Methods And Compositions For Prolonging Elimination Half-times Of Bioactive Compounds

Abstract

Peptide ligands having affinity for IgG or for serum albumin are disclosed. Also disclosed are hybrid molecules comprising a peptide ligand domain and an active domain. The active domain may comprise any molecule having utility as a therapeutic or diagnostic agent. The hybrid molecules of the invention may be prepared using any of a number techniques including production in and purification from recombinant organisms transformed or transfected with an isolated nucleic acid encoding the hybrid molecule, or by chemical synthesis of the hybrid. The hybrid molecules have utility as agents to alter the elimination half-times of active domain molecules. Elimination half-time is altered by generating a hybrid molecule of the present invention wherein the peptide ligand has binding affinity for a plasma protein. In a preferred embodiment, a bioactive molecule having a short elimination half-time is incorporated as or into an active domain of the hybrid molecules of the invention, and the binding affinity of the peptide ligand domain prolongs the elimination half-time of the hybrid as compared to that of the bioactive molecule.

Description

FIELD OF THE INVENTION

[0001] This invention relates to novel compounds termed peptide ligands which bind a predetermined molecule such as a plasma protein. In particular aspects, the invention relates to compositions comprising a hybrid molecule comprising a peptide ligand domain and an active domain such as a biologically active molecule. The active domain may comprise a molecule useful for diagnostic or therapeutic purposes. In preferred embodiments, the hybrid compositions comprising the peptide ligand domain and active domain have improved pharmacokinetic or pharmacological properties. The invention further provides for the research, diagnostic and therapeutic use of the peptide ligand and includes compositions such as pharmaceutical compositions comprising the peptide ligand molecules.

DESCRIPTION OF RELATED DISCLOSURES

[0002] Phage-display provides a means for generating constrained and unconstrained peptide libraries (Devlin et al., (1990) Science 249:404-406; Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; Lowman (1997) Ann. Rev. Biophys. Biomol. Struct. 26:401-424). These libraries can be used to identify and select peptide ligands that can bind a predetermined target molecule (Lowman (1997), supra); Clackson and Wells (1994) Trends Biotechnol. 12:173-184; Devlin et al., (1990) supra). The technique has been used to identify peptide motifs that home to a cellular target (Arap et al., (1998) Science 279:377-380); bind the human type Iinterleukin 1 (IL-1) receptor blocking the binding of IL-.alpha. (Yanofsky et al., (1996) Proc. Natl. Acad. Sci. USA 93:7381-7386); bind to and activate the receptor for the cytokine erythropoietin (EPO) (Wrighton et al., (1996) Science 273:458-463); bind the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO)(Cwirla et al., (1996) Science 276:1696-1699), or to generate affinity improved or matured peptide ligands from native protein binding ligands (Lowman et al., (1991) Biochemistry 30:10832-10838).

[0003] Using structurally constrained peptide libraries generated by monovalent phage display, 14 amino acid peptides that specifically bind to insulin-like growth factor 1 binding proteins (IGFBPs) have been isolated (Lowman et al. (1998), Biochemistry 37:8870-8878). The peptides contain a helix structure and bind IGFBPs in vitro liberating insulin like growth factor-a (IGF-1) activity (Lowman et al., (1998) supra). Utilizing in vivo phage selection peptides capable of mediating selective localization to various organs such as brain and kidney (Pasqualini and Ruoslohti (1996) Nature 380:364-366) as well as peptides that home to particular tumor types bearing .alpha..sub.V.beta..sub.3 or .alpha.V.beta..sub.5 integrins have been identified (Arap et al. (1998), Science 279:377-380). U.S. Pat. No. 5,627,263 describes peptides that are recognized by and selectively bind the .alpha..sub.5.beta..sub.1 integrin. Examples of affinity or specificity improved proteins include human growth hormone, zinc fingers, protease inhibitors, atrial natriuretic factor, and antibodies (Wells, J. and Lowman H. (1992), Curr. Opin. Strides. Biol. 2:597-604; Clackson, T. and Wells, J. (1994), Trends Biotechnol. 12:173-184; Lowman et al., (1991) Biochemistry 30(10):832-838; Lowman and Wells J. (1993), J. Mol. Biol. 234:564-578; Dennis M. and Lazarus R. (1994), J. Biol. Chem. 269(22):137-144).

