as TNFR1 agonists. (See, for example, Belka et al, EMBO, 14 (6): 1156-U65 (1995); Mandik-Nayak et al, J. Immunol, 167: 1920-1928 (2001)). Accordingly, multivalent agents that bind TNFR1 are generally non-effective antagonists of TNFR1 even if they block the binding of TNFa to TNFR1. The extracellular region of TNFR1 comprises an amino terminal segment of thirteen amino acids (amino acids 1-13 of SEQ ID NO: 2 (human); amino acids 1-13 of SEQ TD NO: 4 (mouse)), Domain 1 (amino acids 14- 53 of SEQ ID NO: 2 (human), amino acids 14-53 of SEQ ID NO: 4 (mouse)), Domain 2 (amino acids 54-97 of SEQ ID NO: 2 (human); amino acids 54-97 of SEQ ID NO: 4 (mouse)), Domain 3 (amino acids 98-138 of SEQ ID NO: 2 (human), amino acids 98-138 of SEQ ID NO: 4 (mouse)), and Domain 4 (amino acids 139-167 of SEQ. ID NO: 2 (human): amino acids 139-167 of SEQ ID NO: 4 (mouse)) which is followed by a region close to the membrane (amino acids 168-182 of SEQ ED NO: 2 (human); amino acids 168- 183 SEQ ID NO: 4 (mouse)). (See, Banner et al, Cell 73 (3) 431-445 (1993) and Loetscher er al., Cell 61 (2) 351-359 (1990)). Domains 2 and 3 make contact with ligand binding (TNFß, TNFa). (Banner et al, Cell, 73 (3) 431-445 (1993)). The extracellular region of TNFR1 also contains a region referred to as the pre-ligand binding domain or PLAD domain (amino acids 1-53 of SEQ ID NO: 2 (human); amino acids 1-53 of SEQ ID NO: 4 ( mouse)) (The US Government WO 01/58953; Deng et al., Nature M eddic, doi: 10.1038 / nml304 (2005)). TNFR 1 diffuses from the cell surface in vivo through a process that includes proteolysis of TNFR1 in Domain 4 or in the region near membrane (amino acids 168-182 of SEQ ID NO: 2; amino acids 168-183 of SEQ ED NO: 4), to produce a soluble form of TNFR1. Soluble TNFR1 retains the ability to bind TNFa, and thus functions as an endogenous inhibitor of TNFa activity. In some embodiments, a ligand or dAb monomer that binds soluble TNFR1 can also bind transmembrane TNFR1 and inhibit TNFa-induced clumping of TNFR1 on the cell surface, which precedes signal transduction through the receptor, but does not inhibit binding of TNFa to TNFR 1 of transmembrane and soluble TNFR1. In particular embodiments, the dAb ligand or monomer does not inhibit the TNF-binding activity of soluble and transmembrane forms of TNFR1 but can inhibit the signaling activity of TNFR1. A dAb ligand or monomer of this type can bind Domain 1 or Domain 4 of TNFR1. Such a dAb ligand or monomer can bind soluble TNFR1 and transmembrane TNFR1, but does not inhibit the TNF binding activity of any form of TNFR1. Accordingly, such a ligand or dAb can be administered, for example, to extend the half-life of soluble TNFR1 or to enhance the binding activity of soluble TN1 FR1. Accordingly, administering such a ligand or dAb monomer to a mammal in need thereof can exploit and exploit the endogenous regulatory pathways that inhibit cell surface TNFR1 activity for therapeutic purposes. In other embodiments, the invention provides similar dAbs ligands and monomers that bind other soluble receptors but do not inhibit the binding of endogenous ligands to soluble receptors. In more particular embodiments, the ligand comprises a dAb that binds Domain 1 of TNFR1 and competes with TAR2m-21-23 for binding to mouse TNFR1 or competes with TAR2h-205 for binding to human TNFR1. In more particular embodiments, the ligand is a dimer of such a dAb and, if desired, also comprises a PEG portion. In certain embodiments, the dAb ligand or monomer is substantially resistant to aggregation. For example, in some embodiments, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less about 3%, less than about 2% or less than about 1% of the dAb aggregate ligand or monomer when a solution of 1-5 mg / ml, 5-10 mg / ml, 10-20 mg / ml, 20-50 mg / ml, 50-100 mg / ml, 100-200 mg / ml or 200-500 mg / ml of ligand or dAb in a solvent that it is routinely used for drug formulation such as saline, regulated salt, citrate regulator salt, water, an emulsion, and, any of these solvents with an acceptable excipient such as those approved by FDA, is maintained at approximately 22 °. C, 22-25 ° C, 25-30 ° C, 30-37 ° C, 37-40 ° C, 40-50 ° C, 50-60 ° C, 60-70 ° C, 70-80 ° C, 15-2O ° C, 10-15 ° C, 5-10 ° C, 2-5 ° C, 0-2 ° C, -10 ° C at 0 ° C, -20 ° C at -10 ° C, - 40 ° C to -20 ° C, -60 ° C to -40 ° C, or -80 ° C to -60 ° C, for a period of time of about, for example, 10 minutes, 1 hour, 8 hours, 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, or 2 years. Aggregation can be assessed using any suitable method, such as, by microscopy, assessing turbidity of a solution by visual inspection or spectroscopy or any other suitable method. Aggregation can be assessed using any suitable method, such as, by microscopy, assessing turbidity of a solution by visual inspection or spectroscopy or any other suitable method. Preferably, aggregation is assessed by dynamic light diffusion. Ligands or dAb monomers that are resistant to aggregation provide several advantages. For example, such dAb ligands or monomers can be easily produced in high production as sun proteins by expression using a suitable biological production system, such as E. coli, and can be formulated and / or stored at higher concentrations than conventional polypeptides. , and with less aggregation and loss of activity. In addition, dAb ligands or monomers that are resistant to aggregation can be produced more economically than other antigen or epitope-binding polypeptides (e.g., conventional antibodies). For example, generally, preparation of epitope-binding polypeptides or antigen proposed for in vivo applications includes processes (e.g., gel filtration) that remove aggregated polypeptides. Failure to remove such aggregates can result in a preparation that is not suitable for in vivo applications because, for example, the aggregates of an antigen-binding polypeptide that is proposed to act as an antagonist, can function as an agonist when inducing the degradation or grouping of the target antigen. Protein aggregates can also reduce the efficacy of therapeutic polypeptide by inducing an immune response in the subject to which they are administered. In contrast, the aggregation resistant ligands or dAb monomers of the invention can be prepared for in vivo applications without the need to include process steps that remove aggregates, and can be used in in vivo applications without the aforementioned disadvantages caused by polypeptide aggregates. . In some embodiments, the dAb ligand or monomer reversibly unfolds when heated to a temperature (Ts) and cooled to a temperature (Te), where Ts is greater than the melting temperature (Tm) of dAb, and It is lower than the melting temperature of dAb. For example, the dAb monomer can be reversibly split when heated to 80 ° C and cooled to about room temperature. A poly peptide that unfolded in a reversible manner loses function when it unfolds but regains function when it is doubled again. Such polypeptides are distinguished from polypeptides that are aggregated when they unfold or that are improperly doubled again (poorly folded poly peptides), ie, they do not regain function. The unfolding and redoubling of the polypeptide can be assessed, for example, by directly or indirectly detecting the polypeptide structure using any suitable method. For example, polypeptide structure can be detected by dichroic smoothing (CD) (e.g., away from UV CD, near UV CD), fl uorescence (for example, fl uorescence of tri ptofan side chains), susceptibility to proteolysis, nuclear magnetic resonance (NMR), or when detecting or measuring a poly function peptide that is dependent on proper folding (eg binding to target binding, binding to a generic ligand). In one example, the polypeptide splitting is assessed using a functional assay in which the loss of binding function (eg, binding of a generic and / or target binding, binding a substrate) indicates that the polypeptide is unfold The degree of unfolding and redoubling of a ligand or dAb monomer can be determined using a bend or denaturing curve. A bend curve can be produced by plotting the temperature as the ordinate and the relative concentration of folded polypeptide as the abscissa. The relative concentration of bent ligand or dAb monomer can be determined directly or indirectly using any suitable method (e.g., CD, fluorescence, binding assay). For example, a solution of dAb monomer or ligand can be prepared and the ellipticity of the solution determined by CD. The ellipticity value obtained represents a relative concentration of bent ligand or dAb monomer of 100%. The dAb ligand or monomer in the solution is then split by increasing the temperature of the solution and the elitivity is determined in appropriate increments (eg, after each increase of one degree in temperature). The dAb ligand or monomer in solution is then doubled again as the temperature of the solution decreases and the ellipticity is determined at appropriate increments. The data can be plotted to produce a bend curve and a bend curve. The bending and bending curves have a characteristic sigmoidal shape that includes a portion in which dAb ligand or monomer molecules unfold to several degrees, and a portion in which dAb ligand or monomer molecules unfold. The intercept of the axis and the redouble curve is the relative amount of redouble ligand or dAb monomer recovered. A recovery of at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90% , or at least about 95% is indicative that the dAb ligand or monomer is reversibly split. In a preferred embodiment, the reverse ability of the splitting of the dAb ligand or monomer is determined by preparing a dAb monomer or ligand solution and plotting the redoubled and doubled curves by heat. The dAb monomer or ligand solution can be prepared in any suitable solvent, such as an aqueous buffer having a suitable pH to allow the dAb ligand or monomer to dissolve (e.g., pH which is about 3 units above or below the isoelectric point (pl)). The dAb monomer or ligand solution is concentrated enough to allow the unfolding / bending to be detected. For example, the dAb or ligand monomer solution may be about 0.1 μM to about 100 μM, or preferably about 1 μM to about 10 μM. If the melting temperature (Tm) of the dAb ligand or monomer is known, the solution can be heated to about ten degrees below the Tm (Tm-10) and the bending evaluated by ellipticity or fluorescence (eg, scan away from UV CD 200 nm at 250 nm, fixed wavelength CD at 235 nm or 225 nm; fluorescent emission spectra of tryptophan at 300 to 450 nm with excitation at 298 nm) to provide 100% bent ligand or relative dAb monomer. The solution is then heated to at least ten degrees above Tm (Tm + 10) in predetermined increments (e.g., increments of about 0.1 to about 1 degree), and ellipticity or fluorescence is determined at each increment. Then, the dAb ligand or monomer folds again upon cooling to at least Tm-10 in predetermined increments and ellipticity or fluorescence is determined at each increment. If the melting temperature of the dAb ligand or monomer is not known, the solution can unfold by heating increasingly from about 25 ° C to about 100 ° C and then refolding as it cools increasingly to at least about 25 ° C. C, and ellipticity or fluorescence at each increase in heating and cooling is determined. The data obtained can be plotted to produce a bend curve and a bend curve, in which the intercept of the y axis of the redouble curve is the relative amount of redoubled protein recovered. The dAb ligand or monomer may comprise any suitable immunoglobulin variable domain, and preferably comprises a human variable domain or a variable domain comprising regions of human structure. In certain embodiments, the dAb monomer comprises a universal structure, as described herein. The universal structure can be a VL (V? Or VK) structure, such as a structure comprising the amino acid sequences of the structure encoded by the immunoglobulin gene segment DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24, DPK25, DPK26 or DPK 28 of the human germline. If desired, the VL structure may further comprise the amino acid sequence of the structure encoded by the immunoglobulin gene segment J? 1, J? 2, J? 3, J? 4, or J? 5 of the germline of human. In other embodiments the universal structure can be a VH structure, such as a structure comprising the amino acid sequences of the structure encoded by the immunoglobulin gene segment DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP38, DP45 , DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66, DP67, DP68 or DP69 of the human germline. If desired, the VH structure may further comprise the amino acid sequence of the structure encoded by the human germline immunoglobulin gene segment JH1, JH2, JH3, JH4, JH4b, JH5 and JH6. In particular embodiments, the dAb monomer ligand comprises the VL DPK9 structure, or a VH structure selected from the group consisting of DP47, DP45 and DP38. The dAb monomer may comprise a binding site for a generic ligand, such as protein A, protein L and protein G. In certain embodiments, the dAb monomer comprises one or more structure regions comprising an amino acid sequence that is the same that the amino acid sequence of a corresponding structure region encoded by a human germline antibody gene segment, or the amino acid sequences of one or more of said structure regions collectively comprise up to 5 amino acid differences relative to the amino acid sequence of said corresponding structure region encoded by a human germline antibody gene segment. In other embodiments, the amino acid sequences of FW1, FW2, FW3 and FW4 of the dAb monomer are the same as the amino acid sequences of corresponding structure regions encoded by a human germline antibody gene segment, or the sequences of amino acids of FW1, FW2, FW3 and FW4 collectively contain up to 10 amino acid differences relative to the amino acid sequences of corresponding structure regions encoded by said segment of human germline antibody gene.

In other embodiments, the dAb monomer comprises regions FW1, FW2 and FW3, and the amino acid sequence of said regions FW1, FW2 and FW3 are the same as the amino acid sequence of corresponding structure regions encoded by antibody gene segments of germinal line of human. In some embodiments, the dAb monomer does not comprise a Camelid immunoglobulin variable domain, or one or more structure amino acids that are unique to immunoglobulin variable domains encoded by Camelid germline antibody gene segments. In certain embodiments, the ligands (e.g., dAb monomers) of the invention are effective in disease models when an effective amount is administered. Generally an effective amount is about 1 mg / kg to about 10 mg / kg (e.g., about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg). Many adequate models of diseases exist. The models of chronic inflammatory disease described herein are recognized by those skilled in the art as being predictive of therapeutic efficacy in humans. The prior art does not suggest using ligands comprising a portion that binds an endogenous target compound, such as a soluble cytokine receptor (eg, soluble TNFR1) in these models, or that would be effective. In particular embodiments, the ligand or monomer of dAb is effective in the standard mouse collagen-induced arthritis model For example, administration of an effective amount of the ligand can reduce the average arthritic score of the sum of the four limbs in the standard mouse collagen-induced arthritis model, for example, from about 1 to about 16, about 3 to about 16, about 6 to about 16, about 9 to about 16, or about 12 to about 16, compared to a suitable control. In another example, administering a Effective amount of ligand can delay the onset of arthritis symptoms in the arthritis model induced by standard mouse collagen, for example, for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days or approximately 28 days, compared to adequate control In another example, administration of an effective amount of the ligand can result in an average arthritic score of the sum of the four limbs in the standard collagen-induced mouse arthritis model. Or about 3, about 3 to about 5, about 5 to about 7, about 7 to about 15, about 9 to about 15, about 10 to about 15, about 12 to about 15, or about 14 to about 15. In other embodiments, the dAb ligand or monomer is effective in the model? ARE of arthritis mouse. (Kontoyiannis et al., J Exp Med 196: 1563-74 (2002)). For example, administration of an effective amount of the ligand can reduce the average arthritic score in the arthritis mouse model "ARE", for example, by about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5, or about 2 to about 2.5, compared to an adequate control. In another example, the administration of an effective amount of the ligand can delay the onset of arthritis symptoms in the mouse arthritis model "ARE" by, for example, about 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 10 days, approximately 14 days, approximately 21 days or approximately 28 days, in comparison with an adequate control. In another example, administration of an effective amount of the ligand can result in an average arthritic score in the arthritis mouse model "ARE" from 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1. .5 to about 2, or about 2 to about 2.5. In other embodiments, the ligand or monomer of dAb is effective in the model mouse ARE of non-inflammatory bowel disease (I BD). (Kontoyiannis ef al, J Exp Med 196: 1563-74 (2002)). For example, the administration of an effective amount of the ligand can reduce the average chronic and / or acute inflammation score in the model IRA of mouse BD, for example, by about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5, or about 2 to about 2.5, compared to an adequate control. In another example, the administration of an effective amount of the ligand can delay the onset of symptoms of BD in the model mouse ARE of BD by, for example, about 1 day, about 2 days, about 3 days, about 4 hours. days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 10 days, approximately 14 days, approximately 21 days or approximately 28 days, in comparison with an adequate control. In another example, administration of an effective amount of the ligand can result in an average chronic and / or acute inflammation score in the mouse model of I BD from 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2, or about 2 to about 2.5. In other embodiments, the dAb ligand or monomer is effective in the dextran sulfate (DSS) sodium induced I BD model. See, for example, Okayasu I. et al, Gastroenterology 98: 694-702 (1990); Podolsky K., J Gasteroenterol 38 suppl X \ /: 63-66 (2003)). For example, administration of an effective amount of the ligand can reduce the average severity score in the mouse DSS model of I BD, for example, from about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5, or about 2 to about 2.5, compared to an adequate control. In another example, administration of an effective amount of the ligand can delay the onset of IBD symptoms in the mouse DSS model of I BD by, for example, about 1 day, about 2 days, about 3 days, about 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 10 days, approximately 14 days, approximately 21 days or approximately 28 days, in comparison with an adequate control. In another example, administration of an effective amount of the ligand can result in an average severity score in the mouse DSS model of I BD from 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5. about 1.5 to about 2, or about 2 to about 2.5. In particular embodiments, the dAb ligand or monomer is effective in the mouse tobacco smoke model of chronic obstructive pulmonary disease (COPD). (See, Wright and Churg, Chest, 122: 301-306 (2002), Groneberg, DA er al, Respiratory Research 5: 18 (2004), Coffman RL et al, J. Exp. Med. 207 (12): 1875 -1879 (2001), Van Scott, MR et al, J. App. Physiol. 96: 1433-1444 (2004)). In additional embodiments, the dAb monomer ligand is effective in an animal model of asthma (see, Coffman RL et al, J. Exp. Med. 207 (12): 1875-1879 (2001); Van Scott, MR. , J. App. Physiol. 96: 1433-1444 (2004)), pulmonary fibrosis (eg, Venkatesan, N et al., Lung 287: 1342-1347 (2004), systemic lupus erythematosus (SLE) (Knight et al. (1978) J. Exp. Med., 147: 1653, Reinersten et al. (1978) New Eng. J. Med., 299: 515), myasthenia gravis (Lindstrom er al. (1988) Adv. Immunol., 42: 233), arthritis (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233; Van Eden et al. (1988) Nature, 331: 171), thyroiditis (Marón et al (1980) J. Exp. Med., 152: 1 15), insulin-dependent diabetes mellitus (I DDM) (Kanasawa et al., 1984). ) Diabetologia, 27: 1 13), and the EAE model of multiple sclerosis (see Paterson (1986) Textbook of Immunopathology, Mischer et al., Eds., Grunt and Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al (1987) J. Immunol, 138: 179). Preferably, the dAb ligand or monomer is secreted in an amount of at least about 0.5 mg / L when expressed in E. coli or Pichia species (eg, P. pastoris). In other preferred embodiments, the dAb monomer is secreted in an amount of at least about 0.75 mg / L, at least about 1 mg / L, at least about 4 mg / L, at least about 5 mg / L, at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, or at least about 50 mg / L, or at least about 100 mg / L, or at least about 200 mg / L, or at least about 300 mg / L, or at less about 400 mg / L, or at least about 500 mg / L, or at least about 600 mg / L, or at least about 700 mg / L, or at least about 800 mg / L, at least about 900 mg / L , or at least about 1 g / L when expressed in E. coli or Pichia species (for example, P. pastoris). In other preferred embodiments, the dAb monomer is secreted in an amount of at least about 1 mg / L to at least about 1 g / L., at least about 1 mg / L to at least about 750 mg / L, at least about 100 mg / L to at least about 1 g / L, at least about 200 mg / L to at least about 1 g / L, at least about 300 mg / L to at least about 1 g / L, at least about 400 mg / L to at least about 1 g / L, at least about 500 mg / L to at least about 1 g / L, at less about 600 mg / L to at least about 1 g / L, at least about 700 mg / L to at least about 1 g / L, at least about 800 mg / L to at least about Ig / L, or at least about 900 mg / L to at least about Ig / L when expressed in E. coli species or in Pichia (eg, P. pastoris). Altho dAb ligands and monomers described herein can be secreted when expressed in E. coli or Pichia species (eg, P. pastoris), they can be produced using any suitable method, such as synthetic chemical methods or production methods. that do not use E. coli or Pichia species. In some embodiments, the ligand is an antibody that has binding specificity for an endogenous target compound or an antigen-binding fragment thereof, such as a Fab fragment, Fab 'fragment, F (ab') 2 fragment or Fv fragment ( for example, scFV). In other embodiments, the antagonist or ligand is monovalent, such as a dAb or a monovalent antigen-binding fragment of an antibody, such as a Fab fragment, Fab 'fragment, or Fv fragment. In other embodiments of the invention described throut this description, instead of the use of a "dAb" in a ligand of the invention, it is contemplated that the skilled person can use a domain comprising the CDRs of a dAb that binds an endogenous target compound (eg, CDRs grafted into a suitable protein structure or micro-scaffold, eg, an affibody, a SpA micro-scaffold, a class A domain of the LDL receptor or an EGF domain) or can be a protein domain comprising a binding site for TNFR1, for example, wherein the domain is selected from an affibody, an SpA domain, a class A domain of the LDL receptor or an EGF domain. The description as a whole should be constructed in accordance with the foregoing to provide description of antagonists, ligands and methods used such domains instead of a dAb. In addition, the ligands of the invention can be formatted (eg, extended half-life formats) as described herein. Uses of Ligands that Link an Endogenous Objective Compound and Therapeutic Methods The invention relates to ligands that comprise a portion (e.g., dAb) that has a binding site with binding specificity for an endogenous target compound but not substantially the activity of said endogenous target compound. Preferably, the ligand does not bind to the active site of an endogenous target compound. Additional characteristics of the ligands of the invention are described in the present. For awareness and to avoid repetition, not all of the described characteristics of the ligands are specifically described with respect to the uses and methods of the invention. It is proposed that the uses and methods of the invention include the use, and methods comprising adm or strar, of any of the ligands described herein. The invention relates to compositions comprising a ligand comprising a portion (e.g., dAb) that has a binding site with binding specificity for an endogenous target compound, but not substantially the activity of said endogenous target compound, and a pharmaceutically acceptable carrier, diluent or excipient. The invention also relates to diagnostic and therapeutic methods employing the ligands or compositions of the invention. The ligands described herein can be used for prophylactic and therapeutic applications in vivo, in vivo diagnostic applications and the like. For example, ligands (e.g., dAb monomers) of the present invention will typically find use in preventing, suppressing or treating diseases (e.g., chronic and / or acute inflammatory diseases). For example, antagonists and / or ligands can be administered to treat, suppress or prevent an inflammatory disease (e.g., acute and / or chronic inflammation), cancers and neoplasms (e.g., lymphomas (e.g., B-cell lymphomas). , T cell lymphomas, acute myeloid lymphoma), multiple myeloma, lung cancer, carcinomas), metabolic diseases (eg, diabetes, obesity), allergic hypersensitivity, viral or bacterial infection, autoimmune disorders (including, but not limited to a, Type I diabetes, asthma, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, spondylarthropathy (e.g., ankylosing spondylitis)), systemic lupus erythematosus, inflammatory bowel disease (e.g. of Crohn, ulcerative colitis), myasthenia gravis and Behcet syndrome), psoriasis, endometriosis, abdominal adhesions (for example, post abdominal surgery ), osteoarthritis, chronic obstructive pulmonary disease, or other disease that is suitable for therapy with an endogenous target compound. In the present application, the term "prevention" includes administration of the protective composition prior to the induction of the disease. "Suppression" refers to the administration of the composition after an inductive event, but before the clinical appearance of the disease. "Treatment" includes the administration of the protective composition after the symptoms of the disease manifest. The invention relates to the use of a ligand, as described herein, for the manufacture of a medicament that can be administered to a patient to take advantage of and exploit the beneficial activities of endogenous compounds to produce beneficial effects (eg, therapeutically effects). beneficial, beneficial effects in a diagnosed way). Accordingly, the invention relates to the use of a ligand for the manufacture of a medicament for increasing the amount of said endogenous target compound in a subject (e.g., the amount of the endogenous target compound in the systemic circulation, the amount at a particular site, such as a particular organ, tissue or region (eg, in a joint, in the lung, in a muscle, on the skin site, in the CNS)), to increase the bioavailability of said target compound endogenous in a subject (eg, systemically or at a desired site of action (eg, in a joint, in the lung, in a muscle, on the skin site, in the CNS)), to increase the half-life in vivo of said endogenous target compound, or to treat a disease that is suitable for treatment with an endogenous target compound (eg, such as a disease as described herein), for example. The medication can be for local or systemic administration. The invention relates to the use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the half-life of said endogenous target compound in a subject. The medicament can be administered to a patient to increase the half-life of an endogenous target compound to which the ligand binds by, for example, binding the endogenous target compound and thereby increase the hydrodynamic size of the endogenous target compound, by binding and stabilizing the compound endogenous target, or by binding and decreasing the rate of clearance or metabolism of the endogenous target compound. Generally, the ligand will increase the half-life of the endogenous target compound to which it binds by a factor of at least about 1.5., at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, in relation to the half-life of the endogenous target compound in the absence of ligand. For example, an endogenous target compound may have a half-life of about 1 hour, and after administration of a ligand comprising a portion with a binding site that binds said endogenous target compound, the half-life of the endogenous target compound may increase to approximately 1.5 hours, approximately 10 hours, or more. The invention relates to the use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the amount of said endogenous target compound in a subject. The level or amount of endogenous target compound may increase after administration of a ligand, as described herein, for example, due to the increasing effect of the half-life of the ligands. For example, the level or amount of endogenous target ligand (e.g., in serum) 1 hour, or 2 hours, or 3 hours, or 4 hours, or 5 hours, or 6 hours, or 7 hours, or 8 hours, or 9 hours, or 10 hours, or 11 hours, or 12 hours, or 1 day after administration of a ligand of the invention can be increased by a factor of at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, less about 50,000, or at least about 100,000. Increasing the levels or amounts of an endogenous target compound can be easily detected using any suitable method. For example, a suitable sample (e.g., serum) can be obtained prior to administration of the ligand and at one or more time points after administration, and the level or amount of endogenous target compound in the samples can be determined using any method suitable. The sample can be, for example, a sample of blood, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage, enema or a biopsy. In some embodiments, the amount or level of ligand in such a sample is increased, relative to the amount or level before the ligand is administered. The invention relates to the use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the bioavailability of said endogenous target compound in a subject. The medicament may be administered to a subject to increase the bioavailability of an endogenous target compound systemically, or at a desired site of action (eg, at a site where the ligand is administered locally). For example, a ligand can bind an endogenous target compound that has beneficial activity in the systemic circulation but is rapidly distributed in the tissues or clears (e.g., a steroid and peptide hormone), and decrease the rate of distribution or clearance. Such a ligand may increase the bioavailability of the endogenous target compound in the systemic circulation where the beneficial activity of the endogenous target compound may have a beneficial (e.g., therapeutic) effect. A ligand can increase the bioavailability of an endogenous target compound at a desired site by recruiting the endogenous target compound to that site. For example, a ligand comprising a binding site for a desired endogenous target compound can be administered locally to a patient (e.g., as a deposit formulation). The locally administered ligand can bind the desired endogenous target compound, and recruit the additional endogenous target compound to the site. In this manner, an increased amount or level of endogenous target compound may be present at the site to produce beneficial effects. For example, in one embodiment, the ligand comprises a portion with a binding site for TGF beta 3 isoform. Such a ligand can be administered locally to bind isoform of TGF beta 3 and recly the TGF beta 3 isoform at the site of administration. local, and in this way promote the healing of the wound. Generally, the ligand will increase the bioavailability (eg, in the systemic circulation, at a desired site of action) of the endogenous target compound to which it binds by a factor of at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, relative to the bioavailability of the endogenous target compound in the absence of ligand. For example, when the ligand increases the bioavailability in the systemic circulation, a concentration time curve for the endogenous ligand starting at the time of administration of the ligand can be prepared, and the curve and area under the curve (AUC) can be compared to levels Normal systemic endogenous target ligands to determine the degree of increase in systemic bioavailability. The invention relates to the use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the activity of said endogenous target. Ligands can increase the activity (eg, binding activity) of the endogenous targets to which they bind. For example, a ligand can bind an endogenous target compound such as an endogenous agonist (eg, a cytokine) or a soluble cytokine receptor and enhance the binding activity of the endogenous target compound by increasing the affinity and / or avidity of the compound endogenous target for its endogenous binding partner. Ligands comprising two or more portions having binding sites with binding specificity for a desired endogenous target compound can bind two or more individual endogenous target compound molecules thereby producing a dimer, trimer, oligomer or multimer of endogenous target compound. Such a dimer, trimer, oligomer or multimer will have enhanced binding activity towards, for example, a cell expressing the natural binding partner for the endogenous target compound due to higher avidity. Generally, such a ligand can improve the binding activity of the endogenous target compound. For example, the binding intensity (e.g., affinity or avidity) of a dimer of target compound endogenous to its binding partner can be at least about 10, at least about 100, at least about 1,000, or at least about 10,000 times stronger than the binding intensity of the endogenous target compound as a monomer (ie, not in a complex with a ligand of the invention). In a specific embodiment, the ligand comprises two or more portions (dAbs) that have binding specificity for soluble TNFR 1 but does not bind the active site (the active site of TNFR1 sol uble is contained within Domains 2 and 3). Such a ligand can bind two or more soluble TN1 TN chains to form a soluble TNFR1 dimer, trimer, oligomer or multimer, which has enhanced binding activity towards its trimeric ligand, TNF, due to increased avidity. It should also be appreciated that the half-life of such a soluble TNFR 1 dimer, trimer, oligomer or multimer, which has a hydrodynamic size larger than a single soluble TNFR1 chain, it can also be extended in relation to a single TNFR1. As a result, such a ligand can improve the TNF-binding activity of TN FR 1 sol and improve its therapeutic efficacy. For example, such a ligand can improve the efficacy of soluble TNFR1 by a factor of at least about 10, at least about 100, at least about 1,000, or at least about 10,000. In one example of this embodiment, a ligand that is a dimer of a dAb binding mouse TNFR1 domain 1 is used to demonstrate that ligands that induce soluble TN1 TN dimerization improve the efficacy of soluble TNFR1. For example, such a ligand can be added to an assay containing M cells RC5 from human, TNF from human and TNFR1 from soluble mouse. TNFR 1 of soluble mouse will compete with human TNFR1 in M cells RC5 with human TNF. The ligand that is a dimer of a dAb binding mouse TNFR1 domain 1 binds two chains of soluble mouse TNFR 1 to form a mouse TNFR1 dimer that will be much more effective at inhibiting the effects of TNF in the assay. For example, the IC 50 for mouse TNFR1 can be reduced from the nanomolar range to the picomolar range (approximately 10 or approximately 100 or approximately 1000 fold reduction) by the addition of the ligand that induces the dimerization of soluble TNFR1. Preferably, ligand that induces dimerization (or trimerization or oligomerization) of soluble receptor chains does not agonize substantially the transmembrane or cell surface forms of the receptor in a standard cell assay (ie, when present in a concentration of 1 nM, 10 nM , 100 nM, 1 μM, 10 μM, 100 μM, 1000 μM or 5,000 μM, results in no more than about 5% of the receptor-mediated activity mediated by the receptor's natural ligand in the assay). In a particular embodiment, the invention relates to the use of a ligand comprising two or more portions having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the binding activity of said endogenous target compound, wherein said ligand binds said endogenous target, does not bind the active site of said endogenous target, and does not substantially inhibit the binding activity of said endogenous target compound. The invention also relates to a ligand that binds an endogenous target compound having suitable activity for treating a disease in a subject, wherein said ligand does not bind the active site of said endogenous target compound or substantially inhibits the activity of said endogenous target compound, for use in therapy of a disease suitable for treatment with said endogenous target compound. For example, the ligand can increase the in vivo half-life of said endogenous target compound, increase the amount of said endogenous target compound in a subject, increase the bioavailability of said endogenous target compound, and / or increase the binding activity of said compound. endogenous.