[0004] It has been suggested that the pharmakodynamics of insulin are altered if bound to serum albumin. Acylation of insulin with saturated fatty acids containing 10-16 carbon atoms produces insulin with affinity for albumin (Kurtzhals, P. et al. (1995) Biochem. J. 312:725-731). Differences in albumin binding affinity among acylated insulins were correlated with the timing of the blood-glucose lowering effects of the various molecules after subcutaneous injection into rabbits. Tighter binding to albumin was correlated with a delay in blood glucose lowering, possibly due to acylated insulin binding albumin in the subcutaneous tissue, resulting in a lower absorption rate of the acylated insulins when compared with non-acylated insulin.

[0005] A serum albumin-CD4 conjugate in which the V1 and V2 domains of CD4 were fused with human serum albumin (HSA) has been described (Yeh, P. et al. (1992), Proc. Natl. Acad. Sci. USA 89:1904-1908). The conjugate's elimination half-time was 140-fold that of a soluble CD4 (sCD4) in a rabbit experimental model.

[0006] Extended in vivo half-times of human soluble complement receptor type 1 (sCR1) fused to the albumin binding domains from Streptococcal protein G have been reported (Makrides, S. et al. (1996) J. Pharmacol. Exptl. Ther. 277:532-541). The constructs contained albumin binding domains of protein G having approximately 80 amino acids (fragment BA), and approximately 155 amino acids (fragment BABA).

[0007] The pharmacokinetics of a labeled IgG binding domain derived from the Z domain of protein A having approximately 60 amino acids and of a serum albumin binding domain derived from Streptococcal protein G (B-domain) having approximately 200 amino acids have been described (EP 0 486,525).

SUMMARY OF THE INVENTION

[0008] The present invention provides novel compounds that bind to plasma proteins. The compounds of the present invention (referred to as peptide ligands) are, for example, peptides or peptide derivatives such as peptide mimetics and peptide analogs. According to preferred aspects of the invention, the compounds are non-naturally occurring amino acid sequences that bind plasma proteins such as serum albumin or a portion of an immunoglobulin, as for example, IgG-Fc. Preferably the peptide ligand is a non-naturally occurring amino acid sequence of between about 10 and 20 amino acid residues.

[0009] Such compounds preferably bind a desired plasma protein with an affinity characterized by a dissociation constant, K.sub.d, that is less than about 100 .mu.M, preferably less than about 100 nM, and preferably do not substantially bind other plasma proteins. Specific examples of such compounds include linear or cyclic, especially cyclic peptides, preferably between about 10 and 20 amino acid residues in length, and combinations thereof, optionally modified at the N-terminus or C-terminus or both, as well as their salts and derivatives, functional analogues thereof and extended peptide chains carrying amino acids or polypeptides at the termini of the sequences.

[0010] Preferred peptide ligands bind IgG-Fc and include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following core formula:

[0011] Xaa.sub.i-Cys-Xaa.sub.j-Cys-Xaa.sub.k, wherein Xaa.sub.i is absent or is a peptide of between 1 and 4 amino acids, preferably 4 amino acids; X.sub.j is preferably 9 amino acids having a preferred sequence Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp (SEQ ID NO: 9); or Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10); or Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10), wherein Xaa.sub.1 preferably is Ala, Ser, or Thr; Xaa.sub.2 preferably is Trp or Tyr, Xaa.sub.3 preferably is His, or Trp; Xaa.sub.4 preferably is Leu or Met, and Xaa.sub.k is absent or between 1 and 5 amino acids, preferably 5 amino acids, so long as the cyclic peptide or analog thereof retains the qualitative biological activity of IgG-Fc binding.

[0012] Preferred among this group of compounds are compounds that bind IgG-Fc comprising the sequence:

TABLE-US-00001 (SEQ ID NO: 11) Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu- Val-Trp-Cys-Xaa-Xaa-Xaa-Xaa-Xaa; (SEQ ID NO: 12) Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu- Val-Trp-Cys-Xaa-Xaa-Xaa-Xaa-Xaa; (SEQ ID NO: 13) Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Cys-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-- Xaa.sub.8-Gly- Glu-Leu-Val-Trp-Cys-Xaa.sub.9-Xaa.sub.10-Xaa.sub.11-Xaa.sub.12-Xaa.sub.13, wherein Xaa.sub.5 is Ala, Ser, or Thr; Xaa.sub.6 is Trp or Tyr; Xaa.sub.7 is His or Trp; and Xaa.sub.8 is Leu or Met; and (SEQ ID NO: 14) Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Cys-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-- Xaa.sub.8-Gly- Glu-Leu-Val-Trp-Cys-Xaa.sub.9-Xaa.sub.10-Xaa.sub.11-Xaa.sub.12-Xaa.sub.13 wherein Xaa.sub.4 is Ser, Arg, or Asp; Xaa.sub.5 is Ala, Ser, or Thr; Xaa.sub.6 is Trp or Tyr; Xaa.sub.7 is His or Trp; Xaa.sub.8 is Leu or Met; and Xaa.sub.9 is Glu, Ser, Thr or Val.