The invention relates to the use of a ligand comprising a binding portion having a binding site for an endogenous target compound for increasing the half-life, biodi spontability or activity of said endogenous compound, wherein said binding portion is has a binding site for an endogenous compound is said endogenous compound or a portion or variant thereof, and wherein said ligand binds said endogenous target compound and does not substantially inhibit the activity of said endogenous target compound. The invention relates to the use of a ligand comprising a binding portion that has a binding site for a member of the TNF receptor superfamily to increase the half-life, biodi sponsibility or activity of said member of the receptor superfamily. TN F, wherein said ligand binds said my abro of the TNF receptor superfamily and does not substantially inhibit the activity of said member of the TN receptor superfamily F, and wherein said binding portion having a binding site for a my embryo of the TNF receptor superfamily is a pre-ligand assembly domain (PLAD) or a variant thereof. The invention relates to a method for increasing the half-life of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with specificity. of binding for said endogenous target compound.

The invention relates to a method for increasing the amount of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said endogenous target compound. The invention relates to a method for increasing the bioavailability of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said endogenous target compound. The invention relates to a method for increasing the activity (eg, binding activity) of an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a site. of binding with binding specificity for said endogenous target compound. The invention relates to a method for treating a subject having a disease that is suitable for treatment with an endogenous target compound in a subject, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said endogenous target compound.

The invention relates to a method for increasing the half-life, bioavailability or activity of an endogenous target compound, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a binding site with binding specificity for said endogenous target compound, wherein said binding portion having a binding site for an endogenous compound is said endogenous compound or a portion or variant thereof, and wherein said ligand binds said endogenous target compound and does not substantially inhi- activity of said endogenous target compound. The invention relates to a method for increasing the half-life, bioavailability or activity for a member of the TNF receptor superfamily, comprising administering to a subject in need thereof an effective amount of a ligand comprising a binding portion having a of binding for a member of the TNF receptor superfamily, wherein said ligand binds said member of the TNF receptor superfamily and does not substantially inhibit the activity of said member of the TNF receptor superfamily., and wherein said binding portion having a binding site for a member of the TNF receptor superfamily is a pre-ligand assembly domain (PLAD) or a variant thereof. Prophylactic and therapeutic uses of ligands of the invention include the administration of ligands according to the invention to a recipient mammal, such as a human. Multi-specific and dual-specific ligands (eg, dual-specific antibody formats) bind to multimeric antigen with higher avidity. Dual or multi-specific ligands may allow the degradation of two antigens, for example to produce high-avidity dimers, trimers or multimers of soluble receptors. Substantially pure ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is more preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or homogeneously as desired, the ligands can be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent dyes and the like (Lefkovite and Pernis, (1979 and 1981) I mmunological Methods, Volumes I and II, Academic Press, NY). Generally, the ligands will be used in purified form together with pharmacologically appropriate vehicles. Typically, these vehicles include alcoholic or aqueous / aqueous solutions, emulsions or suspensions, any including saline and / or regulated media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride and lactated Ringer. Physiologically acceptable adjuvants, if necessary to maintain a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates. Intravenous vehicles include fluid and nutrient fillers and electrolyte fillers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition). A variety of suitable formulations can be used, including extended release formulations. The route of administration of pharmaceutical compositions according to the invention can be any of those commonly used by those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the ligands selected therefrom of the invention can be administered to any patient according to standard techniques. The administration can be by any appropriate means, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, through the pulmonary route, or also, appropriately, by direct infusion with a catheter. Parenteral administration can also be by infusion or intraarterial, intrathecal, intraarticular, subcutaneous, or other injection. Additional suitable modes of administration include pulmonary administration, intranasal, intravaginal administration, intrarectal administration (eg, by suppository or enema). The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, contraindications and other parameters to be taken into account by the doctor. Administration may be local (eg, local delivery to the lung by pulmonary administration, eg, intranasal administration) or systemic as indicated. The ligands of the present invention can be used as compositions administered separately or in conjunction with other agents. These may include various immunotherapeutic drugs, such as cyclosporin, methotrexate, adriamycin or cisplatin, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents together with the antagonists (e.g., ligands) of the present invention, or even combinations of ligands according to the present invention having different specificities, such as ligands selected using different epitopes or target antigens, whether or not they are pooled before administration. Generally, the ligand and any additional agents are administered in a manner that provides a coating of therapeutic effect. In certain embodiments, the ligand comprising a portion having a binding site for an endogenous target compound is administered together with a low dose of the endogenous target compound (eg, a recombining form of the endogenous target compound). As described herein, the ligand may, for example, increase the half-life or bioavailability of the endogenous target compound, or increase the activity of the endogenous target compound, by returning to the therapeutically effective low dose. This therapeutic approach is advantageous because the side effects associated with doses of higher agents for treating disease (eg, recombinant forms of endogenous target proteins) can be avoided. In other embodiments, a ligand that has been preloaded with an endogenous target compound (eg, incubated with and that allows a recombinant form of an endogenous target compound to be attached) is administered. The pre-charged ligands are well suited for local administration to provide high bioavailability of an endogenous target ligand that is normally present at sub-therapeutic levels or that is difficult to recruit at a site where the activity of the endogenous target ligand is desired. Ligands according to the invention that are capable of binding to extracellular targets included in endocytosis (eg, Clatrin) can be endocytosed, allowing access to intracellular targets. In addition, dual or multispecific ligands provide a means by which a binding domain (eg, a dAb monomer) that is specifically capable of binding to an intracellular target can be delivered in an intracellular environment. This strategy requires, for example, a specific dual ligand with physical properties that allow it to remain functional within the cell. Alternatively, if the intracellular compartment of final destination is oxidized, a well-bent ligand may not need to be free of disulfide. Advantageously, the dual and multispecific ligands can be used for target cytokines and other molecules cooperating synergistically in therapeutic situations in the body of an organism. The invention therefore provides a method for synergizing the activity of two or more binding domains (e.g., dAbs) that bind cytokines or other molecules, comprising administering a dual or multispecific ligand capable of binding to said two or more molecules (eg. example, cytokines). In this aspect of the invention, the dual or multispecific ligand may be any dual or multispecific ligand, including a ligand composed of complementary and / or non-complementary domains, a ligand in an open conformation, and a ligand in a closed conformation. For example, this aspect of the invention relates to combinations of VH domains and V domains, VH domains only and VL domains only. If nergia in a therapeutic context can be achieved in a number of ways. For example, target combinations can be therapeutically active only if both targets are targeted by the ligand, while the objective of only one objective is not therapeutically effective. In another modality, an objective can only provide a somewhat low or minimal therapeutic effect, but together with a second objective the combination provides a synergistic increase in therapeutic effect Animal model systems that can be used to select the effectiveness of the ligands to protect against or treat the disease are available The appropriate animal model can be used, such as the models described herein. The ingredients of this invention can be lyophilized for storage and reconstituted in a suitable vehicle before use. This technique has been shown effective with conventional immunoglobulins and reconstitution and lyophilization techniques known in the art can be used. by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of loss of antibody activity (eg, with conventional immunoglobulins, IgM antibodies tend to have greater loss of activity than IgG antibodies) and what levels of use may be have to adjust up to compensate The compositions containing the present ligands or one of them can be administered for therapeutic and / or prophylactic treatments In certain therapeutic applications, a suitable amount to perform at least partial inhibition, suppression, modulation, death te, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically effective dose". The amounts needed to achieve this dosage will depend on the severity of the disease and the general state of the patient's own immune system, but will generally vary from 0.005 to 5.0 mg of ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg / dose being used more commonly. For prophylactic applications, compositions containing the present ligands or cocktails thereof can also be administered in similar or slightly lower dosages, to prevent, inhibit or delay the onset of disease (eg, to sustain remission or quiescence, or to prevent the acute). The skilled physician will be able to determine the appropriate dosage range to treat, suppress or prevent the disease. When a TNFR1 antagonist (eg, ligand) is administered to treat, suppress or prevent a chronic inflammatory disease, it can be administered up to four times per day, twice weekly, once weekly, once every two weeks, once a month , or once every two months, at a dose, for example, about 10 μg / kg to about 80 mg / kg, about 100 μg / kg to about 80 mg / kg, about 1 mg / kg to about 80 mg / kg , about 1 mg / kg to about 70 mg / kg, about 1 mg / kg to about 60 mg / kg, about 1 mg / kg to about 50 mg / kg, about 1 mg / kg to about 40 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 10 mg / kg, about 10 μg / kg to about 10 mg / kg, about 10 μg / kg to approximately 5 mg / kg, approximately 10 μg / kg to approximate 2.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg or approximately 10 mg / kg. In particular embodiments, the TNFR1 antagonist (e.g., ligand) is administered to treat, suppress or prevent a chronic inflammatory disease once every two weeks or once a month at a dose of about 10 μg / kg to about 10 mg / kg (eg, about 10 μg / kg, about 100 μg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg or approximately 10 mg / kg.). The ligands may also be administered (eg, systemically or locally), for example, at a dose of about 1 mg / day to about 10 mg / day (eg, 10 mg / day, 9 mg / day, 8 mg / day, 7 mg / day, 6 mg / day, 5 mg / day, 4 mg / day, 3 mg / day, 2 mg / day, or 1 mg / day). Accordingly, the agent can be administered locally to lung tissue at a dose of about 1 μg / kg / day to about 200 μg / kg / day (eg, about 10 μg / kg / day, about 20 μg / kg / day, approximately 30 μg / kg / day, approximately 40 μg / kg / day, approximately 50 μg / kg / day, approximately 60 μg / kg / day, approximately 70 μg / kg / day, approximately 80 μg / day kg / day, approximately 90 μg / kg / day, approximately 100 μg / kg / day, approximately 10 μg / kg / day, approximately 120 μg / kg / day, approximately 130 μg / kg / day, approximately 140 μg / kg / day, approximately 150 μg / kg / day, approximately 160 μg / kg / day, approximately 170 μg / kg / day, approximately 180 μg / kg / day, or approximately 190 μg / kg / day). Treatment or therapy performed using the compositions described herein is considered "effective" if one or more symptoms are reduced (eg, by at least 10% or at least one point on a clinical assessment scale), in relation to such symptoms present before treatment, or in relation to such symptoms in an individual (human or animal model) not treated with such a composition or other suitable control. The symptoms will obviously vary depending on the disease or objective disorder, but can be measured by an ordinary expert technician or doctor. Such symptoms can be measured, for example, by monitoring the level of one or more biochemical indicators of the disease or disorder (eg, of an enzyme or metabolite correlated with the disease, affected cell numbers, etc.), by monitoring physical manifestations (eg, inflammation, tumor size, etc.) or by a accepted clinical rating scale, for example, the Scale of the State of I ncapacity Expanded (for multiple sclerosis), the Irvine Nerve Inflammatory Bowel Disease Questionnaire (32-point assessment evaluates quality of life with respect to bowel function, systemic symptoms, social function and emotional state, ranges of score from 32 to 224, with higher scores indicating a better quality of life), the Arthritis Scale Quality Reumat oide in Life, the St George's Respiratory Questionnaire, or other accepted clinical assessment as known in the subject A sustained reduction (for example, one day or more, preferably longer) in symptoms of disorder or disease by at least 10% or by one or more points on a given clinical scale is indicative of "effective" treatment Similarly, prophylaxis performed using a composition as described herein is "effective" if the onset or severity of one or more symptoms is delayed , reduces or eliminates in relation to such symptoms in a similar individual (animal or human model) not treated with the composition A composition containing a ligand or cocktail thereof according to the present invention can be used in therapeutic and prophylactic establishments to assist in the alteration, inactivation, death or withdrawal of a selected target cell population in a mammal. moreover, the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, eliminate or otherwise effectively remove a target cell population from a heterogeneous collection of cells. The blood of a mammal can be combined extracorporeally with the ligands, for example, antibodies, cell surface receptors or binding proteins thereof by which the unwanted cells die or otherwise remove from the blood to return to the mammal. according to standard techniques. A composition containing a ligand according to the present invention can be used in therapeutic or prophylactic settings to aid in the alteration, inactivation, death or removal of a selected target cell population in a mammal. In addition, the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, eliminate or otherwise effectively remove a target cell population from a heterogeneous collection of cells. The blood of a mammal can be combined extracorporeally with the ligands, for example, antibodies, cell surface receptors or binding proteins thereof by which unwanted cells die or otherwise remove from the blood to return to the mammal in accordance with standard techniques.