[0013] Preferred peptide ligands that bind serum albumin include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following formulae:

and

TABLE-US-00002 (Xaa).sub.x-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa- Xaa-(Xaa).sub.z (SEQ ID NO: 113) (Xaa).sub.x-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser- Cys-(Xaa).sub.z (SEQ ID NO: 114) (Xaa).sub.x-Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe-(Xaa).sub.z (SEQ ID NO: 115) (Xaa).sub.x-Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys-(Xaa).sub.z (SEQ ID NO: 116) (Xaa).sub.x-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu- Trp-(Xaa).sub.z

[0014] Preferred are peptide compounds of the general formulae wherein Xaa is an amino acid and x and z are a whole number greater or equal to 0 (zero), generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5 and more preferably 4 or 5 and Xaa.sub.1 is selected from the group consisting of Ile, Phe, Tyr and Val.

[0015] In particular aspects the invention is directed to combinations of a peptide ligand with a bioactive compound to form a hybrid molecule that comprises a peptide ligand domain and an active domain. The bioactive compounds of the invention include any compound useful as a therapeutic or diagnostic agent. Non-limiting examples of bioactive compounds include polypeptides such as enzymes, hormones, cytokines, antibodies or antibody fragments, as well as organic compounds such as analgesics, antipyretics, antiinflammatory agents, antibiotics, antiviral agents, anti-fungal drugs, cardiovascular drugs, drugs that affect renal function and electrolyte metabolism, drugs that act on the central nervous system and chemotherapeutic drugs, to name but whew.

[0016] In preferred embodiments, the hybrid molecules comprising a peptide ligand domain and an active domain have improved pharmacokinetic or pharmacodynamic properties as compared to the same bioactive molecule comprising the active domain but lacking the peptide ligand domain. The improved pharmacokinetic or pharmacodynamic properties of the hybrids thereby provide for low-dose pharmaceutical formulations and novel pharmaceutical compositions. In certain aspects, the invention provides for methods of using the novel compositions including the therapeutic or diagnostic use of the hybrid molecules.

[0017] In particular aspects, the invention is directed to combinations of peptide ligands with bioactive compounds that have relatively short elimination half-times. The combinations are prepared with various objectives in mind, including improving the therapeutic or diagnostic efficacy of the bioactive compound in aspects of the invention involving in vivo use of the bioactive compound, by for example, increasing the elimination half-time of the bioactive compound. Fusing or linking (i.e., "conjugating") the peptide ligand directed against a plasma protein such as serum albumin, an immunoglobulin, an apolipoprotein or transferrin to a bioactive compound provides compositions with increased elimination half-times. Such combinations or fusions are conveniently made in recombinant host cells, or by the use of bifunctional crosslinking agents.

[0018] Other aspects of the invention include methods and compositions to purify antibodies using peptide ligands having binding affinity for immunoglobulins, such as, for example, the IgG-Fc peptide ligands disclosed herein.

[0019] The present invention further extends to therapeutic and diagnostic applications for the compositions described herein. Therefore, the invention includes pharmaceutical compositions comprising a pharmaceutically acceptable excipient and the hybrid molecules of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1. Phage competitive ELISA assay showing IgG binding of peptide-ligand tagged anti-VEGF Fab-phagemid particles. Four different constructs are shown: pY0192-569 (large filled circles), pY0192-570 (large open circles), PY0317-569 (small filled circles), and pY0317-570 ("x"'s).

[0021] FIG. 2. BIAcore.TM. analysis of IgG binding to peptide-ligand tagged anti-VEGF Fab Y0317-570 (tagged; top panel) Y0317 Fab (control; bottom panel). A cartoon illustration at top shows a model for the binding events observed in the tagged Fab experiment.

[0022] FIG. 3. Group average serum concentration vs. time data (.+-.SD) are presented in the figure for Fab-Y0317-570 and Fab-Y0317.

[0023] FIG. 4. The peptide sequences displayed by phage clones selected for binding to rabbit, human or rat albumin are shown in FIG. 4. Also indicated is the ability of individual phage clones to bind the 3 species of immobilized albumin.