Endogenous Objective Compounds Suitable endogenous target compounds include, for example, soluble cytokine receptors (e.g., soluble TN FR1, soluble TNFR2, soluble I L-1 receptor, soluble I L-4 receptor, soluble I L-13 receptor), endogenous receptor antagonists (I L-1 ra, I L-6ra), enzymes (e.g., beta-glucocerebrosidase (EC 3.2.1 .45)) , blood factors (eg, Factor II, Factor VII), erythropoietin, growth hormone, TPO, interferon-alpha, interferon-beta, interferon-gamma, GLP-1, OXM, sex hormones (eg, testosterone) , estrogen), bone morphogenic proteins (eg, BM P-1, BMP-2, BM P-3, BM P-4, BM P-5, BM P-6, BM P-7), isoform beta 1 Transforming growth factor (TGF-beta), TGF-beta 2 isoform, TGF-beta 3 isoform, I L-10, I L-2, macrophage-granulocyte colony stimulating factor, granulocyte colony stimulating factor, insulin , glucagon, GI P. Suitable endogenous target compounds include cytokines, such as interleukins, interferons, colony stimulating factors and chemokines. Examples of such cytokines include, interleukins IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15 (IL-T), IL16, IL17, IL17B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, IL-31 and IL-32, and interferons IFN-alpha, IFN-beta, IFN-delta, IFN-gamma, IFN-kappa, IFN-lambda-1, IFN-lambda-2, IFN-lambda-3, IFN-omega, and IFN-tau. Suitable endogenous target compounds include chemokines such as CCF18, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, ECEP-1, EDNAP, ENA-78, ENAP, ENAP-alpha, ENAP-beta, endothelial cell growth inhibitor, endothelial IL8, FIC, FDNCF, FINAP, GDCF-2, GCF, GCP-2, GRO-alpha, GRO -beta, GRO-gamma, neutralizing protein of Heparin, Humig, I-309, ILC, ILINCK, MAC, l-TAC, IL8, IP-9, IP-10, Lymphotactin, LAG-1, LARC, LCC-1, LD78-alpha, LD78-beta, LD78-gamma, LDCF, LEC, Lkn-1, CML, LAI, LCF, LA-PF4, LDGF, LDNAP, LIF, LIX, MARC, MCAF, MCIF, Mexiquina, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MDC, MEC, MIP-1-alpha, MIP-1-beta, MIP-1-delta, MIP-1-gamma, MIP-3, MIP- 3-alpha, MIP-3-beta, MIP-4, MIP-4-alpha, MIP-5, Monotactin-1, MPIF -1, MPIF-2, MRP-1, MRP-2, MDGF, MDNAP, MDNCF, megakaryocyte stimulating factor, MGSA, MGSA-alpha, MGSA-beta, Mig, MIP-2, MIP-2-alpha, MIP- 2-beta, MIP-2-gamma, NAF, NAP-1, NAP-2, NAP-3, NAP-4, NCP, Oncostatin A, PARC, PBP, as PBP, PBSF, PF4, PLF, PPBP, RANTES, Regaquina-1, SCM-1-alpha, SCYC1, SCYC2, SCI, SCYA1, SCYA2, SCYA3, SCYA4, SCYA5, SCYA6, SCYA7, SCYA8, SCYA9, SCYA10, SCYA11, SCYA12, SCYA13, SCYA14, SCYA15, SCYA16, SCYA17, SCYA19, SCYA20, SCYA21, SCYA22, SCYA23, SCYA24, SCYA25, SCYA26, SCYA27, SCYA28, SLC, SMC-CF, STCP-1, SCYB1, SCYB2, SCYB3, SCYB4, SCYB5, SCYB6, SCYB7, SCYB8, SCYB9, SCYB9B, SCYB10, SCYB11, SCYB12, SCYB13, SCYB14, SCYB15, SCYB16, SDF-1-alpha, SDF-1-beta, SR-PSOX, SCYD1, SR-PSOX, TARC, TCA-3, TCA-4, TDCF, TECK, TSC-1, TSG-8, TCF, TCK-1, TLSF-alpha, TLSF-beta, TPAR-1, TSG-1, WECHE. Suitable endogenous target compounds include colony stimulating factors (CSFs). CSFs are cytokines that stimulate the proliferation of specific pluripotent germ cells of the bone marrow in adults. Granulocyte-CSF (G-CSF) is specific for proliferative effects in cells of the granulocyte lineage. Macrophage-CSF (M-CSF) is specific for macrophage lineage cells. Granulocyte-macrophage-CSF (GM-CSF) has proliferative effects on both classes of lymphoid cells. Epo is also considered a CSF as well as a growth factor, since it stimulates the proliferation of erythrocyte colony forming units. IL-3 (secreted mainly from T cells) is also known as multi-CSF, since it stimulates germ cells to produce all forms of hematopoietic cells. Suitable endogenous target compounds include Erythropoietin (Epo). Epo is synthesized by the kidney and is the primary regulator of erythropoiesis. Epo stimulates the proliferation and differentiation of immature erythrocytes; it also stimulates the growth of erythroid progenitor cells (e.g., colony forming units and erythrocyte drive formers) and induces the differentiation of erythrocyte column forming units into proeritroblasts. When Epo is given to patients suffering from anemia due to kidney failure, the result is a rapid and significant increase in red blood cell count. Suitable endogenous target compounds include Insulin-like Growth Factor I (IGF-I). IGF-I (originally called somatomedin C) is a growth factor structurally related to insulin. I GF-I is the primary protein included in the cell response to growth hormone (GH): that is, IGF-I is produced in response to G H and then induces subsequent cellular activities, particularly in bone growth. It is the activity of I GF-I in response to G H that gives rise to the term somatomedin. Subsequent studies have shown, however, that IGF-I has paracrine and autocrine activities in addition to the endocrine activities initially observed in bone. The IGF-1 receptor, like the insulin receptor, has intrinsic tyrosine kinase activity. Belonging to its structural similarities, IGF-1 can bind to the insulin receptor, but it does so at a much lower affinity than that of insulin itself. Suitable endogenous target compounds include Growth Factor I Similar to Insulin (IGF-II). IGF-I I is expressed almost exclusively in neonatal and embryonic tissues. After birth, the level of detectable IGF-I protein falls significantly. For this reason I G F-I I is thought to be a fetal growth factor. The IGF-I I receptor is identical to the mannose-6-phosphate receptor that is responsible for the integration of lysozomal enzymes (containing mannose-6-phosphate residues) for lysosomes. Suitable endogenous target compounds include beta-tumor necrosis factor. TNF-beta (also called lymphotoxin) is characterized by its ability to kill a number of different cell types, as well as the ability to induce terminal differentiation in others. A significant non-proliferative response to TNF-beta is an inhibition of lipoprotein lipase present on the surface of vascular endothelial cells. The predominant site of TNF-beta synthesis is T-lymphocytes, in particular the special class of T cells called cytotoxic T-lymphocytes (CTL cells). Induction of TNF-beta expression results from elevations in I L-2 as well as the interaction of antigen with T cell receptors. Suitable endogenous target compounds include alpha tumor necrosis factor (TN F-alpha). TNF-alpha (also called lymphotoxin B, or cachectin) is a pleiotropic inflammatory cytokine and can cause necrosis of some types of tumors. TNF-alpha shares only 36% amino acid sequence homology with TNF-beta. Still, the tertiary structures of the two proteins are remarkably similar and both bind to the TNF receptors, TNFR-1 and TNFR-2. TNF-alpha is a trimeric protein encoded within the major histocompatibility complex. It is first identified in its secreted form 17 kd, but additional search showed then that a non-doubled precursor 27kd also exists in the form of a transmembrane. Stimulated macrophages produce TNF-alpha 27kd, which can either bind directly to TNFR-1 and TNFR-2 receptors through cell-to-cell contact or undergo unfolding and bind in their soluble form. The cytokine is produced by several types of cells, but especially by macrophages. Low levels of TNF-alpha promote the remodeling or replacement of senescent or injured tissue by stimulating fibroblast growth. Additional beneficial functions of TNF-alpha include its role in the immune response to bacterial invasions, and certain fungi, viral and parasites as well as their role in the necrosis of specific tumors. Finally, it acts as a mediatic key in the local inflammatory immune response. TNF-alpha is an acute phase protein that initiates a cascade of cytokines and increases vascular permeability, thus recruiting the macrophage and neutrophils to a site of infection. TNF-alpha secreted by the macrophage causes blood coagulation that serves to contain the infection. TNF-alpha also shows chronic effects (for example, in inflammation) and prolonged overproduction of TNF-alpha also results in a condition known as cachexia, which is characterized by anorexia, net catabolism, weight loss and anemia and which occurs in such a disease as cancer and IF DA. Suitable endogenous target compounds include cytokines and growth factors in hematopoiesis, such as, CD34, CI L (lymphokine inhibiting colony), Daniplestim, GADS (adapter related to GRB2 downstream of shc), Hepatopoietic cell growth factor, Hematopoietic cell kinase, Hematopoietic cell phosphatase, kinase lacking hematopoietic consensus tyrosine, Hematopoietic-309 colony stimulating factor, Hematopoietin-1, Hematopoietin-2, Hemoregulatory peptide, HI M, Hiwi, HnudC, progenipoyetine, progenipoyetin-1, progenipoyetin-2, progenipoyetin-4, Promegapoetin, Promegapoetin-1, Promegapoetin-1a, and Sintoquine. Other haematopoietic growth factors in a broad sense include the various colony stimulating factors (see: CSF, including G-CSF, GM-CSF, M-CSF), Epo, SCF (germ cell factor, SCPF (proliferation factor of germ cell), several Interleukins (IL1, IL3, IL4, IL5, IL6, IL11, IL12), LIF, TGF-beta, MIP-1-alpha, TNF-alpha, also many other low molecular weight factors, and several other Many of these proteins are multifunctional, act in very early or later stages of differentiation, may also act in a lineage-specific manner, or may influence more than one lineage.Proliferation and maturation of committed parents is controlled by factors lineage-specific lineages such as Epo, M-CSF, G-CSF, and IL5 Multipotential progenitors starting active cell proliferation are regulated by several coating cytokines, including IL3, GM-CSF, and IL4. Cyclic activation by dormant primitive progenitors and potential B cell tenance of primitive progenitors seems to require early acting cytokine interactions including IL6, G-CSF, IL11, IL12, LIF, and SCF. Depending on their biological activity, hematopoietins have been classified into several subgroups. The term hematopoietin type 1 is occasionally used to describe these factors included in the regulation of hematopoiesis that act directly in some cell types. This group includes IL3 (multi-CSF) and GM-CSF. Factors that synergize with colony-stimulating factors but that by themselves do not possess intrinsic colony-stimulating activity have occasionally been referred to as hematopoietin type 2. I include I L1, I L4, I L5 and I L6. Hematopoietins 3 are those modulating hematopoietic growth by stimulating the release of colony stimulating factors by their respective producing cells. They include I L1, I L2, TNF-beta and I FN-gamma. Some factors negatively regulate hematopoiesis processes. They can selectively inhibit the proliferation of some types of hematopoietic cells and can even induce cell death. Transforming growth factor TGF-beta, for example, acts predominantly in primitive hematopoietic cells and lymphoid cells. The factor called M I P-1-alpha (inflammatory protein of macrophage-1-alpha) shows similar activity in primitive myelopoietic cells. Suitable endogenous target compounds include endogenous compounds included in angiogenesis. Examples of endogenous compounds included in angiogenesis include, Prolactin na 16K, ADAMTS-1, ADAMTS-8, Adrenomedulin, Angio-associated migratory cell protein, Angiogenin, angiogenin-related protein, Angiomodulin, Angiomotin, Angiopoietin-1, Angiopoietin-2, Angiopoietin -3, Angiopoietin-4, Angiopoietin-5, Angiopoietin-similar to 1, Angiopoietin-similar to 2, Angiopoietin-similar to 3, Angiopoietin-similar to 4, Angiopoietin-related protein-2, Angiostatin, Angiotensin-2, Angiotropin, ARP- 2, B16-F1 melanoma autocrine motility factor, brain-specific angiogenesis inhibitor-1, brain-specific angiogenesis inhibitor-2, brain-specific angiogenesis inhibitor-3 C49a, CAM assay, anti-tumor factor cartilage derivative, cartilage-derived inhibitor, CATF, cCAF, CD55, CDI, CDT6, chondrocyte-derived inhibitor, chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity, chondrodimin-1, growth factor derived from chondrosarcoma, CLAF test chorioallantoic membrane, collagen type 18, connective tissue growth factor, angiogenic factor of luteal habeas, degranulation inhibitor protein, EGF, EG-VEGF, angiogenesis factor-1 derived from kidney embryonic, factor-2 angiogenesis derived from embryonic kidneyvascular endothelial growth factor derived from endocrine gland, Endorepelin, endostatin, endothelial cell-stimulating angiogenic factor, endothelial cell-derived growth factor, endothelial monocyte-activating polypeptide-2, ESAF, f-ECGF, FGF, FGF-4, fibrin E fragment, fibroblast-derived endothelial cell growth factor, fibroblast-inducible secreted protein-12, FrzB2, GFB-1 1 1, glioma-derived angiogenesis inhibitory factor, growth hormone, GSM, HAP, Haptoglobin, factor heparin-binding neurite promoter, hepatocyte growth factor, HGF, HUAF, human angiogenic factor, human inhibitor angiogenesis factor-1, human uterine angiogenesis factor, I FN-alpha, I FN-gamma, IGF, IGF-1, IGF-BP-7, jagged, KAF, kalistatin, K-FGF, kidney angiogenic factor, quinostatin, migration stimulating factor, protein / mitogen regulated proliferin, regulated protein-4 of m Iogen, Monocyto-Angiotropin, M RP / PLF, M RP-4, Neuroleukin, Neuropilin-1, NKG5, NLK, Notch-1, Notch-4, ORF-74, Ovarian growth factor, PAF, Parathyroid hormone-related protein , PD-ECGF, PDGF, PDGF-BB, PEDF, PF4, PGI, placenta growth factor of pigment epithelium-derived factor, placental angiogenic factor, platelet factor-4, platelet-derived endothelial cell growth factor, PIGF , PrGF, Prokinetici n-1, proliferin-related protein, Prostacyclin-stimulating factor, prostatic growth factor, PRR kringle-2 prothrombin domain, PTHrP, RNASE5, Scatter factor, semaphorin-3, Sprouty, TAF, TAM F, TGF -alpha, TGF-beta, thrombin, thrombospondin, TI E-1, TIE-2, tissue factor, TNF-alpha, TNF-beta, inducer of weak apoptosis similar to TNF, Transferrin, TSP, tumor angiogenesis factor, autocrine tumor motility factor, Tumstatin, TWEAK, uterine angiogenesis factor, vascular permeability factor ular, Vasculotropin, VEGF, VEGF-162, VEGF-C, VEGF-D, VEGF-E, VEGI, VPF. Many different growth factors and cytokines have been shown to exert chemotactic, mitogenic, modulatory or inhibitory activities on endothelial cells, smooth muscle cells and fibroblasts and, therefore, can be expected to participate in angiogenic processes in one way or another. The process includes the concerted action of proteolytic enzymes, extracellular matrix components, cell adhesion molecules, and vasoactive factors. The factors modulating the growth, chemotactic behavior and / or functional activities of smooth muscle cells include Activin A, Adrenomedulin, aFGF, ANF, Angiogenin, Angiotensin-2, Betacelulin, bFGF, CLAF, ECDGF (endothelial cell-derived growth factor), ET (Endothelin), Factor X, Factor Xa, HB-EGF, Vascular cell proliferation heart-derived inhibitor, I FN-gamma, I L1, LDGF (Leiomyoma-derived growth factor), MCP-1, M DGF (factor Growth factor-derived macrophage, monocyte-derived growth factor), NPY, Oncostatin M, PD-ECGF, PDGF, Prolactin, Protein S, SDGF (smooth muscle cell-derived growth factor), SDMF (cell-derived migration factor smooth muscle), Taquiquinins, TGF-beta, Trombospondin. Factors modulating growth, chemotactic behavior and / or functional activities of vascular endothelial cells include AcSDKP, aFGF, ANF, Angiogenin, angiomodulin, Angiotropin, AtT20-ECGF, B61, bFGF, bFGF-induced activity, CAM-RF, ChDI, CLAF, ECGF, ECl, EDMF, EGF, EMAP-2, Neurotelin (see: EMM PRI N), Endostatin, endothelial growth factor inhibitor, endothelial cell viability maintenance factor, Epo, FGF-5, IGF-2 (see: growth promoter activity for vascular endothelial cells), HBNF, HGF, HUAF, I FN-gamma, I L1, K-FGF, LI F, M D-ECI, M E E F F, N PY, Oncostatin M, PD-ECGF , PDGF, PF4, PIGF, Prolactin, TNF-alpha, TNF-beta, Transferrin, VEGF. Some of these factors are protein factors detected initially due to some other biological activities and then show promote angiogenesis. The list of angiogenically active protein factors in vivo includes fibroblast growth factors (see: FGF), Angiogenin, Angiopoietin-1, EGF, HGF, NPY, VEGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, I L8, Growth hormone. Fragment E fibrin has also been shown to have angiogenic activity. In addition there are factors such as Angiopoietin-1 that do not behave like classical growth factors for endothelial cells but play a prominent role in angiogenic and vasculogenic processes. PF4 and a 16 kDa fragment of Prolactin are inhibitors in vivo. Suitable endogenous target compounds include endogenous compounds included in the formation and maintenance of the peripheral and / or cel nervous systems, such as neurotrophic factors that improve neuronal differentiation, induce proliferation, influence synaptic functions, and promote the survival of neurons that are normally intended to die during different phases of the development of the peripheral and cel nervous system. Examples of such proteins include neurotrophins, such as BDNF (brain-derived neurotrophic factor), NGF, NT-3 (neurotrophin-3), NT-4, NT-5, NT-6, NT-7, CNTF (ciliary neurotrophic factor ), GDNF (glial cell line derived neurotrophic factor), and Glitter. Growth factors such as bFGF or LI F are also found frequently among neurotrophins due to their trophic activities in a number of neurons. BDNF, NGF and NT-3 are sometimes also collectively referred to as the NGF protein family since NGF is the founding member of this protein family. It has been possible, by combining structural elements of NGF, BDNF and NT-3, to form the multifunctional Pan-Neurotrofin-1 (PNT-1) that efficiently activates all trk receptors and displays multiple neurotrophic specificities. Another factor of neuronal survival is NSE (neuron-specific enolase). Other factors with neurotrophic activities are not normally classified as neurotrophic and frequently possessing a broader spectrum of functions are EGF, HBNF (heparan-binding neurite promoter factor), IGF-2, aFGF and bFGF, PDGF, NSE ( neuron-specific enolase), and Activin A. For antiproliferative growth factors affecting neural cells see also: neural antiprolfierative protein, Astrostatin, GGI F (glial inhibitory growth factor). Depending on their bioactivities, a distinction is made between neurite promoter factors (NPFs) and neuronal differentiation factors. The neurite promoter factors do not promote neuronal survival or general growth by themselves but also require one or more neurotrophic factors to induce the growth of dendritic or axonal processes. Factors with NPF activity include NGF, S 100, GM F-beta (glial maturation factor), proteoglycans, merosin, collagens, cell adhesion molecules, and laminin. Suitable endogenous target compounds include endogenous compounds included in wound healing. Platelets are a rich source of locally active cytokines and growth factors. Among other things, platelet-derived factors include adenosine dinucleotide (which induces platelet aggregation and also stimulates cell migration and proliferation), beta-thromboglobulin, bFGF, CTAP-3 (protein-3 activation of connective tissue), EGF Eosinophilic chemoattractant polypeptide-1 (ie, RANTES), f-ECGF (fibroblast-derived endothelial cell growth factor), fi bronectin (which serves as an early matrix ligand for platelet aggregation), HCl ( inhibitor of collagenase of human), HGF (hepatocyte growth factor), HRF (histamine release factors), IGF-BP-3, NAP-2 (neutrophil activation protein-2), NAP-4 (protein- 4 neutrophil activation), PBP (basic platelet protein), PD-ECGF (platelet-derived endothelial cell growth factor), PDG F, PF4, platelet activation factor (PAF, also included in platelet aggregation), serotonin (which induces vascular permeability and is a chemoattractant for neutrophils), Somatostatin, TG F-alpha, TGF-beta, thromboxane A2 (which is included in vasoconstriction, platelet aggregation, and chemotaxis), Vitronectin. Circulating peripheral blood leukocytes migrate in the wound space. The first cells that appear in the area of the wound are neutrophils. Neutrophil numbers reach maximum levels approximately 24 hours after the injury. Their migration is stimulated by various chemotactic factors and cytokines, including complement factors, I L1, TNF-alpha, TGF-beta, and chemokines such as I L8, GRO-alpha, PF4, MCP-1, I P-10, mig , and also by bacterial polysaccharides. Neutrophils adhere to the endothelium by means of selectins, which function as neutrophil receptors on the endothelial cell surface. Integrin receptors on neutrophil cell surfaces facilitate the binding of neutrophils to the extracellular matrix. Neutrophils do not seem to play a critical role in wound healing in the absence of infection as wounds can be cured in animals in which neutrophils are eliminated. Neutrophils are removed by tissue macrophages when they are no longer needed. Monocytes appear approximately 24 hours after the injury and have maximum values 48 after the injury. Since monocytes mature in macrophages, they can be considered an essential source of cytokines, triggering repair processes. Macrophages and monocytes are also attracted to a variety of chemokines. These myokines contribute to the spatial and temporally different infiltration of leukocyte subsets and thus integrate the reparative and inflammatory processes during wound repair. Tissue macrophages are the cells that essentially control and regulate the healing process of the wound and the wounds can not be cured without the participation of these cells as shown by the experiments including removal of wound macrophages. The differentiation of macrophages is initiated by several specific cytokines. Many cytokines produced and secreted by activated macrophages favor the further migration of inflammatory cells in the wound area. Macrophages also control the degradation of the extracellular matrix and regulate the remodeling of the wound matrix. Macrophages secrete cytokines and growth factors including TGF-beta, FGF, VEGF, and chemokines such as J E. TGF-beta appears to be the main factor responsible for the formation of granulation tissue and the synthesis of extracellular matrix proteins and has thus been referred to as a "wound hormone". TGF-beta is a member of one of the most complex groups of cytokine superfamilies, consisting of several isoforms of TGF-beta and other family members, for example, Activin A and BMP. The complexity of the wound healing process is illustrated by the observation that the manipulation of the proportions of members of the TGF-beta superfamily, particularly the proportion of TG F-beta-l in relation to TGF-beta-3, reduces the scarring and fibrosis Re-epithelialization is mediated by mitogenic and chemotactic growth factors of the EGF family of growth factors. Leptin has been shown to be a potent growth factor for keratinocytes during wound healing. The final phase of wound healing is characterized by the gradual replacement of granulation tissues by connective tissue. This process also requires locally activating the cytokines. However, little is known about the factors and mechanisms that eventually limit tissue growth once the repair process has been completed. The synthesis of collagen and proteinace inhibitors is stimulated, among other things, by TGF-beta and related factors. Closing the wound and the evolution of a scar is associated with a decrease in cellularity, including disappearance of typical myofibroblasts. It has been suggested that cell death by apoptosis is the mechanism responsible for the evolution of granulation tissue in a scar. The healing of the fetal wound is outstanding because of its lack of healing. There is some evidence that adult and fetal fibroblasts display phenotypic differences in terms of migratory activity, motokenic response to cytokines, and the synthesis of motonogenic cytokines, growth factors, and matrix macromolecules. By manipulating the actions of the growth factor and cytokines, it is possible to accelerate or modify the healing of the wound. Animal experiments and also clinical experience have shown that topical administration of several cytokines, including bFGF, EGF, KGF, PDGF, TGF-beta, either alone or in combination, considerably accelerates wound healing by stimulating the formation of granulation tissue and improve epithelization. Suitable endogenous target compounds include proteins of acute phase of inflammation. Suitable examples of such proteins and their functions are given in Table 2. Table 2 The main inducers of acute phase proteins are I L1, I L6, and TNF. The two mediators I L1 and I L6 have been used to classify acute phase proteins into two subgroups. The proteins of acute phase type 1 are those that require the synergistic action of I L6 and I L1 for maximum synthesis. Examples of Type 1 proteins are C-reactive protein, serum amyloid A and alpha-1 glycoprotein. Type 2 acute phase proteins are those that require I L6 only for maximum induction. Examples of Type 2 proteins are fibrinogen, haptoglobin, and alpha-2-macroglobulin chains. Gene expression coding for acute phase 2 proteins is suppressed instead of frequently improved by I L1 (Ramadori et al; Fey et al). The additive, synergistic, co-operative and antagonistic effects between cytokines and other mediating substances that influence the expression of acute phase proteins occur and have been observed in almost all combinations. Many cytokines also show differential effects, inducing the synthesis of one or two acute phase proteins but not others. For example, Activin A induces a subset of acute phase proteins in HepG2 cells. Bacterial lipopolysaccharides and various cytokines (mainly I L1, I L6 and TNF but also LI F, CNTF, oncostatin M, I LI 1, and cardiotrofin-1) are included in the induction of synthesis SAA and some of these cytokines act synergistically (Benigni er al).

I L1 and also I FN-gamma reduce some of the effects of I L6. Some of the effects of I L2 and I L6 are antagonized by TGF-beta. The combined action of two or even more cytokines that can produce effects that do not have their own factor would be able to be achieved. In cultured HepG2 hepatoma cells, I L1, I L6, TNF-alpha and TGF-beta induce the synthesis of anti-chymotrypsin and at the same time repress the synthesis of albumin and AFP (alpha-Fetoprotein). The synthesis of fibrinogen is induced by I L6 and this effect is, in turn, suppressed by I L1 -alpha, TNF-alpha or TGF-beta-1. Suitable endogenous target compounds include endogenous compounds included in hemostasis and coagulation cascades. Apart from being included in the regulation of capillary permeability and vessels, the endothelium of blood vessels play a decisive role in maintaining a non-thrombogenic surface by providing activators and inhibitors of coagulation and fibrinolysis. The endothelium is also included in modulation of several immunological processes. In endothelial cells I L1 induces, among other things, the synthesis of several colony stimulating factors, I L6, TNF-alpha, prostaglandins, platelet activation factor (PAF), plasminogen activator inhibitor (PAI). An important endogenous regulator of I L1 is PGE2 which inhibits secretion of I L1 and TNF-alpha. TNF-alpha, among other things, induces the synthesis of tissue thromboplastin (TPL) that participates in the formation of the factor X activating complex that catalyzes the generation of thrombin. The complex also activates factor IX. TNF-alpha also induces the synthesis of I L1 by endothelial cells and this I L1 can also promote the production of TPL, by endothelial cells. The synthesis of TNF-alpha can also be induced by I L1. TNF-alpha and I L1 also induce the synthesis of cell surface antigens. The expression of adhesion molecules increases the adhesion of lymphocytes and leukocytes in the vessel wall and thus facilitates the transendothelial migration of these cells. TNF-alpha and I L1 down-regulate the inactivation system of Protein C by inhibiting the synthesis of thrombomodulin. Thrombin composed of thrombin activates protein C that can then form complexes with membrane-bound protein S. These complexes inhibit factor Va. Activated protein C also neutralizes the plasminogen activator inhibitor (PAI) by composition. TNF-alpha and also I L1 in this way reduce the inactivation of factor Va. Other suitable endogenous target compounds included in coagulation include Kalikrein, Factor Xl, Factor Xlb, Factor XI, Factor Xla, Factor IX, Factor IXa, Factor Vlll, Factor Villa, Factor X, Factor Xa, Factor Va, Factor XI I, Prothrombin, Thrombin, Fibrinogen, Fibrin, Plasminigen, Plasmin. Suitable endogenous target compounds also include hormones, such as stress hormones (eg, Adrenaline, Adrenocorticotropic hormone, Corticosterone, Epinephrine, Growth hormone, Hydrocortisone), fluid / electrolyte and vascular hormones (eg, Aldosterone, Androstenedione, Vasopressin, Bradykinin , Calcitonin, Neurotensin), digestive and liver hormones (eg, Cholecystokinin, Cholesterol, VI P), reproductive hormones (eg, Gonadotrophin Chronic, Epogen, Estradiol, Estriol, Estrone, Etiocolanolone, FSH, Lactogenic Hormone, Luteinizing Hormone, Testosterone, Oxytocin, Progesterone, Prolactin, Gonadotrophin), sugar-handling hormones (eg, Glucagon, I nsulin), thyroid hormones (eg, T2, T3, T4, Trirotropic hormone, Thyotropic release factor, TSH, Parathyroid hormone ), bone hormones (eg, CSF-1, RANK1, members of the Morphogenic Bone Protein family), and other hormones s, such as thyroid stimulating hormone (TS H), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), growth hormone (GH), adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH) ) (vasopressin), Oxytocin, Thyrotropin releasing hormone (TRH), Gonadotropin releasing hormone (GnRH), Growth hormone releasing hormone (GHRH), Corticotropin releasing hormone (CRH), Somatostatin, Dopamine, Melatonin , Thyroxine (T4), Calcitonin, parathyroid hormone (PTH), Glucocorticoids (eg, Cortisol), Mineralocorticoids (eg, aldosterone), Androgens (eg, testosterone), Adrenaline (epinephrine), Noradrenaline (norepinephrine), Estrogens (eg, estradiol), Progesterone, Human chronic gonadotropin (HCG), Insulin, Gl ucagon, Somatostatin , Amiline, Erythropoietin (EPO), Calcitriol, Calciferol (vitamin D3), Atrial-natriuretic peptide (ANP), Gastrine, Secretin, Cholecystokinin (CCK), Somatostatin, Neuropeptide Y, Ghrelin, PYY3-36, Growth factor-1 insulin-like (IG F-1), Angiotensinogen, Thrombopoietin, Leptin, Retinol Binding Protein 4, and Adiponectin. In addition, suitable hormones are listed in Table 3. Table 3 Table 4 provides a non-exhaustive list of suitable endogenous target compounds and preferred therapeutics for targeting the endogenous compound with a ligand as described herein. The "Identifier" column Exemplary "provides the Registration Numbers of the Chemical Abstracts Services (CAS) (published by the American Chemical Society) and / or Access Numbers to the Genetic Bank ((eg, Locus ID, NP_XXXXX (Reference Sequence Protein), and XP_XXXXX (Model Protein) identifiers available on the website of the National Center for Biotechnology Information (NCBI) www.ncbi.nlm.nih.gov) which correspond to entries in the CAS Registry or databases of the Bank Genetics that contain an amino acid sequence of the endogenous target compound or a fragment or variant of the target endogenous compound The summary pages associated with each of these GenSeq and Genetic Bank Access Numbers and CAS as well as the journalistic publications cited (eg. example, PubMed ID number (PM ID)) are each incorporated for reference in their totalities, particularly with respect to the amino acid sequences described The "biological activity" column describes the biological activities associated with the endogenous target molecule. The column "Preferred Name" describes the disease, disorders, and / or conditions that can be treated, prevented, diagnosed, or improved by a ligand comprising a portion having a binding site with binding specificity for the endogenous target compound The invention also relates to a ligand comprising a portion having a binding site with binding specificity for any of the endogenous target compounds listed in Table 4 for use in the manufacture of a medicament for treating any of the preferred indications. corresponding to Table 4. The invention also relates to a method for treating any of the preferred indications listed in Table 4, comprising administering to a subject in need thereof an effective amount of a ligand comprising a portion having a of binding with binding specificity for the endogenous target compound corresponding to the one listed in Table 4. Table 4 The lists presented of endogenous target compounds by no means are exhaustive. Where a ligand binds to two epitopes (on the same or different antigens), the antigen (s) may be selected from this list. In particular embodiments, the ligand comprises a dAb that binds TNFR1 and a second dAB or epitope binding domain that binds any of these antigens. In such embodiments, the multispecific ligand can comprise any combination of immunoglobulin variable domains (e.g., VHVH, VHVL, V VL).

Ligands and monomers dAb binding serum albumin The invention provides a dAb ligand or monomer (e.g., dual specific ligand comprising such a dAb) that binds to serum albumin (SA) with Kd from 1 nM to 500 μM (ie say, x 10 ~ 9 to 5 x 10 ~ 4), preferably 100 nM to 10 μM. Preferably, for a dual specific ligand comprising a first anti-SA dAb and a second dAb to another target, the affinity (for example Kd and / or Kapagada as measured by surface plasmon resonance, for example using BiaCore) of the second dAb for its objective from 1 to 100000 times (preferably 100 to 100000, more preferably 1000 to 100000, or 10000 to 100000 times) the affinity of the first dAb for SA. For example, the first dAb binds SA with an affinity of about 10 μM, while the second dAb binds its target with an affinity of 100 pM. Preferably, serum albumin is human serum albumin (HSA). In one embodiment, the first dAb (or a dAb monomer) binds SA (eg, HSA) with Kd of about 50, preferably 70, and more preferably 100, 150 or 200 nM. In certain embodiments, the dAb monomer that binds SA resists aggregation, reversibly unfolds, and / or comprises a region of structure as described above for dAb monomers that bind TNFR1.

Ligand Formats Ligands and dAb monomers can be formatted as mono- or multispecific antibodies or antibody fragments or in structures without mono- or multispecific antibody. Suitable formats include, any suitable polypeptide structure in which a variable antibody domain or one or more of the CDRs thereof can be incorporated to confer the binding specificity for antigen in the structure. A variety of suitable antibody formats are known in the art, such as, IgG-like formats, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains; homodimers and heterodimers of heavy chains of antibody and / or light chains, antigen-binding fragments of any of the foregoing (eg, a Fv fragment (e.g., single chain Fv (scFv), Fv linked to disulfide), a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment), a single variable domain (eg, VH, VL, VHH), a dAb, and modified versions of any of the foregoing (eg, modified by the covalent attachment of polyalkylene glycol (for example, polyethylene glycol, polypropylene glycol, polybutylene glycol) or other suitable polymer). See, PCT / GB03 / 002804, filed on June 30, 2003, which designates the United States; (WO 2004/081026) considering PEGylates of unique variable domains and dAbs, suitable methods for preparing them, increases the in vivo half-life of the PEGylated single variable domains and dAB monomers and multimers. The full teaching of PCT / GB03 / 002804 (WO 2004/081026), including the portions referred to above, is incorporated herein by reference. The ligand can be formatted as a dimer, trimer or polymer of desired dAb monomers, for example using a suitable linker such as (Gly4S er) n, where n = from 1 to 8, for example, 2, 3, 4, 5, 6 or 7. If desired, ligands, including dAb monomers, dimers and trimers, can be linked to an antibody Fe region, comprising one or both of the CH2 and CH3 domains, and optionally a scaffold region. For example, vectors encoding ligands bound as a single nucleotide sequence to an Fe region can be used to prepare such polypeptides.

The ligands and monomers of dAB can also be combined and / or formatted into multiple non-antibody ligand structures to form multivalent complexes, which bind target molecules with the same antigen, thereby providing superior avidity. For example, natural bacterial receptors such as SpA can be used as scaffolds for the grafting of CDRs to generate ligands that specifically bind to one or more epitopes. The details of this procedure are described in the USA. UU 5,831, 012. Other suitable scaffolds include those based on fibronectin and affibodies. Details of suitable procedures are described in WO 98/58965. Other suitable scaffolds include lipocalin and CTLA4, as described in van den Beuken et al. , J. Mol. Biol. 310: 591-601 (2001), and scaffolds such as those described in WO 00/69907 (Medical Research Council), which are based for example on the bacterial GroE L ring structure or other chaperone polypeptides. Protein scaffolds can be combined; for example, CDRs can be grafted onto a CTLA4 scaffold and used in conjunction with VH or VL immunoglobulin domains to form a ligand. Similarly, fi bronectin, lipocalin and other scaffolds can be combined. A variety of suitable methods for preparing any desired format are known in the art. For example, antibody chains and formats (eg, IgG type formats, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, heavy antibody chains), light chains of antibody, homodimers and heterodimers of heavy chains of antibody and / or light chains) can be prepared by expression of suitable expression construct and / or culture of suitable cells (for example, hybridomas, heterohybridomas, recombinant host cells which contain recombinant constructs that encode the format). In addition, formats such as antibody antigen binding fragments or antibody chains (eg, an Fv fragment (e.g., single Fv chain (scFv), a disulfide linked Fv), a Fab fragment, a Fab 'fragment , a fragment F (ab ') 2), can be prepared by expression of suitable expression constructs or by enzymatic digestion of antibodies, for example, using papain or pepsin. The ligand can be formatted as a dual specific ligand or a multi-specific ligand, for example, as described in WO 03/002609, the full teachings of which are incorporated herein by reference. The specific dual ligands comprise unique variable immunoglobulin domains that have different binding specificities. Such dual specific ligands may comprise combinations of light and heavy chain domains. For example, the dual specific ligand may comprise a VH domain and a VL domain, which may be joined together in the form of a scFv (eg, using a suitable linker such as Gly4Ser), or formatted into a bispecific antibody or fragment of antigen binding thereof (eg, F (ab ') 2 fragment). The specific dual ligands do not comprise complementary VH / VL pairs that form an antigen binding site of conventional two-chain antibody that binds the antigen or epitope cooperatively. In turn, the dual format ligands comprise a complementary pair Vj / VL, wherein the V domains have different binding specificities. In addition, the dual specific ligands may comprise one or more desired CH or CL domains. A scaffolding region domain can also be included if desired. Such combinations of domains can, for example, mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F (ab ') 2 molecules. Other structures, such as a single arm of an IgG molecule comprising VH, VL, CHI and CL domains, are contemplated. Preferably, the dual specific ligand of the invention comprises only two variable domains although several such ligands can be incorporated together in the same protein, for example, two such ligands can be incorporated into an IgG or a multimeric immunoglobulin, such as IgM. . Alternatively, in another embodiment a plurality of dual specific ligands combine to form a multimer. For example, two different dual specific ligands combine to create a tetra-specific molecule. It will be appreciated by a person skilled in the art that the heavy and light variable regions of a dual specific ligand produced according to the method of the present invention may be on the same polypeptide chain, or alternatively, on different polypeptide chains. In case the variable regions are on different polypeptide chains, then they can be linked by means of a linker, generally a flexible linker (such as a polypeptide chain), a chemical linker group, or any other method known in the art. The multispecific ligand possesses more than one binding specificity per epitope. Generally, the multispecific ligand comprises two or more epitope-binding domains, such dAbs or non-antibody protein domain comprising a binding site for an epitope, eg, a affibody, an SpA domain, a domain or class A LDL receptor, an EGF domain, an avimer. The multispecific tags can also be formatted as described herein. In some modalities, the ligand is an IgG type format.

Such formats have the structure of four conventional chains of one I gG molecule (2 heavy chains and two light chains), in which one or more of the variable regions (VH and V) have been replaced with a dAb or domi Unique vari able arm of a desired specificity. Preferably, each of the variable regions (2 VH regions and 2 V regions) is replaced with a single variable dAb or domain. Unique dAb (s) or variable domain (s) that are included in an IgG type format can have the same specificity or different specificities. In some embodiments, the IgG type format is tetravalent and may have one, two, three or four specificities. For example, the IgG type format can be monospecific and comprises 4 dAbs that have the same specificity; bispecific and comprises 3 dAbs that have the same specificity and another dAb that has a different specificity; bispecific and comprises two dAbs that have the same specificity and two dAbs that have the same specificity a third dAbs with a different specificity and a fourth dAb with a different specificity of the first, second and third dAbs; or tetraespecific and comprises four dAbs that each have a different specificity. Antigen binding fragments of the IgG type formats (eg, Fab, F (ab ') 2, Fab', Fv, scFv) can be prepared. Preferably, type I gG formats or antigen binding fragments thereof do not degrade TNFR 1.

Extended Medium Life Formats Ligands can be formatted to extend in vivo the serum half-life. The increased half life in vivo is useful in in vivo applications of immunoglobulins, especially antibodies and more especially small antibody fragments such as dAbs. Such fragments (Fvs, Fvs linked by disulfide, Fabs, scFvs, dAbs) are rapidly cleared from the body, which can severely limit clinical applications. A ligand (for example, a dAb monomer) can be formatted to have a larger hydrodynamic size, for example, by linking a polyalkylene glycol group (for example, polyethylene glycol (PEG) group, polypropylene glycol group), to the serum butane , transferrin, transferrin receptor or at least the transferrin binding part thereof, an antibody Fe region, or by conjugation to an antibody domain. In some embodiments, the ligand is PEGylated. Preferably, the PEGylated ligand binds an endogenous target compound with substantially the same affinity as the same ligand that is not PEGylated. For example, the ligand may be a PEGylated dAb monomer that binds an endogenous target compound, wherein the PEGy monomer dAb binds said endogenous target compound with an affinity that differs from the affinity of dAb in non-PEGylated form by not more than a factor of about 1000, preferably not more than a factor of about 100, more preferably not more than a factor of about 10, or with substantially no affinity without changing in relation to a non-PEGylated form. Small ligands, such as dAb monomer, can be formatted as a larger antigen binding fragment of an antibody or as an antibody (e.g., formatted as a Fab, Fab ', F (ab) 2, F (ab') 2, IgG, scFv). The hydrodynamic size of an antagonist (e.g., ligand, dAb monomer) and its serum half-life can also be increased by conjugating or binding the antagonist to a binding domain (e.g., antibody or antibody fragment) that binds an antigen or epitope that increases the half-life in vivo, as described herein. For example, the ligand (e.g., dAb monomer) may be conjugated or bound to an anti-neonatal serum or anti-neonatal Fe receptor, Fab, Fab 'or scFv, or to an anti-SA receptor or Fe receptor affibody. anti-neonatal The hydrodynamic size of the ligands (e.g., dAb monomers and multimers) of the invention can be determined using methods that are well known in the art. For example, gel filtration chromatography can be used to determine the hydrodynamic size of a ligand. Gel filtration matrices suitable for determining the hydrodynamic sizes of ligands, such as degraded agarose binders, are well known and readily available. The size of a ligand format (e.g., the size of a PEG portion attached to a unit portion having a binding site for an endogenous target compound, can be varied depending on the desired application, for example, wherein the ligand it is intended to leave the circulation and enter peripheral tissues, it is desirable to keep the hydrodynamic size of the ligand low to facilitate the extravasation of the blood stream, alternatively, where it is desired to have the remaining ligand in the systemic circulation for a longer period of time the size of ligand can be increased, for example, by formatting an Ig-like protein or by adding a PEG portion of 30 to 60 kDa Examples of suitable albumin, albumin fragments or albumin variants to be used in ligand according to The invention is described in WO 2005 / 077042A2, which is incorporated herein by reference in its entirety.

In particular, the following albumin, albumin fragments or albumin variants can be used in the present invention: SEQ ID NO: 1 (as described in WO 2005 / 077042A2, this sequence being incorporated explicitly in the present description for reference); albumin fragment or variant comprising or consisting of amino acids 1-387 of SEQ ID NO: 1 in WO 2005 / 077042A2; • albumin, or fragment or variant thereof, comprising an amino acid sequence selected from the group consisting of: (a) amino acids 54 to 61 of SEQ ID NO: 1 in WO 2005 / 077042A2; (b) amino acids 76 to 89 of SEQ ID NO: 1 in WO 2005 / 077042A2; (c) amino acids 92 to 100 of SEQ ID NO: 1 in WO 2005 / 077042A2; (d) amino acids 170 to 176 of SEQ ID NO: 1 in WO 2005 / 077042A2; (e) amino acids 247 to 252 of SEQ ID NO: 1 in WO 2005 / 077042A2; (f) amino acids 266 to 277 of SEQ ID NO: 1 in WO 2005 / 077042A2; (g) amino acids 280 to 288 of SEQ ID NO: 1 in WO 2005 / 077042A2; (h) amino acids 362 to 368 of SEQ ID NO: 1 in WO 2005 / 077042A2; (i) amino acids 439 to 447 of SEQ ID NO: 1 in WO 2005 / 077042A2 (j) amino acids 462 to 475 of SEQ ID NO: 1 in WO 2005 / 077042A2; (k) amino acids 478 to 486 of SEQ ID NO: 1 in WO 2005 / 077042A2; and (1) amino acids 560 to 566 of SEQ ID NO: 1 in WO2005 / 077042A2. Additional examples of suitable albumin, fragments and analogs for use in a ligand according to the invention are described in WO 03/076567A2, which is incorporated herein by reference in its entirety. In particular, the following albumin, fragments or variants can be used in the present invention. human serum albumin as described in WO 03/076567 A2, for example, in Figure 3 (this sequence information being incorporated explicitly in the present description for reference); • human serum albumin (HA) consisting of a single non-glycosylated polypeptide chain of 585 amino acids with a formula molecular weight of 66,500 (See, Meloun, et al, FEBS Letters 58: 136 (1975); Behrens, et al, Fed. Proc. 34: 591 (1975); Lawn, et al., Nucleic Acids Research 9: 6102-6114 (1981); Minghetti, er al., J. Biol. Chem.261: 61 Al (1986)); A polymorphic or analogous variant or fragment of albumin as described in Weitkamp, et al, Ann. Hum. Genet 37: 219 (1973); A fragment of albumin or variant as described in EP 322094, for example, HA (1-37-3., HA (1-388), HA (1-389), HA (I-369), and HA (1-419). ) and fragments between 1 -369 and 1 -419; A fragment of albumin or variant as described in EP 399666, eg, HA (1-177) and HA (I -200) and fragments between HA (IX), where X is any number from 178 to 199. Where a portion extending the half-life (one or more) (eg, albumin, transferin and fragments and analogs thereof) is used in the ligand of the invention, it can be conjugated using any method suitable, such as, by direct fusion to the binding portion having a binding site for an endogenous target compound, for example, by using a single nucleotide construct that encodes a fusion protein, wherein the fusion protein is encoded as a single polypeptide chain with the portion extending the half-life located N- or C-terminally to the TNFR1 binding portion. Alternatively, conjugation can be achieved by using a peptide linker between the portions, for example, a peptide linker as described in WO 03/076567A2 or WO 2004/003019 (these descriptions of the linker being incorporated by reference in the present description to provide examples for use in the present invention).

Typically, a polypeptide that improves serum half-life in vivo is a polypeptide that occurs naturally in vivo and that resists degradation or removal by endogenous mechanisms that remove unwanted material from the organism (eg, human). For example, a polypeptide that improves the serum half-life in vivo can be selected from extracellular binder proteins, proteins found in the blood, proteins found in the blood brain barrier or in neural tissue, proteins located towards the kidney, liver, lung, heart, skin or bone, tension proteins, disease-specific proteins, or proteins included in transport Fe. Suitable polypeptides that improve serum half-life in vivo include, for example, receptor-specific neuropharmaceutical neuropharmaceutical fusion proteins (see U.S. Patent No. 5,977,307, the teachings of which are incorporated herein by reference). the present for reference), cerebral capillary endothelial cell receptor, transferin, transfer receptor (e.g., soluble transferrin receptor), insulin, insulin-like growth factor-1 receptor (IGF 1), growth factor receptor 2 type insulin (IGF2), insulin receptor, blood coagulation factor X, 1-antitrypsin and HNF 1 a. Suitable polypeptides that improve serum half-life also include alpha 1 glycoprotein (orosomucoid; AAG), alpha 1 antichymotrypsin (ACT), alpha 1 microglobulin (HC protein, AI M), antithrombin III (AT III), apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin (Cp) , component C3 complement (C3), component C4 complement (C4), esterase inhibitor C1 (C1 NHC), reagent protein C (CRP), ferritin (FER), hemopexin (HPX), lipoprotein (a) (Lp ( a)), mannose binding protein (M BP), myoglobin (Myo), prealbumin (transthyretin; PAL), retinol binding protein (RBP), and rheumatoid factor (RF). Suitable proteins of the extracellular binder include, for example, collagens, laminins, integrins and fibronectin. Collagens are the main proteins of the extracellular binder. Approximately 15 types of collagen molecules are currently known, found in different parts of the body, for example, type I collagen (counting for 90% of body collagen) found in bones, skin, tendon, ligaments, cornea, internal organs or collagen type II found in cartilage, spinal disc, notocordia, and vitreous humor of the eye. Suitable proteins in the blood include, for example, plasma proteins (e.g., fibrin, macroglobulin-2, serum albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin, profilin , ubiquitin, uteroglobulin and β-2-microglobulin), enzymes and enzyme inhibitors (eg, plasminogen, lysozyme, cystatin aC, antitrypsin alpha 1 and pancreatic trypsin inhibitor), proteins of the immune system, such as immunoglobulin proteins (eg, example, IgA, IgD, IgE, IgG, IgM, immunoglobulin light chains (kappa / lambda)), transport proteins (eg, retinol binding protein, microglobulin -1), defensins (eg, beta-defensin 1) , neutrophil defensin 1, neutrophil defensin 2 and neutrophil defensin 3) and the like. Suitable proteins found in the blood brain barrier or neural tissue include, for example, melanocortin receptor, myelin, ascorbate transporter, and the like. Suitable polypeptides that improve the measured life of serum in vivo also include proteins located in the kidney (eg, polycystin, type IV collagen, K1 organic anion transporter, Heymann antigen), proteins located in the liver (e.g. alcohol dehydrogenase, G250), proteins located in the lung (for example, secreting component, which binds IgA), proteins located in the heart (for example, HSP 27, which is associated with dilated cardiomyopathy), proteins located on the skin (for example, keratin), bone-specific proteins such as morphogenic proteins (BMPs), which are a subgroup of the transforming ß-transforming growth factor proteins that demonstrate osteogenic activity (e.g., BMP-2, BMP- 4, BMP-5, BMP-6, BMP-7, BMP-8), tumor-specific proteins (e.g., trophoblast antigen, herceptin receptor, estrogen receptor, cathepsins (e.g., cathepsin B, which can be found in liver and spleen)). Suitable disease-specific proteins include, for example, antigens expressed only on activated T cells, including LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL, see Nature 402, 304-309 (1999)), OX40 (a member of the TNF receptor family, expressed on activated T cells and specifically regulated in producer cells (HTLV-1) type I of the human T leukemia virus, see Immunol.165 (1): 263-270 (2000)) . Suitable disease specific proteins also include, for example, metalloproteases (associated with arthritis / cancers) including CG6512 Drosophila, human paraplegina, human FtsH, human AFG3L2, murine ftsH; and antiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor / vascular permeability factor (VEGF / VPF), transforming growth factor a (TGF a), tumor necrosis factor-alpha (TNF-a), angiogenin, interleukin-3 (IL-3), interleukin-8 (I L-8), endothelial growth factor-derived platelets (PD-ECGF), placental growth factor (P1 GF), growth factor BB derived from platelets midkine (PDGF), and fractalkine. Suitable polypeptides that improve serum half-life in vivo also include strain proteins such as heat shock proteins (HSPs). HSPs are usually found intracellularly. When they are found extracellularly, it is an indicator that a cell has died and spilled its contents. Unscheduled cell death (necrosis) occurs when as a result of trauma, disease or injury, extracellular HSPs activate a response of the immune system. The binding to extracellular HSP can result in localization of the compositions of the invention to a disease site. Suitable proteins included in Fe transport include, for example, Brambell receptor (also known as FcRB). This Fe receptor has two functions, both of which are potentially useful for delivery. The functions are (1) the transport of IgG from mother to girl through placental protection (2) of IgG from degradation thereby prolonging its serum half-life. It is considered that the endosome IgG receptor recycles. (See, Holliger et al, Nat Biotechnol 15 (7): 632-6 (1 997).) Methods for pharmacokinetic analysis and determination of the half-life of ligand will be familiar to those skilled in the art. Details can be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi &; D Perron, published by M arcel Dekker, 2nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as half-lives of alpha t and beta t and area under the curve (AUC).

Assays for Assessing the Endogenous Objective Compound Function The following assays, suitable variations thereof and other suitable assays can be used to assess the activity of endogenous target compounds, and assess whether a ligand substantially inhibits the activity of a target compound endogenous to the which joins The ABC-1 function can be assayed by measuring the polyprotein-mediated lipid flow of cultured cells (J Clin Ivest 1999Oct; 104 (8): R25-31). The chimera exotoxin function of pseudonomas aFGF1 can be assayed in vitro using a cytotoxicity assay (Proc Nati Acad Sci USA 1989 Jun; 86 (11): 4215-4219). An activity of inhibin A can be assayed in vitro by measuring its inducing activity by differentiation to erythroleukemia cells Mouse friend (M EL) and human K-562 cells (Proc Nati Acad Sci USA 1988 Apr; 85 (8): 2434- 2438); or its suppression of secretion of pituitary-stimulating hormone (Biol Reprod 2000 Sep; 63 (3): 865-871). The activity of inhi bi na beta C can be tested in vitro by measuring its differentiating inducing activity towards the cells (M EL) of erythroleukemia Mouse friend and human K-562 cells (Proc Nati Acad Sci USA 1988 Apr; 85 (8) : 2434-2438); or its suppression of the secretion of pituitary-stimulating hormone (Biol Reprod 2000 Sep; 63 (3): 865-871). The adenosine deaminase activity can be assayed in vitro by measuring purine catabolism (Mol Cell Biol 1985 Apr; 5 (4): 762-767). The adiposine function can be tested in vitro by measuring the accumulation of cAM P in mouse melanoma cells stimulated by adiposin (Hum Mol Genet 1995 Feb; 4 (2): 223-230). The ASP function can be tested in vitro by measuring the inhibition of cAM P accumulation stimulated by alpha-MSH in mouse melanoma cells (Hum Mol Genet 1995 Feb, 4 (2): 223-230). Myelin can be tested in vitro by measuring its phosphorylation by MAP kinase (J Neurochem 1999 Sep; 73 (3): 1090-1097); or its effect on synaptic transmission between neurons (Eur Neurol, 1988; 28 (2): 57-63). The myelin basic protein can be tested in vitro by measuring its phosphorylation by MAP (J Neurochem 1999 Sep; 73 (3): 1090-1097); or its effect on synaptic transmission between neurons (Eur Neurol, 1988; 28 (2): 57-63). The collagen function can be measured using a collagen fibril stability assay (Cell Mol Life Sci 2000 May; 57 (5): 859-863); or an in vitro cell adhesion assay (J Cell Biochem Oct. 1, 1997; 67 (1): 75-83). The alpha glucosidase can be assayed by hydrolysis of p-Nitrophenyl a-d-glucoside from chromogenic artificial substrate. Bergmeyer, H. U. (ed) Methods of Enzymatic Analysis, Second English Edition, 1, 459 (1974). Alpha-galactosidase can be assayed by hydrolysis of p-Nitrophenyl aD-galactosidase from chromogenic artificial substrate to p-Nitrophenyl and D-galactose-Rietra et al., (1975) "Properties of the residual alpha-galactosidase activity in the tissues of a Fabry hemizygote. " Clin Chim Acta; 62 (3): 401 -13. Alpha-L iduronidase can be assayed by substrate hydrolysis 4-methylumbelliferyl alpha-L-iduronide is followed in a fluorometric assay (Hopwood er al. (1979), Clin Chim Acta.; 92: 257-65); other assays are found in Thompson (1978) "Substrates for the assay of alpha-L-iduronidase". Clin Chim Acta; 89 (3): 435-46. The activity of angiopoietin 1 can be tested in vitro using a capillary bud test (Curr Biol Apr. 23, 1998; 8 (9): 529-532). The activity of Angiopoeitin 2 can be tested in vitro using a cell-shoot assay (Curr Biol Apr. 23, 1998; 8 (9): 529-532). Angiostatin can be tested in an endothelial proliferation assay (Kringle domains of Human Angiostatin, Cao er al (1996) J. Biol. Chem. 271 29461 -29467. The activity of ADM P can be tested in vitro by its ability to regulate the dorsalizing factors noggin, goosecoid or follistati n. (Development 1995 Dec; 121 (12): 4293-4301). TRAI L can be assayed in an apoptosis assay (TRAI L-R2: a novel apoptosis-mediating receptor for TRAI L, Walczak et al. (1996) EM BOJ 16: 5386-5397). The arrest function can be tested in vitro by measuring its ability to inhibit endothelial cell proliferation, migration, tube formation, and neovascularization of Matrigel (Cancer Res May 1, 2000; 60 (9): 2520-6). The activity of arylsulfatase B can be tested by the in vitro measurement of hydrolysis of sulfates of N-Acetyl-D-galactosamine (J Biol Chem Feb. 25, 1990; 265 (6): 3374-3381). Asparaginase activity can be tested in vitro using an asparaginase enzymatic assay (Anal Biochem Apr. 10, 2000; 280 (1): 42-45). The activity of rBPI can be tested in vitro using an antibacterial assay (J Biol Chem Nov. 5, 1987; 262 (31): 14891-14894). BDNF can be tested in neuronal growth and synaptic activity assay (BDNF improves neural growth and synaptic activity in hippocampal cell cultures Bartrupef al (1997) Neuroreport 1; 8 (17): 3791 -4 Daniels LB, Glew RH, Radi n DK, Vunnam RR). B-glucocerebrosidase can be assayed using a fluorometric assay for Gaucher disease using conduritol-beta-epoxide with liver as the source of Beta-glucosidase (Clin Chim Acta. Sep. 25, 1980; 106 (2): 155-63; Johnson WG , Gal AE, Miranda AF, Pentchev PG The diagnosis of adult Gaucher disease: use of a new chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-gl ucopyranoside, in cultured skin fibroblasts Clin Chim Acta. 14, 1980, 102 (1): 91 -7). BM P-2 can be assayed as described by Wang, E.A et al. Recombinant humanbone morphogenetic protein induces boneformation. Proc. NatAcad. Sci. 87: 2220-2224, 1990. The BT-SD function can be tested in vitro by using a superoxide dismutase assay (Nucleic Acids Res Mar. 25, 1985; 13 (6): 2017-34). The activity of BRCAI can be assayed by measuring alterations in expression of p21 WAFI / PI PI (Oncogene Jun. 1 1, 1998; 16 (23): 3069-82). The BRCA2 activity can be assayed by measuring alterations in p21 WAF1 / CI P1 expression (Oncogene Jun. 1 1, 1998; 16 (23): 3069-82). The vasodilatory activity of CGRP can be assayed using the aortic ring vasodilation assay described in Pharmacol Res. 1999 Mar; 39 (3): 217-20; the proliferation activities of endothelial cell and osteoblast in vitro (Eur J Pharmacol Dec. 15, 2000; 409 (3): 273-8; Proc Nati Acad Sci US A. 1990 May; 87 (9): 3299-303 ). The calreticulin activity can be measured in vitro using calcium imaging assays (Cell, Sep. 8, 1995; 82 (5): 765-71). The CD4 function can be tested in vitro by measuring the gpl20 binding (Viral Immunol 2000; 13 (4): 547-554); or tomocytokines of monocyte responses following the binding of gp120 (J I mmunol Oct. 15, 1998; 161 (8): 4309-4317). Ligand CD40 can be assayed as described by Hollenbaugh D, et al. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EM BO J. 1992 Dec; 1 1 (12): 4313-21. Chemokine binding proteins can be assayed using receptor binding assays (J Biol Chem May 9, 1997; 272 (19): 12495-12504). CNTF can be tested in vitro using neuronal proliferation and survival assays (EM BO J. Apr. 2, 2001; 20 (7) 1692-1703). The activity of contortrostatin can be measured in vitro by measuring binding to integrins alphavbeta 3 and nalfabvbetad, inhibition of platelet aggregation ofn, and inhibition of cancer cell adhesion to fibronectin and vitronectin (Arch Biochem Biophys Mar. 15, 2000; 375 (2) : 278-288). The CRF binding protein activity can be measured using a CRF binding assay (Peptides Jan. 22, 2001; 22 (1): 47-56). The activity of CTLA4 activity can be measured using a T cell activation assay (J I mmunol Mar. 1, 2001; 166 (5): 3143-3150). The Decorin function can be measured using a collagen fibril stability assay in vitro (Cell Mol Life Sci 2000 May; 57 (5): 859-863); or an in vitro cell adhesion assay (J Cell Biochem Oct. 1, 1997; 67 (1): 75-83). The function of Del-I can be tested in vitro using an alphavbeta3 integrin adhesion assay. (Genes Dev Jan. 1, 1998; 12 (1): 21 -33). Desmoteplase can be assayed as described by Wallen, P., plasminogen Biochemistry. ln: Kline D. L., Reddy, K. N. N. , eds. Fibrinolysis. Boca Ratón, FL: CRC Press, 1980: 1 -25; Saksela, O., Rifki n, D. B., Cell-associated plasminogen activation: Regulation and physiological functions.Annu Rev Cell Biol 1988; 4: 93-126; Womack C J, Ivey F M, Gardner A W, Macko R F, Fibrinolytic response to acute exercise in patients with arterial arterial disease. Med Sci Sports Exerc 2001 Feb; 33 (2): 214-9. Dnasa activity can be measured using the DNA degradation assay in J Biochem (Tokyo). 1982 Oct; 92 (4): 1297-303. The activity of Ectoapirasa can be tested in vitro using an ectoapirasa assay (J Biol Chem Sep. 18, 1998; 273 (38): 24814-24821). EGF can be assayed using a cell growth assay (J Biol Chem Mar. 31, 1995; 270 (13): 7495-500). EMAP II activity can be assayed in vitro using a cell-shoot assay (Curr Biol Apr. 23, 1998; 8 (9): 529-532). FGF-I can be assayed using a cell proliferation assay: Cell, vol.50, no.5, pp.729-737 (Aug. 1987). Proc Nati Acad Sci U.S. A, vol.86, no.3, pp.802-806 (1989). FGF-2 can be assayed using a proliferation assay using NR6R-3T3 cells (Rizzino 1988 Cancer Res. 48: 4266). Fibrolase activity can be assayed in vitro using a fibrinolytic assay. (Thromb Res May 15, 1994; 74 (4): 355-367). FLT3 can be assayed in a proliferation assay using a Flt-3 transformed pro B cell line (Hannum 1994 Nature 368: 643). The activity of Follitropin can be tested in vitro by measuring the production of cAMP in cells expressing the FSH receptor (J Reprod Immunol 2001 Jan; 49 (1): 1-19). GDNF activity can be assayed in vitro by measuring increases in Ret tyrosine phosphorylation in response to GDNF treatment (Mol Cell Biol Mar. 15, 1995; (3): 1613-1619). The activity of Gelsolin can be tested in vitro by measuring actin proteolysis (Nature 1987 Jan 22-28; 325 (6102): 362-364). The activity of GGF2 can be tested in vitro by measuring the activation of tyrosine kinases of the ERBB receptor in human rhabdomyosarcoma cells (Int J Cancer Jul. 1, 2000; 87 (1): 29-36). The glucogenic activity of Glucagon is mediated by a high affinity glucagon receptor. The biological activity of recombinant glucagon can be assessed by direct binding assays (Science Mar. 12, 1993; 259 (5101): 1614-6). The HBNF activity can be tested in vitro using a neurite overgrowth assay. (J Biol Chem Oct. 27, 1995; 270 (43): 25992-25999). The binding of hCG receptor and activation can be measured to assess the biological activity of recombinant hCG (J Biol Chem Oct. 5, 1993; 268 (28): 20851-4). Heat shock proteins can be assayed by the administration of HSP peptide complexes in two models of UV-induced carcinoma in mice (US Pat. No. 5,837,251). Interleukin 1 can be assayed by 1) binding receptor IL-1 receptors in YT-NCI or C3H / HeJ cells (Cárter et al., Nature 344: 633-638, 1990); 2) induction of endothelial cell leukocyte adhesion (Cárter et al., Nature 344: 633-638, 1990); 3) proliferation assays in A375-C6 cells (Murai T et al., J. Biol. Chem. 276: 6797-6806, 2001); D10S proliferation: Orencole & Dinarello (1989) Cytokine 1, 14-20. I-Ira can be assayed by 1) competition for the binding of I L-1 to IL-I receptors in YT-NCI or C3H / HeJ cells (Cárter et al., Nature 344: 633-638, 1990); 2) inhibition of endothelial cell leukocyte adhesion induced by I L-1 (Cárter et al., Nature 344: 633-638, 1990); 3) proliferation assays in A375-C6 cells, a human melanoma cell line susceptible to the antiproliferative action of IL-I (Murai T et al., J. Biol. Chem. 276: 6797-6806, 2001). I L-10 can be assayed by 1) binding of I L-10 to NK cells (carson WE et al., Blood 85: 3577-3585, 1995); 2) inhibition of TNF-alpha production by macrophages (Riley J K et al., J. Biol. Chem. 274: 16513-16521, 1999); 3) inhibition of macrophage deproliferation (O'Farrell A-M et al., EM Bo 17: 1006-1018, 1998); MC-9 proliferation: Thompson- Sni peser al (1991) J. Exp. Med. 173, 507-510 I L-1 1 can be assayed in hematopoietic cell proliferation assay. «Interleukin-1 1 human enhancement megakaryocytopoiesis in vitro." Blood Jan. 15, 1992; 79 (2): 327-31. "Synergistic effects of stem cell factor and interleukin 6 or interleukin 1 1 on the expansion of murine hematopoietic progenitors in liquid suspension culture. "Stem Cells, 1995 Jul; 13 (4): 404-13; B9-11 proliferation: Lu et al (1994) J immunol Methods 173, 19. I L-12 can be tested in a natural killer cell (NK) cytotoxicity and interferon-gamma (IFN-gamma) release assay. "Requirement for type 2 NO synthase for IL-12 signaling in innate immunity." Science 284: 951-955, 1999; 225 proliferation: Hori et al (1987), Blood 70, 1069-1078. The activity of I L18 binding protein can be tested in vitro by measuring its ability to inhibit the early Th1 response (I mmunity 1999 Jan; 10 (1) : 127-136) The function of diphtheria toxin I L2 chimera in vitro using a cytotoxicity assay (Exp Hematol 2000 Dec; 28 (12): 1390-1400) I L-4 can be tested in a cytotoxicity assay on normal T lymphocytes. (PMI D: 8144944); Increase RAMOS of expression CD23: Siegel & Mostowski (1990) J Immunol Methods 132, 287-295. The activity of receptor I L-4 can be measured by the ability to inhibit the proliferation of I-L-4 dependent TF-1 cells (Kitamura, T et al., 1989, J. Cell, PPhysiol, 140: 323). I L-8 can be assayed in an assay that monitors the reflux of calcium in cells carrying I L8R (Holmes ef al (1991) Science 253, 1278-80). Gamma interferon can measure activity in antiviral assay using Hela cells infected with EMC virus (Meager, A. 1987, Lymphokines and Interferons, A Practical Approach, Clemens, MJ et al., Eds., IRL Press, p.129); can measure the modulation of class I I M HC expression in COLÓ 205 human colorectal carcinoma cell line (Gibson and Kramer, 1989, J. I mmunol.Methods, 125: 105-1 13). The interferon-omega activity can be measured using an in vitro antiviral assay (J Med Microbiol 1998 Nov; 47 (11): 1015-8). The activity of I P-10 can be measured in vitro using a rat artery smooth muscle cell chemotaxis assay (J Biol Chem Sep. 27, 1996; 271 (39): 24286-24293). The activity of IL-3 can be measured in vitro using a hematopoietic cell differentiation assay (Science 1985; 228 (4701): 810-815). The activity of KGF-1 can be tested in vitro using an epithelial cell proliferation assay. (Proc Nati Acad Sci U.S.A. 1989 Feb; 86 (3): 802-806). Kistrin activity can be tested in vitro by assaying thrombolysis, reoculation, and bleeding associated with the administration of recombinant tissue-type plasminogen activator (rt-PA) in a canine model of coronary artery thrombosis (Circulation 1991 Mar, 83 (3 ) 1038-1047) The activity of Kunitz protease inhibitor 1 can be assayed in vitro by measuring the inhibitory activity towards the HGF activator (J Biol Chem Mar 7, 1997, 272 (10) 6370-6376) Lactotransferpna activity can be measured using an in vitro viral inhibition assay (J Med Microbiol 1998 Nov, 47 (11) 1015-8), and antimicrobial assays (J Clin Invest Sep 1, 1998, 102 (5) 874-80) Leptin can be assayed by modulation m vivo of dietary intake, reduction in body weight, and decrease in insulin and glucose levels in ob / ob mice, radioimmunoassay (RIA) and activation of leptin receptor in a cell-based assay Protein Expr Pupf 1998 Dec, 14 ( 3) 335-42 The activity of LI F can be measured in vitro using a hematopoietic cell differentiation assay (Science 1985, 228 (4701) 810-815) The activity of LFA-3 can be tested in vitro by measuring its ability to inhibit T cell function (J Exp Med Jul 1, 1993, 178 (1) 21 1 -222) Lys plasminogen activity can be measured using a m vitro fibpnolysis assay (J Biol Chem Dec 25, 1992, 267 (36) 26150-6) M aspin activity can be measured using m vitro angiogenesis assays (Nat Med 2000 Feb, 6 (2) 196-9) Metionmase activity can be assayed m vitro as described by Ito ef al (J Biochem (Tokyo), 1975 Nov, 78 (5) 1 105-1 107) MTP activity can be assayed in vitro by using a lipoprotein secretion assay containing apoB. (J Biol Chem Sep. 2, 1994; 269 (35): 21951-21954). The activity of NG F can be assayed by measuring the activation of CREB transcription factor in sympathetic neurons in culture (Science Dec. 17, 1999; 286 (5448): 2358-2361). Neutral endopeptidase activity can be assayed in vitro by measuring proteolysis of bombesin-like peptides (Proc NatlmAcad Sci U.S.A. Dec. 1, 1991; 88 (23): 10662-10666). The activity of NI F can be tested in vitro using a polymorphonuclear leukocyte adhesion assay (Mol Pharm 1999 Nov; 56 (5): 926-932). Noggin activity can be assayed by measuring TNF receptor responses to TNF stimulation of HeLuna cells. (J Biol Chem Feb. 28, 1997; 272 (9): 5861-5870). The activity of NT-3 can be assayed in vitro by measuring its ability to proliferate cultured NC progenitor cells grown in a serum-free defined medium (Proc Nati Acad Sci U.S. Mar. 1, 1992; 89 (5): 1661-1665). OP-I activity can be assayed in vitro by measuring VEGF expression in fetal rat calvarial cells (Mol Cell Endocrinol Jul. 20, 1999; 153 (1-2): 1 13-124). The osteoprotegerin activity can be tested in vitro using a co-culture assay for osteoclastogenesis, a bone resorption assay using a long fetal bone organ culture system, a dentin resorption assay, or a fibroblast proliferation assay ( FASEB J. 1998; 12: 845-854). The acetylhydrolase activity of plasma PAF can be determined by the method of Staffopni et al (Stroke 1997 Dec, 28 (12) 2417-20). The patched function can be tested in vitro by measuring the binding of its ligand, sonic hedgehog (Nature Nov 14 , 1996, 384 (6605) 129-134) PDGF activity can be assayed in vitro by measuring its ability to induce tyrosine phosphorylation at the PDGF receptor (J Biol Chem May 25, 1989, 264 (15) 8905-8912) The PEDF function can be assayed in vitro using a neurite overgrowth assay (J Biol Chem Oct 27, 1995, 270 (43) 25992-25999). The plasminogen activator inhibitor can be assayed as described by Wallen, P, Biochemistry of plasminogen, I n Klme DL, Reddy, KNN, eds Fibpnolysis Boca Raton, FL CRC Press, 1980 1 -25, Saksela, O, Rifkm, DB, Cell-associated plasminogen activation Regulation and physiological functions Annu Rev Cell Biol 1988, 4 93 -126, Womack CJ, Ivey FM, Gardner AW, Macko RF, Fibpnolytic r sponge to acute exercise patients with peppheral arterial disease Med Sci Sports Exerc 2001 Feb, 33 (2) 214-9 PGP activity can be assayed in vitro by measuring the agonism of tyrosine 3 kinase and granulocyte colony stimulating factor ( Exp Hematol 2001 Jan, 29 (1) 41 -50) PM P activity can be assayed in vitro by measuring the ability to induce megacapocytopoiesis in purified CD43 + cells from the bone marrow, mobilized peripheral blood progenitor cells, or umbilical cord (J Hematother 1999 Apr, 8 (2) 199-208) The efficacy of prosaptide can be measured through multiple dose regimens using diabetic rats (Anesthesiology 2000 Nov; 93 (5): 1271-8). The activity of Ranpirnase in vitro can be tested by using ribonuclease and cytotoxic activity assays (J Mol Biol Apr. 19, 1996; 257 (5): 992-1007). The Relaxin activity can be tested in vitro using an inhibition of in vitro test of rat uterine contractions induced by KCl (Can J Physiol Pharmacol 1981 May; 59 (5): 507-12). S-Retinal Antigen activity can be tested in vitro by measuring the activity of cyclic GMP opening channels in photoreceptor cells (J Biol Chem Nov. 25, 1994; 269 (47): 29765-29770). RhLH activity can be assayed in vitro by measuring Fura-2 fluorescence in isolated thecal cells in response to RhLH stimulation (Endocrinology 2000 Jun; 141 (6): 2220-2228). The activity of Saruplasa can be measured in vitro using a plasminogen cleavage assay (J Biol Chem Jan. 18, 2001, epub ahead of print). The complementary type 1 receptor can be tested in vitro by measuring the C3b and C4b junction. (J Exp Med Nov. 1, 1988; 168 (5): 1699-1717). The function of the sonic hedgehog can be tested in vitro by measuring its binding to patching (Nature 1996 Nov. 14; 384 (6605): 129-134). Staphylokinase activity can be measured in vitro using a plasminogen cleavage assay (J Biol Chem Jan. 18, 2001, epub ahead of print). The activity of SCF can be assayed by measuring mast cell survival, proliferation, or production of pro-inflammatory cytokines (I mmunol Rev 2001 Feb; 179: 57-60). The activity of etreptokinase can be measured in vitro using a plasminogen activation assay (J Biol Chem Jan. 18, 2001, epub ahead of print). Superoxide dismutase activity can be tested in vitro using a superoxide dismutase assay (Nucleic Acids Res Mar. 25, 1985; 13 (6): 2017-34). The T 1 / ST2 function can be tested in vitro using a Th2 cell activation assay (J Immunol Mar. 1, 2001; 166 (5): 3143-3150). The activity of TGF beta 1 can be tested in vitro by measuring the induction of renal fibroblast proliferation by the induction of FGF-2 (Kidney Int 2001 Feb; 59 (2): 579-592). The TGF Beta 2 function can be tested in vitro by measuring the inhibition of iNOS transcription (I nflamm Res 1997 Sep; 46 (9): 327-331). TGF-beta 3 can be tested for stimulation of prostaglandin E2 (PGE2) production and bone resorption in neonatal mouse calvary in organ culture (PNAS USA 1985 Jul; 82 (13): 4535-4538), or stimulation of synthesis of collagen, osteopontin, osteonectin, and alkaline phosphatase, and the ability to stimulate duplication in osteoblast-like cells (J Biol Chem 1987; 262: 2869, J Biol Chem 1988; 263: 13916, J Cell Biol 1988; 106: 915, J Cell Physiol 1987; 133: 426, Endocrinology 1987; 121: 212, Endocrinology 1986; 19: 2306, and J Cell Biol 1987; 105: 457). Thrombopoietin (TPO) can be assayed for the regulation of growth and differentiation of megakaryocytes (Mol Cell Biol 2001 Apr; 21 (8): 2659-2670; Exp Hematol 2001 Jan; 29 (1): 51-58; Leukemia 2000 Oct; 14 (10): 1751-1756). The function of Tie-2 / Tek can be assayed by measuring its phosphorylation in response to stimulation by angiopoietin (I nt Immunol 1998 Aug; 10 (8): 1217-1227). The tissue factor trajectory inhibitor can be assayed for the inactivation of factor Xa and inhibition of the tissue factor complex Vl that of the extrinsic coagulation pathway (J Biol Chem May 5, 1998; 263 (13): 6001-6004; Thromb Haemost 1998; 79 (2): 306-309). The TNF binding protein activity can be assayed by measuring the inhibition of PI P5K activation in response to TNF stimulation of HeLuna cells. (J Biol Chem Feb. 28, 1997; 272 (9): 5861-5870). The TNF binding protein activity can be assayed by measuring the inhibition of PI P5K activation in response to TNF stimulation of HeLuna cells. (J Biol Chem Feb. 28, 1997; 272 (9): 5861-5870). TNF receptor activity can be assayed by measuring increases in PI P5K activation in response to TNF stimulation of HeLuna cells (J Biol Chem Feb. 28, 1997; 272 (9): 5861-5870). t-PA can be assayed as described in Wallen, P., Biochemistry of plasminogen. I n: Kl ine D. L., Reddy, K. N. N., eds. Fibrinolysis. Boca Ratón, FL: CRC Press, 1980: 1 -25; Saksela, O., Rifkin, D.B., Cell-associated plasminogen activation: Regulation and physiological functions. Annu Rev Cell Biol 1988; 4: 93-126; Womack C J, IveyFM, Gardner A W, Macko R F, Fibri nolytic response to acute exercise in patients with arterial arterial disease. Med Sci Sports Exerc 2001 Feb; 33 (2): 214-9. The IFN-tau activity can be tested in vitro by measuring the IFN-tau-induced production of a 70 kD acid protein in cultured endometrial explants prepared from female ewes on day 13 of the estrous cycle. (Mol Endocrinol 1990 Oct; 4 (10): 1506-1514). The activity of Troponin I can be tested in vitro using a myofibril binding assay (J Muscle Res Cell Motil 1999 Nov; 20 (8): 755-760). The oxidase urate activity can be tested in vitro using an uricase-catalyzed oxidation assay of uric acid (Anal Chem May 15, 1999; 71 (10): 1928-1934); or by recombinant urate oxidase immunoassay (J Pharm Sci 1996 Sep; 85 (9): 955-959). The urokinase activity can be measured in vitro using a plasminogen cleavage assay. Sazonova ef al. (J Biol Chem Jan. 18, 2001, electronic publication prior to print). The activity of VEGF-I can be tested in vitro using an endothelial cell proliferation assay. (Proc Nati Acad Sci U.S.A. 1989 Feb; 86 (3): 802-806). Viscumin activity can be assayed using granulocyte and neutrophil activation assays. (Anticancer Res 1999 Jul-Aug; 19 (4B): 2925-2928; and J Leukoc Biol 2000 Dec; 68 (6): 845-853).

Preparation of Immunoglobulin-based Ligands The binding ligands (e.g., dAbs) as described herein can be prepared according to previously established techniques used in the field of making antibodies, for the preparation of scFv, "phage" antibodies and other elaborated antibody molecules. Techniques for the preparation of antibodies are described, for example, in the following reviews and references cited therein: Winter & Milstein, (1991) Nature 349: 293-299; Pluckthun (1992) I mmunological Reviews 130: 151-188; Wright ef al, (1992) Crti. Rev. Immunol .12: 125-168; Holliger, P. & Winter, G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, ef al. (1995) J. Hematother. 4, 463-470; Chester, K.A. & Hawkins, R.E. (1995) Trends Biotechn. 13, 294-300; Hoogenboom, H. R. (1997) Nature Biotechnol. 15, 125-126; Fearon, D. (1997) Nature Biotechnol. 15, 618-619; Pluckthun, A. & Pack, P. (1997) Immunotechnology 3, 83-105; Carter, P. & Merchant, A. M. (1997) Curr. Opin. Biotechnol. 8,449-454; Holliger, P. & Winter, G. (1997) Cancer Immunol. Immunother. 45, 128-130. Suitable techniques employed for the selection of variable antibody domains with a desired specificity employ library and selection procedures that are known in the art. Natural libraries (Marks et al (1991) J. Mol. Biol, 222: 581; Vaughan et al (1996) Nature Biotech., 14: 309) which use relocated V genes collected from human B cells are well known to those skilled in the art. in the matter. Synthetic Libraries (Hoogenboom &Winter (1992) J. Mol. Biol, 227: 381; Barbas et al. (1992) Proc. Nati. Acad. ScL USA, 89: 4457; Nissim ef al. (1994) EM BO J 13: 692; Griffiths et al. (1994) EM BO J, 13: 3245; De Kruif et al. (1995) J. Mol. Biol, 248: 97) are prepared by cloning immunoglobulin V genes, usually using PCR. Errors in the PCR process can lead to a high degree of random selection. The VH and / or VL libraries can be screened against target antigens or epitopes separately, in which case the single domain binding is directly selected for, or together.

Library Vector Systems A variety of selection systems are known in the art that are suitable for use in the present invention. Examples of such systems are described below. The bacteriophage lambda expression systems can be selected directly as bacteriophage plaques or as colonies of lysogens, both as previously described (Huse et al (1989) Science, 246: 1275; Cato and Koprowski (1990) Proc. Nati. Acad. ScL USA, 87; Mullinax ef al. (1990) Proc. Nati Acad. ScL USA, 87: 8095; Persson ef al. (1991) Proc. Nati. Acad. ScL US A., 88: 2432) and are of use in the invention. While such expression systems can be used to select up to 106 different members of a library, they are not easily adapted for selection of larger numbers (more than 106 members). Of particular use in the construction of libraries are selection deployment systems, which allow a nucleic acid to bind to the polypeptide it expresses. As used herein, a selection deployment system is a system that allows the selection, by means of appropriate deployment, of the individual members of the library by linking the generic and / or objective ligands. Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques. Such systems, in which various peptide sequences are deployed on the surface of filamentous bacteriophage (Scott and Smith (1990) Science, 249: 386), have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encode them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen (McCafferty ef al, WO 92/01047). The nucleotide sequences encoding the variable regions bind to gene fragments encoding the leader signals directing them to the periplasmic space of E. coli and as a result the resulting antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage goat proteins (for example, pl llo pVII I). Alternatively, the antibody fragments are displayed externally in lambda phage capsids (phagobodies). An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells.

In addition, since the nucleotide sequence encoding the polypeptide library member is contained in a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively forward. Methods for the construction of bacteriophage antibody display libraries and lambda phage display libraries are well known in the art (McCafferty et al. (1990) Nature, 348: 552; Kang et al (1991) Proc. Nati Acad. Sci.

U.S. A., 88: 4363; Clackson ef al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton ef al. (1991) Proc.

Nati Acad. Sci U.S.A., 88: 10134; Hoogenboom ef al. (1991) Nucleic Acids Res., 19: 4133; Chang ef al. (1991) J. Immunol, 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks ef al. (1991) supra; Barbas ef al. (1992) supra; Hawkins and Winter (1992) J Immunol, 22: 867; Marks ef al, 1992, J. Biol. Chem., 267: 16007; Lerner et al (1992) Science, 258: 1313, incorporated herein by reference). A particularly advantageous method has been the use of phage scFv libraries (Huston et al, 1988, Proc. Nati. Acad.

Sci U.S. A., 85: 5879-5883; Chaudhary ef al (1990) Proc. Nati Acad. Sci U.S. A., 87: 1066-1070; McCafferty et al (1990) supra; Clackson ef al (1991) Nature, 352: 624; Marks ef al. (1991) J. Mol.

Biol, 222: 581; Chiswell ef al (1992) Trends Biotech. , 10: 80; Marks ef al (1992) J. Biol. Chem., 267). Several modalities of the scFv libraries deployed in bacteriophage goat proteins have been described. Refinements of phage display procedures are also known, for example as described in WO96 / 06213 and WO92 / 01047 (Medical Research Council ef al.) And WO97 / 08320 (Morphosys), which are incorporated herein by reference. for reference. Other systems for generating polypeptide libraries include the use of cell-free enzymatic machinery for the in vitro synthesis of library members. In one method, the RNA molecules are selected by alternative rounds of selection against a target ligand and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar technique can be used to identify the DNA sequences that bind to the predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92 / 05258 and WO92 / 14843). In a similar manner, the in vitro translation can be used to synthesize the polypeptides as a method to generate the large libraries. These methods, which generally comprise established polysome complexes, are further described in WO88 / 08453, WO90 / 05785, WO90 / 07003, WO91 / 02076, WO91 / 05058, and WO92 / 02536. Alternate non-phage display systems, such as those described in WO95 / 22625 and WO95 / 1 1922 (Affymax) use polysomes to display polypeptides for selection. Yet an additional category of techniques includes the selection of repertoires in artificial compartments, which allow the linking of a gene with its gene product. For example, a screening system in which the nucleic acids encoding the desirable gene products can be selected in microcapsules formed by water-in-oil emulsions is described in WO99 / 02671, WO00 / 40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16 (7), 652-6. The genetic elements that encode a gene product having a desired activity are divided into microcapsules and then transcribed and / or translated to produce their respective gene products (RNA or protein) within the microcapsules. The genetic elements that produce the gene product having desired activity are subsequently chosen. This method chooses the gene products of interest by detecting the desired activity by a variety of means.

Construction of Library The libraries intended for selection, they can be constructed using techniques known in the art, for example as set forth above, or can be purchased from commercial sources. The libraries that are useful in the present invention are described, for example, in WO 99/20749. Once a vector system is chosen and one or more nucleic acid sequences encoding interleukin polypeptides are cloned into the library vector, one can generate diversity within the molecules cloned by taking the mutagenesis before The expression; alternatively, the encoded proteins can be expressed and selected, as described above, before the utagenesis and additional rounds of selection are performed. The mutagenesis of the nucleic acid sequences encoding structurally optimized poly peptides is carried out by standard molecular methods. Of particular use is the polymerase chain reaction, or PCR, (M ullis and Faloon (1 987) Methods Enzymol, 1 55: 335, hereby incorporated by reference). PCR, which uses multiple cycles of DNA duplication catalyzed by a thermostable, DNA-dependent DNA polymerase to amplify the target sequence of interest, is well known in the art. The construction of several antibody libraries has been discussed in Wi nter ef al. (1 994) Ann Rev. I mmunology 12, 433-55, and references cited therein. PCR is performed using hardened DNA (at least 1 fg, more usefully, 1 - 1000 ng) and at least 25 pmol of oligonucleotide primers, it may be advantageous to use a larger amount of primer when the primer group is highly heterogeneous , as such sequence is represented by only a small fraction of the group molecules, and the quantities become limiting in the last amplification cycles. A typical reaction mixture includes: 2 μl of DNA, 25 pmol of oligonucleotide primer, 2.5 μl of 10X of regulator 1 PCR (Perkin-Elmer, Foster City, CA), 0.4 μl of 1.25 μM dNTP, 0.15 μl ( or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, CA) and demineralized water to a total volume of 25 μl. The mineral oil is overcoated and the PCR is performed using a programmable thermal cycler. The length and temperature of each stage of a PCR cycle, as well as the number of cycles, is adjusted according to the stringency requirements in effect. The kneading temperature and timing are determined both by the efficiency at which a primer is expected to be kneaded for tempering and the degree of inequality that will be tolerated; obviously, when nucleic acid molecules are amplified and mutagenized simultaneously, inequality is required, at least in the first round of synthesis. The ability to optimize the stringency of the primer kneading conditions is well within the knowledge of one skilled in the art. A kneading temperature of between 30 ° C and 72 ° C is used. The initial denaturation of the tempered molecules normally occurs at between 92 ° C and 99 ° C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94-99 ° for 15 seconds at 1 minute), kneading (temperature determined as discussed above, 1-2 minutes) and extension (72 ° C for 1 -5 minutes, depending on the length of the amplified product). The final extension is usually 4 minutes at 72 ° C, and can be followed by an infinite stage (0-24 hours) at 4 ° C.

Single Variable Domains in Combination The domains useful in the invention, once selected, can be combined by a variety of methods known in the art, including covalent and non-covalent methods. Preferred methods include the use of polypeptide linkers, as described, for example, in relation to scFv molecules (Bird et al, (1988) Science 242: 423-426). The discussion of suitable linkers is provided in Bird ef al. Science 242, 423-426; Hudson et al, Journal I mmunol Methods 231 (1999) 177-189; Hudson et al, Proc Nat Acad Sci USA 85, 5879-5883. The linkers are preferably flexible, allowing the two unique domains to interact. A linker example is a linker (Gly Ser) n, where n = 1 to 8, for example, 2, 3, 4, 5 or 7. Linkers used in antibodies, which are less flexible, can also be used (Holliger ef al , (1993) PNAS (USA) 90: 6444-6448). In one embodiment, the linker employed is not an immunoglobulin articulation region. The variable domains can be combined using methods other than linkers. For example, the use of disulphide bridges, provided through the elaborated or naturally occurring cysteine residues, can be exploited to stabilize VH'VH'V 'VL or VR-VL dimers (Reiter et al, (1994) Protein). Eng. 7: 697-704) or by remodeling the interface between the variable domains to improve "fit" and thus stability of interaction (Ridgeway et al., (1996) Protein Eng. 7: 617-621; al, (1997) Protein Science 6: 781-788). Other techniques for binding or stabilizing the variable domains of inmnoglobulins, and in particular VH antibody domains, can be employed as suitable.

Characterization of Ligands The binding of a ligand (eg, dAb monomer, dual specific ligand) to its specific antigen (s) or epitope (s) can be tested by methods that will be familiar to those experts in the field and include ELISA. In a preferred embodiment of the invention, the binding is tested using E LISA monoclonal phage. Phage ELISA can be performed according to any suitable procedure: an exemplary protocol is established below. The phage populations produced in each round of selection can be selected for binding by ELISA to the selected antigen or epitope, to identify "polyclonal" phage antibodies. The phage of infected bacterial colonies unique to these populations can thus be selected by ELISA to identify "monoclonal" phage antibodies. It is also desirable to select the soluble antibody fragments to bind antigen or epitope, and this can also be taken by ELISA using reagents, for example, against an N or C terminal tag (see for example Winter et al (1994) Ann. Rev. Immunology 12, 433-55 and references cited herein The diversity of selected phage monoclonal antibodies can also be assessed by gel electrophoresis of PCR products (Marks ef al 1991, supra; Nissim ef al 1994 supra), testing (Tomlinson et al, 1992) J. Mol. Biol. 227 ', 776) or to the sequence the DNA vector.

Structure of ligands In the case that the variable domains are selected from V gene repertoires selected for example using deployment technology as described herein, then these variable domains comprise a region of universal structure, such that they can be recognized by a specific generic ligand as defined herein. The use of universal structures, generic ligands and the like is described in WO 99/20749. Where the V gene repertoires are used, the variation in the polypeptide sequence is preferably located within the structural cycles of the variable domains. The variable domain polypeptide sequences can be altered by DNA change or by mutation in order to improve the interaction of each variable domain with its complementary pair. The DNA change is known in the subject and taught, for example, by Stemmer, 1994, Nature 370: 389-391 and U.S. Pat. U U No. 6,297,053, both of which are incorporated herein by reference. Other methods of mutagenesis are well known to those skilled in the art. In general, the nucleic acid molecules and vector constructs required for selection, preparation and formatting of ligands can be constructed and manipulated as set forth in standard laboratory manuals, such as Sambrook et al. (1989,) Molecular Cloning: A Laboratory M annual, Cold Spring Harbor, USA. The manipulation of nucleic acids useful in the present invention are typically carried out in recombinant vectors.

As used herein, the vector refers to a discrete element that is used to introduce heterologous DNA into cells for expression and / or duplication thereof. The methods by which it is selected or constructed and, consequently, use such vectors, are well known to a person skilled in the art. The numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes and episomal vectors. Such vectors can be used for simple cloning and mutagenesis; the gene expression vector is alternatively used. A vector to be used according to the invention can be selected to accommodate a polypeptide coding sequence of a desired size, typically 0.25 kilobase (kb) to 40 kb or more in suitable host cell of length A transformed with the vector after the in vitro cloning manipulations. Each vector contains several functional components, which generally include a cloning site (or "polylinker"), a duplication source and at least one eligible marker gene. If the given vector is an expression vector, it additionally possesses one or more of the following: enhancer element, promoter, transcription termination and signal sequences, each placed in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a ligand according to the invention. Both cloning and expression vectors generally contain nucleic acid sequences that allow the vector to be duplicated in one or more selected host cells. Typically in cloning vectors, this sequence is one that allows the vector to duplicate independently of the host chromosomal DNA and includes origins of duplication or sequences of autonomous duplication. Such sequences are well known in the art by a variety of bacteria, yeast and viruses. The duplication origin of plasmid pBR322 is suitable for most gram-negative bacteria, the plasmid origin of 2 micras is suitable for yeast, and several viral origins (eg SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of duplication is not necessary for mammalian expression vectors unless they are used in mammalian cells capable of duplicating high levels of DNA, such as COS cells. Advantageously, an expression or cloning vector may contain a selection gene also referred to as an eligible marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, complementary auxotrophic deficiencies, or critical nutrient supply not available in the growth medium. Since the duplication of vectors encoding a ligand according to the present invention is most preferably performed in E. coli, an E. coli-eligible marker, for example, the β-lactamase gene which confers resistance to antibiotic ampicillin, is use. These can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19. Expression vectors usually contain a promoter that is recognized by the host organism and is operably linked to the coding sequence of interest. Such a promoter can be inducible or constitutive. The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. Promoters suitable for use with prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the coding sequence. Preferred vectors are expression vectors that allow the expression of a nucleotide sequence corresponding to a polypeptide library member. In this manner, selection with the first and / or second antigen or epitope can be performed by separate propagation and expression of a single clone expressing the polypeptide library member or by use of any selection display system. As described above, the preferred selection deployment system is the display of bacteriophage. In this manner, phage or phagemid vectors can be used, for example, plT 1 or plT2. The leader sequences useful in the invention include pe1 B, stll, ompA, phoA, bla and pelA. An example are fagomido vectors having an origin of duplication E. coli (for duplication of double strand) and also a phage origin of duplication (for the production of single strand DNA). The manipulation and expression of such vectors is well known in the art (Hoogenboom and Winter (1992) supra).; Nissim ef al. (1994) supra). Briefly, the vector contains a β-lactamase gene to confer selectivity in fagomido and a lac promoter upstream of an expression cassette consisting (terminal N or C) of a leader sequence pe1 B (which directs the expressed polypeptide into space periplasmic), a multiple cloning site (for cloning the nucleotide version of the library member), optionally, one or more peptide tags (for detection), optionally, one or more stop codon TAG and the phage protein pl ll. In this way, using several suppressive and non-suppressive strains of E. coli and with the addition of glucose, iso-propyl thio-β-D-galactoside (I PTG) or an auxiliary phage, such as VCS M 13, the vector is capable of being duplicated as a plasmid without expression, producing large quantities of the polypeptide library member alone or producing phage, some of which contain at least one copy of the polypeptide p11 fusion on its surface. The construction of vectors encoding the ligands according to the invention employs conventional ligation techniques. The isolated vectors or DNA fragments are divided, cut, and re-ligated in the desired shape to generate the required vector. If desired, the analysis to confirm that the correct sequences are presented in the constructed vector can be performed in a known manner. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing assays to assess expression and function are known to those skilled in the art. The presence of a gene sequence in a sample is detected, or its amplification and / or expression quantified by conventional methods, such as Southern or Northern analysis, Western blotting, dot blotting of DNA, RNA or protein, in situ hybridization, immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily contemplate how these methods can be modified, if desired.

Structures Structures may be based on immunoglobulin molecules or may be non-immunoglobulin in origin as set forth above. Preferred immunoglobulin structures as defined herein include any one or more of those selected from the following: an immunoglobulin molecule comprising at least (i) the CL (kappa or lambda subclass) domain of an antibody; or (ii) the CH1 domain of an antibody heavy chain; an immunoglobulin molecule comprising the CH 1 and CH 2 domains of an antibody heavy chain; an immunoglobulin molecule comprising the CH 1, CH 2 and CH 3 domains of an antibody heavy chain; or any of subgroup (ii) together with the CL domain (kappa or lambda subclass) of an antibody. A domain of articulation region can also be included. Such combinations of domains, for example, can mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F (ab ') 2 molecules. Those experts in the field will be aware that this list is not presented that is exhaustive.

Protein scaffolds Each epitope binding domain comprises a protein scaffold and one or more CDRs that are included in the domain-specific interaction with one or more epitopes. Advantageously, an epitope binding domain according to the present invention comprises three CDRs. Suitable protein scaffolds include any of those selected from the group consisting of the following: those based on immunoglobulin domains, those based on fibronectin, those based on affibodies, those based on CTLA4, those based on chaperones such as GroEL, those based on in lipocalin and those based on bacterial Fe receptors SpA and SpD. Those experts in the field appreciate that this list is not intended to be exhaustive.

Scaffolds for use in Ligands Under Construction Selection of the main chain conformation The members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain. For example, although the antibodies are highly diverse in terms of their primary sequence, the comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen-binding cycles of antibodies (H1, H2 , L1, L2, L3) adopt a limited number of main chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol, 196: 901; Chothia ef al. (1989) Nature, 342: 877) . The analysis of cycle lengths and key residues has therefore allowed the majority of human antibodies (Chothia et al. (1992) J. Mol. Biol, 227: 799; Tomlinson et al. (1995) EMSO J, 14: 4628 Williams et al. (1996) J. Mol. Biol, 264: 220). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main chain conformations for short cycle lengths that depend on the length and the presence of particular residues, or types of residues, at key positions in the antibody cycle and structure (Martin et al (1996) J. Mol Biol, 263: 800; Shirai et al (1996) FEBS Letters, 399: 1 ). The libraries of ligands and / or domains can be designed in which certain cycle lengths and key residues have been chosen to ensure that the main chain conformation of the members is known. Advantageously, these are actual conformations of the immunoglobulin superfamily molecules found in nature, to minimize non-functional changes, as discussed above. The germ line gene V segments serve as a suitable basic structure for building the antibody or T cell receptor librarians; other sequences are also of use. Variations can occur at a low frequency, in such a way that a small number of functional members can have an altered main chain conformation, which does not affect its function. The canonical structure theory is also of use to assess the number of different main chain conformations encoded by ligands, to predict the main chain conformation based on the ligand sequences and to choose the residues for diversification that do not affect the structure canonical It is known that, in the domain V? human, the cycle L1 can adopt one of four canonical structures, the cycle L2 has a unique canonical structure and that 90% of the domains V? humans adopt one of four or five canonical structures for the L3 cycle (Tomlinson et al (1995) supra); in this way, in the V domain? alone, different canonical structures can be combined to create a range of different main chain conformations. Since the domain V? codifies a different range of canonical structures for cycles L1, L2 and L3 and that domains V «and V? they can be paired with any VH domain that can encode various canonical structures for the H 1 and H2 cycles, the number of canonical structure combinations observed for these five cycles is very large. This implies that the generation of diversity in the main chain conformation can be essential for the production of a wide range of binding specificities. However, by constructing an antibody library in a unique known backbone conformation it has been found, contrary to expectation, that the diversity in the main chain conformation is not required to generate sufficient diversity to target substantially all of the antigens. . Even more surprisingly, the single-stranded conformation does not need to be a consensus structure - a naturally occurring unique conformation can be used as the basis for an entire library. Thus, in a preferred aspect, the dual specific ligands of the invention possess a unique known backbone conformation. The single-strand conformation chosen is preferably of common place among the molecules of the superfamily type of immunoglobulin in question. A conformation is of common place when a significant number of molecules that occur naturally are observed to adopt it. According to the foregoing, in a preferred aspect of the invention, the natural occurrence of the different main chain conformations for each binding cycle of an immunoglobulin domain are considered separately and then a naturally occurring variable domain is chosen. , which has the desired combination of main chain conformations for the different cycles. If neither is available, the closest equivalent can be chosen. It is preferable that the desired combination of main chain conformations for the different cycles is created by selecting the germline gene segments that encode the desired main chain conformations. It is more preferable that the selected germline gene segments are frequently expressed in nature, and more preferably that they are the most frequently expressed of all natural germline gene segments. In the design of ligands (eg, dAbs) or libraries thereof, the occurrence of the different primary chain conformations for each of the six antigen binding cycles can be considered separately. For H 1, H 2, L 1, L 2 and L 3, a given conformation that is adopted for between 20% and 100% of the antigen binding cycles of naturally occurring molecules is chosen. Typically, its observed incidence is above 35% (ie, between 35% and 100%) and, ideally, above 50% or even above 65%. Since the vast majority of H3 cycles do not have canonical structures, it is preferable to choose a main chain conformation that is commonplace among those cycles that do not deploy canonical structures. For each of the cycles, the conformation that is observed more frequently in the natural repertoire is therefore selected. In human antibodies, the most popular canonical structures (CS) for each cycle are as follows: H 1 - CS 1 (79% of the expressed repertoire), H2 - CS 3 (46%), Ll - CS 2 of V? (39%), L2 - CS 1 (100%), L3 - CS 1 of V? (36%) (calculation assumes a relation ?:? Ratio of 70:30, Hood et al. (1967) Cold Spring Harbor Symp. Quant. Biol, 48: 133). For the H3 cycles that have canonical structures, a length of CDR3 (Kabat ef al. (1991) Sequences of proteins of immunological interest, U .S. Department of Health and Human Services) of seven residues with a salt bridge from residue 94 to residue 101 seems to be the most common. There are at least 16 human antibody sequences in the EM BL data library with the required length H3 and the key residues to form this conformation and at least two crystallographic structures in the protein data bank that can be used as a basis for molding of antibody (2 cgr and 1 tet). The most frequently expressed germline gene segments that this combination of canonical structures are segment VH 3-23 (DP-47), segment JH J H4b, segment V? 02/012 (DPK9) and segment J? J? 1 . The segments VH DP45 and DP38 are also suitable. These segments can therefore be used in combination as a basis to build a library with the single main chain conformation. Alternatively, instead of choosing the single main chain conformation based on the natural occurrence of the different main chain conformations for each of the binding cycles in isolation, the natural occurrence of combinations of main chain conformations is used as the basis for choosing the single main chain conformation. In the case of antibodies, for example, the natural occurrence of combinations of canonical structure by any of two, three, four, five or all six of the antigen binding cycles, can be determined. Here, it is preferable that the chosen conformation be of common place in antibodies that occur naturally and more preferably than is observed more frequently in the natural repertoire. Thus, in human antibodies, for example, when the natural combinations of the five antigen binding cycles, H 1, H 2, L 1, L 2 and L 3, are considered, the most frequent combination of the canonical structures is determined and then it is combined with the most popular conformation for the H3 cycle, as a basis for choosing the single main chain conformation.

Diversification of the Canonical Sequence Having selected several known main chain conformations or, preferably, a single known major backbone conformation, the ligands (e.g., dAbs) or libraries for use in the invention can be constructed by varying the binding site of the molecule in order to generate a repertoire with structural and / or functional diversity. This means that the variants are generated in such a way that they have sufficient diversity in their structure and / or function in such a way that they are able to provide a range of activities. The desired diversity is typically generated by varying the selected molecule in one or more positions. The positions to be changed can be chosen at random or are preferably selected. The variation can be achieved either by random selection, during which the resident amino acid is replaced by an amino acid or analogue thereof, natural or synthetic, producing a very large number of variants or by replacing the resident amino acid with one or more of a subset of defined amino acids, producing a more limited number of variants. Several methods have been reported to introduce such diversity. Error-prone PCR (Hawkins et al (1992) J. Mol. Biol, 226: 889), chemical mutagenesis (Deng ef al (1994) J. Biol. Chem., 269: 9533) or bacterial mutator strains ( Low ef al (1996) J. Mol. Biol, 260: 359) can be used to introduce mutations at random into the genes encoding the molecule. Methods for mutating selected positions are also well known in the art and include the use of unequal oligogonucleotides or degenerate ohgonucleotides, with or without the use of PCR. For example, several synthetic antibody libraries have been created by having Target Mutations to Antigen Binding Cycles The H3 region of Fab binding by human tetanus toxoid has been selected at random to create a range of novel binding specificities (Barbas et al (1992) Proc Nati Acad ScL USA, 89 4457) The random or semi-random H3 and L3 regions have been annexed to the germline V gene segments to produce large libraries with unmutated structure regions (Hoogenboom &Winter (1992) J Mol Biol, 227 381, Barbas ef al (1992) Proc Nati Acad Sci USA, 89 4457, Nissim ef al (1 994) EM BOJ, 1 3 692, Gpffiths ef al (1 994) EM BO J, 1 3 3245, De Kruif et al (1 995) J Mol Biol, 248 97) Such diversification has been extended to include certain or all other antigen binding cycles (Cramep ef al (1996) Nature Med, 2 1 00, Riechmann ef al (1995) Bio / Technology, 1 3 475, Morphosys, WO97 / 08320, supra) Since the selection At random the cycle has the potential to create approximately more than 1015 structures for H3 sol and a large number of variants for the other cycles, it is not easy to use current transformer technology or even to use cell-free systems to reduce a library representing all possible combinations For example, in one of the largest libraries built to date, 6 x 1010 different antibodies, which is only a fraction of the potential diversity for a library of its design, were generated (Griffiths ef al. (1994) supra). Preferably, only the residues that are directly included in the creation or modification of the desired function of the molecule are diversified. For several molecules, the function will be to join a target and therefore the diversity should be concentrated in the target binding site, while avoiding changing the residues that are crucial to the global packaging of the molecule or to maintain the chosen main chain conformation. .

Diversification of the Canonical Sequence as it applies to the Antibody Domains In the case of antibody-based ligands (e.g., dAbs), the binding site for the target is most frequently the antigen-binding site. In this way, preferably only those residues at the antigen binding site are varied. These residues are extremely diverse in the human antibody repertoire and are known to make contacts in high resolution antibody / antigen complexes. For example, in L2 it is known that positions 50 and 53 are diverse in antibodies that occur naturally and are observed to make contact with the antigen. In contrast, the conventional procedure could have been to diversify all the residues in the corresponding Complementarity Determining Region (CDR 1) as defined by Kabat ef al. (1991, supra, some seven residues compared to the two diversified in the library to be used according to the invention.) This represents a major improvement in terms of the functional diversity required to create a range of antigen-binding specificities. , antibody diversity is the result of two processes: somatic recombination of the germline V, D and J gene segments to create a natural primary repertoire (so-called diversity as a whole and germline) and somatic hypermutation of V genes Resulting rearrangements The analysis of human antibody sequences has shown that the diversity in the primary repertoire is focused on the center of the antigen binding site while somatic hypermutation spreads the diversity to regions at the periphery of the binding site of antigen that are highly conserved in the primary repertoire (see Tomlinson et al (1996) J. Mol. Biol, 256: 813) This complementarity has probably occurred as an efficient strategy to search for the sequence space and, despite being apparently unique to the antibodies, it can easily be applied to other polypeptide repertoires. The residues that are varied are a subgroup of those that form the binding site for the target. The different subgroups (including coating) of residues at the target binding site are diversified at different stages during the selection, if desired. In the case of an antibody repertoire, an initial "natural" repertoire can be created where certain, but not all, residues in the antigen-binding site are diversified. As used herein in this context, the term "natural" refers to antibody molecules having a non-predetermined objective. These molecules resemble those that are encoded by the immunoglobulin genes of an individual who has not overcome immune diversification, as is the case with newborn and fetal individuals, whose immune systems have not yet been changed by a wide variety of antigenic stimuli. . This repertoire is thus selected against a range of antigens or epitopes. If required, additional diversity can thus be introduced outside the diversified region in the initial repertoire. This mature repertoire can be selected for the modified function, specificity or affinity. The natural repertoires of the binding domains for the construction of ligands in which some or all of the residues in the antigen binding site are varied are known in the art. (See, WO 2004/058821, WO 2004/003019, and WO 03/002609). The "primary" library mimics the natural primary repertoire, with diversity restricted to residues in the center of the binding antigen site that are diverse in the V-gene segments of the germinal line (germinal line diversity) or diversified during the recombination process (conjunctive diversity). Those residues that diversify include, but are not limited to, H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96. In the "somatic" library, diversity is restricted to residues that are diversified during the recombination process (diversional binding) or highly mutated somatically. Those residues that diversify include, but are not limited to: H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96. All the residues previously listed as suitable for diversification in these libraries are known to make contacts in one or more antibody-antigen complexes. Since in both libraries, not all residues in the antigen binding site are varied, the additional diversity is incorporated during selection by varying the remaining residues, if desired to do so. It should be apparent to those skilled in the art that any subgroup of any of these residues (or additional residues comprising the antigen binding site) can be used for the initial and / or subsequent diversification of the antigen binding site. In the construction of libraries for use in the invention, the diversification of the chosen positions is typically achieved at the nucleic acid level, by altering the coding sequence that specifies the polypeptide sequence in such a way that a number of possible amino acids (all or a subset of them) can be incorporated in that position. Using the I UPAC nomenclature, the most versatile codon is NNK, which encodes all amino acids as well as the TAG stop codon. The NNK codon is preferably used in order to introduce the required diversity. Other codons that span the same ends are also of use, including the N NN codon, which leads to the production of the additional stop codons TGA and TAA. A characteristic of the side chain diversity at the antigen binding site of human antibodies is a pronounced derivation that favors certain amino acid residues. If the amino acid composition of the ten most diverse positions in each of the regions VH, V? and V >; in addition, more than 76% of the single chain diversity becomes only seven different residues, these being, seri na (24%), tyrosine (14%), asparagi na (11%), glycine ( 9%), to the ani na (7%), aspartate (6%) and threonine (6%). This derivation makes the hydrophilic residues and the small residues that can provide the main chain flexibility likely reflect the evolution of the surfaces that predispose to the binding of a wide range of antigens or epitopes and can help explain the required promiscuity of antibodies in the primary repertoire. Since it is preferable to mimic this amino acid distribution, the distribution of amino acids at the positions to be varied preferably mimics that observed at the antigen-binding site of antibodies. Such a derivation in amylocyte substitution that allows the selection of certain polypeptides (not only antibody polypeptides) against a range of target antigens is easily applied to any polypeptide repertoire. There are several methods for deriving the amino acid distribution in the position to be varied (including the use of tri-nucleotide mutagenesis, see WO 97/08320), of which the preferred method, due to the ease of synthesis, is the use of conventional degenerate codons. By comparing the amino acid profile encoded by all combinations of degenerate codons (with single, double, triple and quadruple degeneration in equal relationships in each position) with the use of natural amino acid it is possible to calculate the most representative codon. The codons (AGT) (AGC) T, (AGT) (AGC) C and (AGT) (AGC) (CT) - that is, DVT, DVC and DVY, respectively using nomenclature I UPAC- are those closest to the profile of desired amino acid: they encode 22% serine and 1 1% tyrosine, asparagine, glycine, alanine, aspartate, threonine and cysteine. Preferably, therefore, the libraries are constructed using the DVT, DVC or DVY codon in each of the diversified positions.

L929 Cytotoxicity Assay TNFR1 antagonists (eg, ligands, dAb monomers) can be identified by the ability to inhibit TNF-induced cytotoxicity in L929 mouse fibroblasts (Evans, T. (2000) Molecular Biotechnology 15, 243- 248). Briefly, L929 cells laminated to microconcentration plates are incubated overnight with TNFR1 antagonist, 100 pg / ml TNF and 1 mg / ml actinomycin D (Sigma, Poole, UK). Then the cell viability is measured by reading the absorption at 490 nm following an incubation with 3- (4,5-di methylo tiolol-2-yl) -5- (3-carboxy methoxy feni I) -2- (4 -sulfofenyl) -2-H-tetrazolium (Promega, Madison, USA). The TNFR 1 antagonists will inhibit cytotoxicity and therefore produce an increase in absorption compared to TNF only control.

HeLa I L-8 Assay TNFR1 antagonists (e.g., dAb monomers) can be identified by the ability to inhibit TNF-induced secretion of I L-8 by human HeLa cells (method adapted from that of Akeson, L. ef al (1996) Journal of Biological Chemistry 271, 30517-30523, which describes the induction of I L-8 by I L-1 in HUVEC, here we observe induction by human TNF alpha and use HeLa cells instead of HUVEC cell line ). Briefly, HeLa cells can be laminated in microconcentration plates and incubated overnight with the TNFR1 antagonist and 300 pg / ml TNF. Post-incubation, the supernatant is aspirated from the cells and the I-L-8 concentration is measured using an intercalated ELISA (R & D Systems), or another suitable method. TNFR 1 antagonists inhibit the secretion of I L-8, and less I L-8 is detected in the supernatant compared to the control of TNF alone.

EXAMPLES Example 1. Extent of serum half-life of tumor necrosis factor (TN F) Transgenic mice for human TNF (Tg197) were administered in groups of 10 animals with weekly intraperitoneal doses of anti-TNF dAbs that had been reformatted as dimers with a 40k branched PEG (referred to as PEG TAR1-5-19) or as a fusion with an anti-serum-binding dAb (termed TAR1 trimer -5-19 / MSA). Saline administration was used as a control. After 7 weeks of drug administration, the serum of the animals was analyzed to circulate the TNF levels using an ELISA kit interspersed with anti-TNF. The animals administered with saline had a circulating TNF level of 25.6 pg / ml while the animals receiving TAR1 -5-19 / MSA trimer had levels of 2315 pg / ml. This clearly demonstrates that dAbs with extended serum half-life can increase the circulating levels of a molecule with a short serum half-life such as a cytokine and thus increase serum levels.

EXAMPLE 2. Extension of serum half-life of soluble tumor necrosis factor receptor 1 (sTN FRP.) CD1 mice were given a single intravenous dose of 1 mg / kg of an anti-mouse ddb TNFR1 that had been reformatted ( extended half-life formats) with either a 40k branched PEG (termed anti-TNFR1 dAb PEG) or as a fusion with an anti-serum albumin dAb (termed anti-TNFR1 / anti-SA dimer). evaluated at different time points for levels of soluble TNFR1 using an interspersed E LISA anti-TNFR1 as detailed below.

Measurement of TNFR1 The Maxisorp F96 96-well flat bottom immunoplates (Nunc, Catalog No. 439454) were coated with anti-mouse sTNF R1 / TNFRSF1 A antibody (50 ul per well at 1 ug / ml in PBS). Plates were incubated for 1 hr. at room temperature. The plates were washed three times with 200 ul of PBS. The non-specific binding was blocked with n3% BSA / PBS for 1 hr. at room temperature. The plates were washed three times with 200 ul of PBS / 0.05% of Tween-20. Dilutions of recombinant mouse sTNF R1 (R &D Systems, # cat.4254-R1) were prepared in PBS / 1% BSA at 5000ng mi '1, 500ng ml "1, 50ng mi' 1 and 5ng ml" 1 and 50ul added to the cavity. Alternatively, serum from mice immunized with branched PEG TAR2m-21 -23 / 40K was prepared at 1/5, 1/50, 1/500 and 1/5000 dilutions in PBS / 1% BSA and 50ul added to the well. The plates were incubated at 1 hour, room temperature. The plates were washed three times with 200 ul of PBS / 0.05% of Tween-20. The biotinylated anti-mouse sTNF R1 antibody (R &D) Systems, # cat. AHFOI) was prepared in 1 ug / ml and 50ul added to the well. Plates were incubated for 1 hour, room temperature. Plates were washed three times with 200ul PBS / 0.05% Tween-20. The monoclonal mouse anti-biotin fraction I gG conjugated with peroxidase (Stratech, Cat. No.: 200-032-096) was prepared at 160ng ml-1 and 50 ul added to the cavity. Plates were incubated for 1 hour, room temperature. The plates were washed three times with 200ul PBS / 0.05% Tween-20 then three times with PBS. The microcavity peroxidase substrate component TM B-1 SureBlue (KPL, Cat. No.: 52-00-00) was added to all wells. The plate was incubated at room temperature for 15 minutes and the reaction was stopped with 1 M HCl. The absorption was read at 450 nM. All samples were tested in duplicate. Cavities without sample were included. The OD results for the 1/5 dilutions of serum from the mice dosed with the anti-TNFR1 PEG dAb were plotted against time (Figure 1). The results clearly show the increasing levels of TNFR1 in the serum before falling back to basic levels following the authorization of dAb.

Example 3 dAbs that bind the Receptor Molecules but not Un @? P the Union Site. This example illustrates a method for identifying dAbs that bind a desired receptor molecule but do not bind to the active site of the receptor. The procedure is illustrated using the tumor necrosis factor receptor 1 (TNFR1), and is generally applicable to the receptor molecules. Chimeric receptor molecules made from different murine TNFR1 domains and human TNFR1 domains (Banner DW, ef al Cell, 75 (3): 431-45 (1993).) Such that the molecule contains the four defined extracellular domains of TNFR 1 but that these vary in origin between human and mouse TNFR1 proteins, were produced. The produced chimeric receptors shared properties of both human TNFR1 and mouse TNFR1 according to the different roles of domain and functionality. The molecules provided a means for titrating the domain specificity of dAbs, antibodies and antigen-binding fragments thereof and other molecules (e.g., protein domains such as affibodies, LDL receptor domains, or EGF domains) that bind Human or mouse TNFR1.

Methods The mouse and human TNFR1 sequences were previously cloned into the Pichia expression vector pPicZalpha (Invitrogen) by means of the restriction endonuclease sites EcoRI and Notl. The mouse TNFR1 DNA was hardened (and consequently, the chimeric receptor constructs that end with a 4 mupno domain) contained a 3 '6x human TNFR1 histidine tag (and consequently the chimeric receptor constructs that end with a human 4 domain) contained both Myc tags and 6x Histidine in sequence at the 3' end. The PCRs initials were performed according to the standard PCR conditions using RubyTaq DNA polymerase (USB Corporation, Cniveland, Ohio), 100 ng of tempered DNA (comprising the mined tempering of relevant DNA either full-length mTNFRI or hTNFRI DNA) Typical PCR assays were established as follows 25 μl of 10X RubyTaq PCR regulator containing polymerase, 2 μl of first priming r (10 μM base), 2 μl of second primer (10 μM base), 1 μl (100 ng) of full length annealed DNA TNFR1, 20 μl of dH2O (to a final volume of 50 μl) fixed in thin-walled tubes and placed in a thermal cycler where the reaction was performed according to the following parameters Initial denaturation | 3 minutes 94X Denaturation 30 seconds 94 ° C Kneading (25 cycles) 30 seconds 55 ° C Extension 1 minute 72 ° C Final Extension | 10 minutes 72 ° C Summary of PCR Reactions Started Used © r. the generation of chimeric constructions * Annotation: H = Human domain; M = mouse domain; for example, M HHH = Mouse domain 1, human domains 2-4. The PCR products generated these initial PCRs, were cut from 1% agarose gel and were purified using a gel purification kit (Qiagen) before elution in 50 μl of dH2O.

Primers used for the construction generation of TNFR 1 Chimeric PCR SOE The PCR assembly (also known as "hal ar through" or SOE Coating Extension Division see Gene, 75; 77 (1): 61-8 (1989)) allows primary PCR products to be brought along with digestion or ligation, making use of the complementary ends of primary PCR products. During this process the primary products are brought together and denatured before their complementary ends are allowed to knead together in the presence of Taq DNA polymerase and dNTPs. The various cycles of kneading and extension result in a filling in the complementary filaments and the production of a full-length tempering. The primers that now flank the full-length construction cassette are added and a conventional PCR was run to amplify the installed product. The SOE PCRs were performed in order to reduce together and amplify the various TNFR1 domains derived from the initial PCRs described above. The nested SOE PCRs were set as follows: 40 μl 1 0x PCR regulator containing MgCl 2; ~ 2 μl (100 ng) of initial product of initial PCR 1; ~ 2 μl (100 ng) of clean PCR 2 product; 36 μl of dH2O (at final volume of 80 μl). The SOE primer mixture was added after the installation step as follows: 2 μl of 5 'flanking primer (Primer 1); 2 μl of flanking primer 3"(Primer 2), 10 μl 1 0x PCR regulator, 6 μl of dH2O (final volume 20 μl) PCR reactions were performed using the program described above. they required approximately 45 minutes of which the thermocycler was set to pause at 94 ° C. 20 μl of primer mixture was added to each reaction and mixed.

Amplification conditions (Pause at 94 ° C, mix of primers added later) The PCR products were checked by running 3-5 μl of each reaction on a 1% agarose gel.

Cloning of TNFR1 chimeras installed in the Pichia expression vector. The pPicZalpha vector (Invitrogen) was sequentially ingested with EcoRI and Notl enzymes before purification of the Chromaspina TE-1000 gel filtration column (Clontech, Mountain View, CA).

Transformation of chimeric TNFR1 constructs in E. coli The ligated chimeric constructs were transformed into electrocompetent E. coli HB2151 cells and recovered for one hour in low LB medium to be laminated in low salt LB agar with 0.25 μg / ml ZEOCIN, formulation of antibiotic containing Pleomycin D (Cayla, Toulouse, France) for 24 hrs. at 37 ° C. The individual colonics were thus verified by sequence to ensure the correct sequence of the chimeric construct within the expression vector and the large-scale Maxiprep plasmid preparations made of each chimeric construct vector. The nucleotide sequences of chimeric constructs prepared are presented below. The chimeric constructions were named according to the origin of their domains (running from Domain 1 on the left to Domain 4 on the right). For example, HMMM contains Human Domain 1 and Mouse Domains 2-4. Chimeric proteins containing mouse domain 4 have only Hag tags and lack the separating region between the transmembrane region and Domain 4. HMMM AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG GGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGCCTCAGTTGC AAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTGACAAGGACA CGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTG CGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAAC ACCGTGTGTAACTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCC ACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAGCGCTCATCATCATCATCATCA TTAATGA (SEQ ID NO: 19) HHHM AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACA CCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC ACCGTGTGCACCTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCC ACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAGCGCTCATCATCATCATCATCA TTAATGA (SEQ ID NO: 20) HHMH AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACA CCGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTG CGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAAC ACCGTGTGTAACTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAA GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA (SEQ ID NO: 21) HMHH AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG GGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAGTTGC AAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTGACAAGGACA CGGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC ACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAA GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA (SEQ ID NO: 22) MHHH AGCTTGTGTCCCCAAGGAAAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGT GCCACAAAGGAACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAG GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACA CCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC ACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAA GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA (SEQ ID NO: 23) Preparation of chimeric TNFR1 construction and transformation in Pichia pastoris. The plasmid DNA generated by each maxiprep was ingested with the uncommonly cut restriction endonuclease Pmel in order to linearize the DNA before the pichia transformation. The linearized DNA was subsequently cleaned by phenol / chloroform extraction and ethanol precipitation, before resuspension in 30 μl of dH2O. 10 μl of the linearized DNA solution was mixed with 80 μl of electro-competent PICHIA KM71H cells for 5 minutes before electroporation at 1.5 kV, 200 μl. , 25 μF. The cells were immediately recovered with YPDS and incubated for 2 hours at 30 ° C before plating in agar agar and PDS containing 100 μg / ml ZEOCI N, antibiotic formulation containing Fleomicin D (Cayla, Toulouse, France), for 2 hours. days.

Expression of constructions in Pichia A colony of individual transformant for each construction was collected in 5 ml of BMGY as a starter culture and grown for 24 hrs. at 30 ° C. This culture was used to inoculate 500 ml of BMGY medium that grew for 24 hrs. at 30 ° C before the cells were collected by centrifugation at 1500-3000 g for 5 minutes at temp. ambient. The cells were resuspended in 100 ml of BM MY and grew for 4 days with graduated increases in methanol concentration (0.5% day 1, 1% day 2, 1.5% day 3 and 2% day 4). Then the expression supernatant was coated after centrifugation of the 3300 g cultures for 15 minutes.

Purification of TNFR1 chimeric constructions using nickel resin. The Culture supernatants were initially regulated through the addition of 10 mM final imidazole concentration and 2x PBS. The His-tagged protein was absorbed per group for 4 hours (shaking) at room temperature through the addition of Nickel-NTA resin. The supernatant / resin mixture was thus flowed on a poly-prep column (Biorad). The resin was thus washed with 10 column volumes of 2xPBS before elution using 250 mM imidazole 1x PBS. After regulatory exchange, chimeric construct expression was deglycosylated using the EndoH deglicosylase before verification by SDS-PAGE.

Temperate DNA sequences used during Human TNFR1 PCR (Homo sapiens) (access extracellular region) Genbank 33991418) CTGGTCCCTCACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGG AAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAAGG AACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGG AGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCA GCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAG TGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGG AGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTG CACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTC TTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAG TGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGCACTGAGGACTCA GGCACCACA (SEQ ID NO: 1) The extracellular region encoded by human TNFR1 has the following amino acid sequence. LVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECE SGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLF QCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTK1CLP QIENVKGTEDSGTT (SEQ ID NO: 2) Murine (Mus musculus) TNFR1 (extracellular region Genbank access 31,560,798) CTAGTCCCTTCTCTTGGTGACCGGGAGAAGAGGGATAGCTTGTGTCCCCAAGGA AAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGTGCCACAAAGGA ACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAGGGA GTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAG TTGCAAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGC TGACAAGGACACGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGA GTGAGACACACTTCCAGTGCGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGA CAATCCCCTGTAAGGAGACTCAGAACACCGTGTGTAACTGCCATGCAGGGTTCT TTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCCACTGCAAGAAAAATGAGGAG TGTATGAAGTTGTGCCTACCTCCTCCGCTTGCAAATGTCACAAACCCCCAGGAC TCAGGTACTGCG (SEQ ID NO: 3) The extracellular region encoded by murine (Mus musculus) TNFR1 has the following amino acid sequence. LVPSLGDREKRDSLCPQGKYVHSKNNSICCTKCHKGTYLVSDCPSPGRDTVCRJECE KGTFTASQNYLRQCLSCKTCRKEMSQVEISPCQADKDTVCGCKENQFQRYLSETHF QCVDCSPCFNGTVTIPCKETQNTVCNCHAGFFLRESECVPCSHCKKNEEC K1CLP PPLANVTNPQDSGTA (SEQ ID NO: 4) Domain specificity of anti-TNFR1 dAbs The domain specificity of anti-TNFR1 dAbs was determined using surface plasmon resonance (SPR) ('Detection of immuno-complex formation via surface plasmon resonance on gold-coated diffraction gratings. Biosensors, 1987-88; 3 (4): 211-25.) To determine the ability of antibodies to bind mouse or completely human biotinylated TNFR1 which was immobilized on a SPR chip surface, after the antibodies had been incubated and equilibrated with an excess of the chimeric receptors described above. In this test, the flow of an anti-TNFR1 dAb on the TNFR1 surface generates a SPR signal indicating the amount of dAb binding immobilized TNFR1 on the SPR chip. If the dAb is pre-incubated and equilibrated with a chimeric molecule comprising the domain (s) of TNFR1 that the particular dAb binds, then the flux of this mixture on the surface of TNFR1 will produce a smaller SPR signal in relation to dAb only. However, if the dAb is pre-incubated and equilibrated with a chimeric molecule that does not comprise the domain (s) of TNFR1 that the particular dAb binds, then the flux of this mixture on the surface of TNFR1 will produce a signal SPR which is approximately the same as the signal obtained during the dAb alone.

Method Generation of a TN FR1 surface chip SPR The surface choice of TNFR1 is determined by the spice specificity of the anti-TNFR1 dAb to be tested. Therefore, the anti-human TNFR1 dAbs were evaluated using a human TNFR1 coated surface and the anti-mouse TNFR1 dAbs were evaluated using a chip coated with mouse TNFR1. The biotinylated TNFR1 was diluted in the appropriate SPR regulator and run through a streptavidin (SA) sensor chip in a SPR BIACORE 3000 instrument (Biacore International AB, Uppsala, Switzerland). A low flow velocity (5-10 μl / minute) was used in order to maximize the contact time between the biotinylated TNFR1 and the streptavidin surface. The flow continued until the estraptividin surface was saturated with biotinylated material, in order to generate a chip with maximum TNFR 1 surface. The chip typically linked several hundred to several thousand SPR response units of the biotinylated material.

Concentration of an anti-TNFR1 response on the SPR chip A successful competition experiment requires the initial optimization of the anti-TNFR1 dAb concentration in such a way that the minimum amount of dAb flows over the surface giving an important SPR signal. Within a certain concentration range, the dAb will bind the surface in a dose dependent manner such that the number of united dAb RUs reflects the concentration of fluid dAb across the chip surface. In order to achieve the concentration range of this dose dependency, the anti-TNFR 1 dAbs were concentrated in a 10-fold dilution series of Biacore regulator ranging from 1 in 10 dilution to 1 in 1,000,000 dilution. Dilutions were thus injected individually and sequentially through the chip surface of TNFR1, starting with the most dilute sample. The maximum number of RUs achieved in each dilution was measured. After each injection the TNFR 1 surface was regenerated to remove bound anti-TNFR1 dAb where necessary using an appropriate SPR regeneration buffer. Using this method the maximum concentration of anti-TNFR1 dAb required to generate a signal representing approximately 100RU was determined. Pre-equilibration of dAbs / chimeric anti-TNFR1 Once the optimal anti-TNFR1 dAb concentration was determined, the TNFR1 mixtures of dAb / chimeric anti-TNFR1 were fixed. The mixtures were fixed in such a way that the final concentration of anti-TNFR1 dAb was identical to the previously determined optimum concentration. The reactions were typically fixed in 100 μl volumes containing 50 microliters of a 2 x concentrate of anti-TNFR1 dAb, 40 microliters of biacor buffer and 10 microliters of purified purified chimeric protein. Typical concentrations for the final mixture were about 10-100 μM of chimeric protein and about 10-100 nM of anti-TNFR1 dAb. The mixtures were left to equilibrate for 30 minutes at room temperature.

Biacore Competition Experiment After equilibration, each TNFR1 anti-TNFR1 / chimeric TNFR1 mix was run sequentially on the SPR surface of TNFR1 and the number of response units was measured. After each mixture was injected, the surface was regenerated to remove the bound anti-TNFR1 dAb before the next mixture was injected. The different responses generated using the different chimerics allowed the determination of the TNFR1 domains bound by particular dAbs. These studies revealed that TAR2m-21-23 binds Mouse TNFR1 Domain 1, and that TAR2h-205 binds Domain 1 of human TNFR1. ? TAR2m-21 -23 EVQLLESGGGLVQPGGSLRLSCAASGFTFN-RYSMG-WLRQAPGKG LEWVS- ID SYGRGTYYEDPVKG R-R FSISR D NSKNTLYLQMN SLRAEDTAV YCAK 1 SQ FG SNA FDY WGQGTQVTVSS (S EQ ID NO: 24) TAR2h-205 EVQ LLESGGG LVQPGGSLR LSCA KYSMG SGFTFV ~ ~ WVRQA? PGKGLEWVS ~~ QI S NTGGHTYYADSVKG- R FTISRDNSKNTLYLQ N SLRAEDTAVYYCAK YTGRW EP F DY WGQGTLVTVSS (SEQ ID NO: 25) In the amino acid sequences of TAR2M-21 -23 and TAR2h-205 CDR1 is flanked by ~, CDR2 is flanked by -, and CDR3 is flanked by ~ -. The amino acid sequences presented are continuous without openings.

Example 4. Methods of Selection Chimeric receptor proteins such as chimeric TNFR1 proteins described herein can be used in assays or selections to isolate agents (e.g., antibodies, dAbs) that bind to particular domains within a desired receptor. Using TNFR1 as a model for a desired receptor protein, these methods describe the addition of chimeric proteins to crude antibody preparations of their selection for receptor binding either by ELISA or surface plasmon resonance. Additionally, they describe the use of coated chimeric proteins on a surface (e.g., ELISA plate or SPR chip) and the selection of antibodies through proof of their binding to chimeric proteins on this surface. Similar methods can be used to identify agents (e.g., antibodies, dAbs) that bind to desired domains (e.g., off-site active) of other receptors.

Selection of ELI SA Soluble This method can be used to rapidly isolate antibodies or antibody fragments (e.g., dAbs) that bind specific domains of TNFR1 from a large repertoire of antibodies or antibody fragments of unknown specificity. A 96-well test plate will be coated overnight at 4 ° C with 100 μl per well of chimeric TNFR1. The cavities were washed 3 times with 0.1% TPBS (salt regulated by phosphate containing Tween-20 at a concentration of 0.1%). 200 μl per 1% cavity of TPBS will be added to block the plate, and the plate will be incubated 1-2 hours at room temperature. The wells will then be washed 3 times with PBS before the addition of 50 μl of bacterial or periprep supernatant, containing the soluble antibody or antibody fragment (containing the c-Myc epitope tag), in 50 μl of 0.2% TPBS . The plate will then be incubated for 1 hour at room temperature. After this plate will be washed 5 times with 0.1% TPBS (0.1% Tween-20 in PBS). 100 μl of a primary anti-c-Myc mouse monoclonal will thus be added in 0.1% of TPBS to each well and the plate will be incubated for 1 hour at room temperature. This primary antibody solution will be discarded and the plate will be washed 5 times with 0.1% TPBS. 100 μl of goat conjugate HRP of pre-diluted anti-mouse IgG (Fe specific) will be added as follows (Sigma Cat. No. A0168) and the plate will be incubated for 1 hour at room temperature. The secondary antibody will be discarded as such and the plate washed 6 times with 0.1% TPBS followed by 2 washes with PBS. 50 μl of TM B peroxidase solution will be added to each well and the plate will be left at room temperature for 2-60 minutes. The reaction will be detected by the addition of 50 μl of 1 M hydrochloric acid. The OD at 450 nm of the plate will be read in a 96-well plate reader within 30 minutes of acid addition. Those antibodies present in crude bacterial supernatant or peripreps that bind the TN1 FR domains present within the chimeric protein will give a stronger ELISA signal than those which do not.

ELI SA Competitive Assay This method can be used to rapidly select a diverse pool of crude antibody or antibody fragment preparations that bind TNFR1 in order to determine its domain binding specificity. A 96-well assay plate will be coated overnight at 4 ° C with 100 μl per well of human or murine TNFR1 (either human or mouse). The cavities will be washed 3 times with 0.1% TPBS (phosphate regulated salt containing Tween 20 at a concentration of 0.1%). 200 μl per cavity of 1% TPBS (1% Tween-20 in PBS) will be added to block the plate, and the plate will be incubated for 1-2 hours at room temperature. The wells will then be washed 3 times with PBS. At the same time the bacterial supernatants or peripreps will pre-equilibrate with a pre-optimized concentration of chimeric TNFR1 protein in solution. 50 μl of this crude bacterial preparation / chimeric protein mixture, containing the soluble antibody or antibody fragment will thus be added to the ELISA plate. The plate will be incubated for 1 hour at room temperature. Afterwards, the plate will be washed 5 times with 0.1% TPBS (0.1% Tween-20 in PBS), and 100 μl of a primary detection antibody (or Protein A-HRP or Protein L HRP) will be added in 0.1% of TPBS, to each well and the plate will be incubated for 1 hour at room temperature. This primary antibody solution will be discarded and the plate washed 5 times with 0.1% TPBS. If 100 μl of a HRP / goat pre-diluted secondary antibody conjugate is required, it will be added as such, and the plate will be incubated for 1 hour at room temperature. The secondary antibody will be discarded as such and the plate washed 6 times with 0.1% TPBS followed by 2 washes with PBS. 50 μl of peroxidase solution TM B will be added to each well and the plate will be left at room temperature for 2-60 minutes. The reaction will be stopped by the addition of 50 μl of 1 M hydrochloric acid. The OD at 450 nm of the plate will be read in a 96-well plate reader within 30 minutes of acid addition. A reduction in ELISA signal will be indicative of antibody binding of the chimeric TNFR 1 domains instead of full TNFR1 coated on the plate, and therefore, that the antibody be one of the domains within the chimeric protein.

Competitive ELISA Assay for Antibodies and Antibody Fragments Competing with a Reference Antibody or Antibody Fragment for TNFR1 Binding This method can be used to rapidly select various crude antibody groups or antibody fragment preparations that bind TNFR1 for those antibodies or antibody fragments that compete with a reference antibody or antibody fragment (e.g., TAR2m-21-23) to bind TNFR1 or bind a desired domain of TNFR1 (e.g., domain 1). The method uses a reference antibody or antibody fragment and test antibody or antibody fragment (eg, a population of antibodies to be selected) that contain different detectable labels (epitope tags). A 96-well assay plate will be coated overnight at 4 ° C with 100 μl per well of human or murine TNFR1. The cavities will be washed 3 times with 0.1% TPBS (phosphate regulated salt containing Tween 20 at a concentration of 0.1%). 200 μl per cavity of 1% TPBS (1% Tween-20 in PBS) will be added to block the plate, and the plate will be incubated for 1-2 hours at room temperature. The wells will then be washed 3 times with PBS. At the same time the crude antibody preparations to be tested will be mixed with a pre-optimized concentration of reference antibody or antibody fragment (e.g., domain binding antibody, TAR2m-21-23) in solution. As already stated it is important that this antibody does not include the same detection tags as present in the antibodies being selected for domain binding specificity. 50 μl of this crude bacterial preparation / chimeric protein mixture will be added to the ELISA plate. The plate will be incubated for 1 hour at room temperature. Afterwards, the plate will be washed 5 times with 0.1% TPBS, 100 μl of a primary detection antibody (which binds the label present only in the selected antibody population) will be added in 0.1% of TPBS to each cavity and the plate will be incubated for 1 hour at room temperature. This primary antibody solution will be discarded and the plate washed 5 times with 0.1% TPBS. 100 μl of a HRP conjugate / secondary antibody that recognizes the primary detection antibody will be added as such, and the plate will incubate for 1 hour at room temperature. The secondary antibody solution will be discarded as follows and the plate washed 6 times with 0.1% TPBS followed by 2 washes with PBS. 50 μl of TMB peroxidase solution will be added to each well and the plate will be left at room temperature for 2-60 minutes. The reaction will stop by the addition of 50 μl of 1 M hydrochloric acid. The OD at 450 nm of the plate will be read in a 96-well plate reader within 30 minutes of acid addition An ELI SA prepared and separated using this method but if the addition of the reference antibody or antibody fragment should be done in parallel A reduction in ELISA signal in the presence of reference antibody or antibody fragment, compared to the ELISA signal for the same antibody preparation without competing the reference antibody or antibody fragment, will indicate that the particular antibody or antibody fragment competes with the reference antibody or antibody fragment to bind to TNFR 1, and bind the same domain of TNFR 1 as the reference antibody or antibody fragment SPR Selection The ELISA methods described above can be easily adapted to a format using surface plasmon resonance, for example, using a SPR BIACORE 3000 instrument (Biacore International AB, Uppsala, Switzerland). Generally, the chimeric protein will be equilibrated with supernatant. Bacterial crude contained antibodies ant? -TN FR 1 or antibody fragments, and the resulting mixture flowed on a chi p SPR coated with full length human TNFR1 or murine TNFR1.

Example 5. PEGylation of dAbs The effects of PEGylation of dAbs on the ability of dAbs to bind TNFR1 in a cell-free receptor binding assay and to inhibit TNFR1 function on the cell surface in a cell-based assay it was studied.

Cell Assay Release Assay I L-8 M RC-5 The activities of certain dAbs that bind human TNFR1 were assessed in the following M-cell RC-5 assay. The assay is based on the induction of I L-8 secretion by TNF in M RC-5 cells and is adapted from the method described in Alceson, L. ef al. Journal of Biological Chemistry 27.7: 30517-30523 (1996), which described the induction of I L-8 by I L-1 in HUVEC. The activity of the dAbs was tested by evaluating the induction of I L-8 by human TNFa using M RC-5 cells instead of the HUVEC cell line. Briefly, the MRC-5 cells were laminated in microconcentration plates and the plates were incubated overnight with human dAb and TNFa (300 pg / ml). Following the incubation, the culture supernatant was aspirated and the concentration of I L-8 in the supernatant was measured by means of an intercalated ELISA (R &D Systems). The activity of anti-TNFR1 dAb resulted in a reduction in the secretion of I L-8 in the supernatant compared to the control cavities that were incubated with TNFa alone.

Receptor Binding Assay Anti-TNF dAbs were tested for the ability to bind TNF binding to recombinant TNF receptor 1 (p55) In short, Maxisorp plates were incubated overnight with 30 mg / ml antibody monoclonal mouse anti-human Fe (Zymed, San Francisco, USA) Cavities were washed with phosphate-buffered saline (PBS) containing 005% Tween-20 and then blocked with 1% BSA in PBS before being incubated with 100 μl. ng / ml TNF 1 receptor fusion protein Fe (R &D Systems, Mmneapohs, USA) Anti-TNF dAb was mixed with TNF that was added to the washed cavities at a final concentration of 10 ng / ml TNF binding was detected with 02 mg / ml biotinylated anti-TNF antibody ((HyCult biotechnology, Uben, The Netherlands) followed by 1 in 500 dilution streptavidin labeled with horseradish peroxidase (Amersham Biosciences, UK) and then incubation with substrate TMB (KPL, Gaithersburg, USA) The reaction was stopped by the addition of HCl and the absorption was read at 450 nm The activity of anti-TNF dAb led to a reduction in TNF binding and therefore a reduction in absorption compared to the single control of TNF The comparative effects of dAbs PEGylation in Cell surface TNFR1 are illustrated in the data presented in Table 5. Table 5 The receptor binding assay (RBA), which uses a recombinant form of receptor, containing only the extracellular part of the receptor in a non-cell-based assay format, is shown on the left, and where the effects of PEGylation are minimal and in the case of 5K PEG, no difference in the union was observed. In contrast, as observed in the data obtained in the cell assay, PEGylation of dAbs substantially reduced the effectiveness of the dAbs in the assay. In some cases, effectiveness with reduction up to a factor of 33 times. The data collected in the cell-based culture assay where it was based on the ability of dAbs to block receptor activity to the cell surface. The results of the study show that PEGylation of dAs had a much greater effect on the ability of dAbs to inhibit the function of TNFR1 on the cell surface than on the ability to bind TNFR1 in a cell-free assay.

Example 6. dAbs That Join Endogenous Combatant Molecules But Do Not Join The Union Site. This example illustrates a method for identifying the dAbs that bind a desired endogenous combatant molecule but do not bind the active site of the endogenous combatant. The procedure is illustrated using erythropoietin (epo), and endogenous combatants are generally applicable. EPO binding domain antibodies can be identified and isolated using any suitable screening method. A cell line that expresses the recombinant E PO receptor and that is dependent on hemoatopoietic factors for growth can be used in an assay to identify dAbs that bind EPO but do not bind in the active site, and therefore do not prevent the binding of EPO to the EPO receptor. For example, Ba / F3 cells, which is a murine I L-3-dependent cell line that has been transfected with human EPO receptor and overcome epo-dependent growth, can be used. (D'Andrea et al., Blood 82: 46-52 (1993).) Ba / F3 cells can be cultured in a suitable culture dish, such as PBS.The cells can be cultured thus for approximately 48 hours in culture medium that contains EPO The domain antibodies to be tested can be added to individual cultures (eg, individual wells of a suitable cell culture assay plate.) After about 48 hours, cell proliferation can be assayed using any suitable method, such as diphenyl-tetrazole dimethylthiazole (MTT) bromide reduction assay A lack of inhibition of cell proliferation is from cavities containing a dAb in relation to the control cavities containing culture medium but no dAb will indicate that the dAb does not bind the Epo active site All publications mentioned in this specification, and references cited in such publications, are incorporated herein for reference. Modifications and variations of the methods and system of the invention described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to the specific preferred embodiments, it should be understood that The invention as claimed should not be unduly limited to such specific embodiments. In turn, various modifications of the described modes of carrying out the invention, which are obvious to those skilled in molecular biology or related fields, are intended to be found within the scope of the following claims

Claims (98)

1. CLAIMING IS 1. Use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the amount of said endogenous target compound in a subject, wherein said ligand binds said endogenous target compound and does not substantially inhibit the activity of said endogenous target compound.
2. 2. Use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the bioavailability of said endogenous target compound in a subject, wherein said ligand binds said endogenous target compound and does not inhibit substantially the activity of said endogenous target compound.
3. 3. Use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the in vivo half-life of said endogenous target compound, wherein said ligand binds said endogenous target compound, and does not substantially inhibit the activity of said endogenous target compound.
4. 4. Use of a ligand comprising a portion having a binding site for an endogenous target compound for the manufacture of a medicament for local administration that increases the amount of said endogenous target compound at the site of administration, wherein said ligand binds said endogenous target compound, and does not substantially inhibit the activity of said endogenous target compound.
5. 5. The use according to any of claims 1 to 4 wherein the amount of endogenous target ligand in said subject approximately 4 hours after administration of said medicament is increased by at least 1.5 times, relative to the amount prior to administration of said medication. The use of claim 5 wherein the amount of endogenous target ligand in said subject about 4 hours after the administration of said medicament is increased by at least 10 times, relative to the amount prior to the administration of said medicament. The use according to any of claims 1 to 6 wherein said ligand binds said endogenous target compound and increases the in vivo half-life of said endogenous target compound by at least 1.5 times. The use of claim 7 wherein said ligand binds said endogenous target compound and increases the in vivo half-life of said endogenous target compound by at least 10-fold. The use according to any of claims 1 to 6 wherein said ligand inhibits the activity of said endogenous target compound by not more than about 10%. 10. The use according to any of claims 1 to 6 wherein the inhibitory concentration 50 (IC 50) of said ligand is at least 1 micromolar. eleven . The use according to any of claims 1 to 10 wherein said ligand binds said endogenous target compound but does not bind to the active site of said endogenous target compound. 12. The use according to any of claims 1 to 1 wherein said ligand comprises two or more copies of said portion having a binding site for an endogenous target. The use of claim 12 wherein said ligand is a dimer of said portion having a binding site for an endogenous target. The use according to any of claims 1 - 1 3 wherein said portion having a binding site for an endogenous target compound is selected from the group consisting of an affibody, an SpA domain, a class A domain of the LDL receptor, an EGF domain, and an avimer. 15. The use according to any of claims 1-14 wherein said portion having a binding site for an endogenous target compound is an antibody or antibody fragment. 16. The use of claim 15 wherein said portion having a binding site for an endogenous target compound is an antibody fragment selected from the group consisting of a Fv fragment., a single chain Fv fragment, a disulfide linked Fv fragment, a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, a diabody, a single immunoglobulin variable domain. The use of claim 16 wherein said portion having a binding site for an endogenous target compound is a unique immunoglobulin variable domain. 18. The use of claim 17 wherein said unique immunoglobulin variable domain is selected from the group consisting of a human VH, and a human L19. The use according to any of claims 1 to 18 wherein said ligand further comprises a portion extending the half-life. The use of claim 19 wherein said portion extending the half-life is a portion of polyalkylene glycol, serum albumin or a fragment thereof, transferrin receptor or a transferrin binding portion thereof, or a portion comprising a binding site for a polypeptide that improves the half-life in vivo. The use of claim 20 wherein said portion extending the half-life is an antibody or antibody fragment that has a binding site for a polypeptide that improves the half-life in vivo. 22. The use of claim 21 wherein said antibody or antibody fragment is a dAb. 23. The use of claim 20 wherein said portion extending the half-life is a portion comprising a binding site for a polypeptide that improves the in vivo half-life selected from the group consisting of an affinity, an SpA domain, a domain of class A of the LDL receptor, an EG F domain, and an avimer. 24. The use according to any of claims 21 to 23 wherein the polypeptide that improves the half-life in vivo is to the neonatal Fe or serum receptor. 25. The use of claim 20, wherein said portion extending the half-life is a portion of polyethylene glycol. 26. The use according to any of claims 1 to 25 wherein said medicament is substantially non-immunogenic. 27. The use according to any of claims 1 to 26 wherein said medicament is a depot formulation. 28. The use according to any one of claims 1 to 27 wherein said endogenous target compound is selected from the group consisting of a soluble cytokine receptor, an endogenous receptor antagonist, an enzyme, a cytokine, a growth factor, and a hormone. 29. The use of claim 28 wherein said endogenous target compound is a soluble cytokine receptor. 30. The use of claim 28 wherein said soluble cytokine receptor is soluble TN FR 1 31. The use according to any one of claims 1 -30 wherein the portion having a binding site for an endogenous target compound binds said endogenous target compound with Kd of 1 x 10'7 M or less. 32. The use according to any of claims 1 -30 wherein the ligand binds said endogenous target compound with Kd of 1 x 10"7 M or less 33. Use of a ligand comprising a portion having a binding site for a endogenous target compound for the manufacture of a medicament for increasing the activity of said endogenous target, wherein said ligand binds said endogenous target and does not substantially inhibit the activity of said endogenous target compound 34. The use of claim 33 wherein said ligand does not bind to the active site of said endogenous target compound 35. The use of claim 33 or claim 34 wherein said ligand inhibits the activity of said endogenous target compound by no more than about 10%. 33 or claim 34, wherein the inhibitory concentration 50 (IC 50) of said ligand is at least 1 micromolar. 37. Use according to any of claims 33-36. wherein said ligand comprises two or more portions having a binding site for said endogenous target. 38. The use according to any of claims 33-36 wherein said ligand comprises two or more copies of said portion having a binding site for an endogenous target. 39. The use of claim 38 wherein said ligand is a dimer of said portion having a binding site for an endogenous target. 40. The use according to any of claims 33-39 wherein said portion having a binding site for an endogenous target compound is selected from the group consisting of an affibody, an SpA domain, a class A domain of the LDL receptor, a EGF domain, and an avimer. 41 The use according to any of claims 33-40 wherein said portion having a binding site for an endogenous target compound is an antibody or antibody fragment. 42. The use of claim 41 wherein said portion having a binding site for an endogenous target compound is an antibody fragment selected from the group consisting of an Fv fragment, a single chain Fv fragment, a disulfide attached Fv fragment. , a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, a diabody, a single immunoglobulin variable domain. 43. The use of claim 42 wherein said portion having a binding site for an endogenous target compound is a unique immunoglobulin variable domain. 44. The use of claim 43 wherein said unique variable immunoglobulin domain is selected from the group consisting of a human VH, and a human VL-45. The use according to any of claims 33 to 44 wherein said ligand comprises in addition a portion extending the half life. 46. The use of claim 45 wherein said portion extending the half-life is a portion of polyalkylene glycol, serum albumin or a fragment thereof, transferrin receptor or a transferrin binding portion thereof, or a portion comprising a binding site for a polypeptide that improves the half-life in vivo. 47. The use of claim 46 wherein said portion extending the half-life is an antibody or antibody fragment having a binding site for a polypeptide that improves the half-life in vivo. 48. The use of claim 47 wherein said antibody or antibody fragment is a dAb. 49. The use of claim 46 wherein said portion extending the half-life is a portion comprising a binding site for a polypeptide that improves the in vivo half-life selected from the group consisting of an affinity agent., an SpA domain, a class A domain of the LDL receptor, an EGF domain, and an avimer. 50. The use according to any of claims 47 to 49 wherein the polypeptide that improves the half-life in vivo is serum albumin or neonatal Fe receptor. 51 The use of claim 46 wherein said portion extending the half-life is a polyethylene glycol portion. 52. The use according to any of claims 33 to 51 wherein said medicament is substantially non-immunogenic. 53. The use according to any of claims 1 to 27 wherein said medicament is a depot formulation. 54. The use according to any of claims 33 to 53 wherein said endogenous target compound is selected from the group consisting of a soluble cytokine receptor, an endogenous receptor antagonist, an enzyme, a cytokine, a growth factor, and a hormone. . 55. The use of claim 54 wherein said endogenous target compound is a soluable cytokine receptor. 56. The use of claim 55 wherein said soluble cytokine receptor is soluble TNFR1. 57. The use according to any of claims 33-56 wherein the portion having a binding site for an endogenous target compound binds said endogenous target compound with Kd of 1 x 10"7 M or less. of claims 33-56 wherein the ligand binds said endogenous target compound with Kd of 1 x 10"7 M or less. 59. Use of a ligand comprising two or more portions having a binding site for an endogenous target compound for the manufacture of a medicament for increasing the binding activity of said endogenous target compound, wherein said ligand binds said endogenous target, binds the active site of said endogenous target, and does not substantially inhibit the binding activity of said endogenous target compound. 60. The use of claim 59 wherein said ligand comprises two copies of a portion having a binding site for an endogenous target. 61 The use of claim 61 wherein said ligand is a dimer of said portion having a binding site for an endogenous target. 62. The use according to any of claims 59-61 wherein said binding activity is increased by at least a factor of 10. 63. The use according to any of claims 59-63 wherein said endogenous target compound is a receptor of soluble cytokine. 64. The use of claim 64 wherein said soluble cytokine receptor is soluble TNFR1, and said portions having a binding site for an endogenous target compound independently unite in a TNFR1 domain selected from the group consisting of Domain 1 and Domain 4. 65. The use according to any of claims 59-64 wherein said portion having a binding site for an endogenous target compound is selected from the group consisting of an affibody, an SpA domain, a class A domain of the LDL receptor, a EGF domain, and an avimer. 66. The use according to any of claims 59-64 wherein said portion having a binding site for an endogenous target compound is an antibody or antibody fragment. 67. The use of claim 66 wherein said portion having a binding site for an endogenous target compound is an antibody fragment selected from the group consisting of an Fv fragment, a single chain Fv fragment, a disulfide attached Fv fragment. , a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, a diabody, a single immunoglobulin variable domain. 68. The use of claim 67 wherein said portion having a binding site for an endogenous target compound is a unique immunoglobulin variable domain. 69. The use of claim 68 wherein said unique immunoglobulin variable domain is selected from the group consisting of a human VH, and a human VL-70. The use according to any of claims 59 to 69 wherein said ligand comprises in addition a portion extending the half life. 71 The use of claim 70 wherein said portion extending the half-life is a portion of polyalkylene glycol, serum albumin or a fragment thereof, transferrin receptor or a transferrin binding portion thereof, or a portion comprising a binding site for a polypeptide that improves the half-life in vivo. 72. The use of claim 71 wherein said portion extending the half-life is an antibody or antibody fragment having a binding site for a polypeptide that improves the half-life in vivo. 73. The use of claim 72 wherein said antibody or antibody fragment is a dAb. 74. The use of claim 71 wherein said portion extending the half-life is a portion comprising a binding site for a polypeptide that improves the in vivo half-life selected from the group consisting of an affibrant, an SpA domain, a class domain A of the LDL receptor, an EG F domain, and an avimer. 75. The use according to any of claims 72 to 74 wherein the polypeptide that improves the half-life in vivo is serum albumin or neonatal Fe receptor. 76. The use of claim 71 wherein said portion extending the half-life is a polyethylene glycol portion. 77. The use according to any of claims 59 to 76 wherein said medicament is substantially non-immunogenic. 78. The use according to any of claims 59 to 77 wherein said medicament is a depot formulation. 79. The use according to any of claims 59-78 wherein the portion having a binding site for an endogenous target compound binds said endogenous target compound with Kd of 1 x 10"7 M or less.80. Use according to any of the claims 59-78 wherein the ligand binds said endogenous target compound with Kd of 1 x 10"7 M or less. 81 A ligand that binds an endogenous target compound having suitable activity to treat a disease in a subject, wherein said ligand does not bind the active site of said endogenous target compound or substantially inhibits the activity of said endogenous target compound, for use in therapy of a disease suitable for treatment with said endogenous target compound. 82. The ligand of claim 81 wherein said ligand increases the in vivo half-life of said endogenous target compound. 83. The ligand of claim 81 or claim 82 wherein said ligand increases the amount of said endogenous target compound in a subject. 84. The ligand according to any of claims 81-83 wherein said ligand increases the bioavailability of said endogenous target compound. 85. The ligand according to any of claims 81-84 wherein said ligand increases the binding activity of said endogenous compound. 86. The ligand according to any of claims 81-85 wherein said ligand is as described in any of claims 1-80. 87. A pharmaceutical composition comprising a ligand that binds an endogenous target compound having suitable activity for treating a disease in a subject and a physiologically acceptable carrier, wherein said ligand does not bind the active site of said endogenous target compound or substantially inhibits the activity of said endogenous target compound. 88. The pharmaceutical composition of claim 87 wherein said ligand increases the in vivo half-life of said endogenous target compound. 89. The pharmaceutical composition of claim 87 or claim 88 wherein said ligand increases the amount of said endogenous target compound in a subject. 90. The pharmaceutical composition according to any of claims 87-89 wherein said ligand increases the bioavailability of said endogenous target compound. 91 The pharmaceutical composition according to any of claims 87-90 wherein said ligand increases the binding activity of said endogenous compound. 92. The pharmaceutical composition according to any of claims 87-91 wherein said ligand is as described in any of claims 1-80. 93. A drug delivery device comprising a pharmaceutical composition according to any of claims 87-92. 94. The drug delivery device of claim 93 wherein said drug delivery device is selected from the group consisting of a parenteral delivery device, intravenous delivery device, intramuscular delivery device, intraperitoneal delivery device, transdermal delivery, pulmonary delivery device, intraarterial delivery device, intrathecal delivery device, intra-articular delivery device, subcutaneous delivery device, intranasal delivery device, vaginal delivery device, and rectal delivery device. 95. The drug delivery device of claim 94 wherein said device is selected from the group consisting of a syringe, a transdermal delivery device, a capsule, a tablet, a nebulizer, an inhaler, an atomizer, an aerosolizer, a vaporizer, a dry powder inhaler, a metered dose inhaler, a metered dose sprayer, a metered dose vaporizer, a metered dose atomizer, a catheter. 96. Use of a ligand comprising a binding portion having a binding site for an endogenous target compound for increasing the half-life, bioavailability or activity of said endogenous compound, wherein said binding portion having a binding site for a Endogenous compound is said endogenous compound or a portion or variant thereof, and wherein said ligand binds said endogenous target compound and does not substantially inhibit the activity of said endogenous target compound. 97. The use of claim 96 wherein said endogenous compound is a soluble receptor that is a member of the TNF receptor superfamily. 98. Use of a ligand comprising a binding portion having a binding site for a member of the TNF receptor superfamily to increase the half-life, bioavailability or activity of said member of the TNF receptor superfamily, wherein said ligand binds said member of the TNF receptor superfamily and does not substantially inhibit the activity of said member of the TNF receptor superfamily, and wherein said binding portion having a binding site for a member of the TNF receptor superfamily is a domain of pre-ligand assembly (PLAD) or a variant thereof.