[0024] FIGS. 5A and 5B. Sequences identified following soft randomization are shown in FIG. 5 along with their species specificity as determined by phage ELISA.

[0025] FIG. 6. Clones originating from the RB soft randomization library were found by ELISA to bind each of these species of albumin and were specific for albumin based upon their lack of binding to ovalbumin and casein.

[0026] FIG. 7. Clones that bind to multiple species of albumin (multi-species binders) are listed in FIG. 7.

[0027] FIGS. 8A, 8B and 8C. Sequences from libraries selected against rat, rabbit and human albumin are shown in FIGS. 8A, 8B, and 8C respectively.

[0028] FIG. 9. Peptides corresponding to identified phage sequences were synthesized and their affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay.

[0029] FIG. 10 A peptides corresponding to the SA06 identified phage sequence was synthesized and its affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay.

[0030] FIG. 11. The SA06 sequence was added to the carboxy terminus of either light chain (D3H44-L) or heavy chain (D3H44-Ls) of the Fab. In addition, identical constructs were made with the intra-chain disulfide replaced by alanines (D3H44-Ls and D3H44-Hs, respectively) as depicted in FIG. 11.

[0031] FIG. 12. Purified D3H44 fusions retained their ability to bind TF as measured using a FX activation assay.

[0032] FIG. 13. Purified D3H44 fusions retained their ability to bind TF as measured using a prothrombin time assay that measures prolongation of tissue factor dependent clotting.

[0033] FIG. 14 Unlike D3H44 lacking the albumin binding sequence (WT), both D3H44-L and D3H44-Ls are able to bind to albumin as measured in the SA08b binding assay.

[0034] FIG. 15 Both D3H44 albumin-binding fusions are capable of binding TF and albumin simultaneously as judged by a biotin-TF binding assay.

[0035] FIG. 16 Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters.

[0036] FIG. 17. Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

[0037] The term "peptide ligand" within the context of the present invention is meant to refer to non-naturally occurring amino acid sequences that function to bind a particular target molecule. Peptide ligands within the context of the present invention are generally constrained (that is, having some element of structure as, for example, the presence of amino acids which initiate a .beta. turn or .beta. pleated sheet, or for example, cyclized by the presence of disulfide-bonded Cys residues) or unconstrained (linear) amino acid sequences of less than about 50 amino acid residues, and preferably less than about 40 amino acids residues. Of the peptide ligands less than about 40 amino acid residues, preferred are the peptide ligands of between about 10 and about 30 amino acid residues and especially the peptide ligands of about 20 amino acid residues. However, upon reading the instant disclosure, the skilled artisan will recognize that it is not the length of a particular peptide ligand but its ability to bind a particular target molecule that distinguishes the peptide ligand of the present invention. Therefore peptide ligands of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 amino acid residues, for example, are equally likely to be peptide ligands within the context of the present invention.

[0038] A peptide ligand of the present invention will bind a target molecule with sufficient affinity and specificity if the peptide ligand "homes" to, "binds" or "targets" a target molecule such as a specific cell type bearing the target molecule in vitro and preferably in vivo (see, for example, the use of the term "homes to," "homing," and "targets" in Pasqualini and Ruoslahti (1996) Nature, 380:364-366 and Arap et al., (1998) Science, 279:377-380). In general, the peptide ligand will bind a target molecule with an affinity characterized by a dissociation constant, K.sub.d, of less than about 1 .mu.M, preferably less than about 100 nM and more preferably less than about 10 nM. However, peptide ligands having an affinity for a target molecule of less than about 1 nM and preferably between about 1 pM and 1 nM are equally likely to be peptide ligands within the context of the present invention. In general a peptide ligand that binds a particular target molecule as described above can be isolated and identified by any of a number of art-standard techniques as described herein.

[0039] Peptides ligands are amino acid sequences as described above which may contain naturally as well as non-naturally occurring amino acid residues. Therefore, so-called "peptide mimetics" and "peptide analogs" which may include non-amino acid chemical structures that mimic the structure of a particular amino acid or peptide may be peptide ligands within the context of the invention. Such mimetics or analogs are characterized generally as exhibiting similar physical characteristics such as size, charge or hydrophobicity present in the appropriate spacial orientation as found in their peptide counterparts. A specific example of a peptide mimetic compound is a compound in which the amide bond between one or more of the amino acids is replaced by, for example, a carbon-carbon bond or other bond as is well known in the art (see, for example Sawyer, in Peptide Based Drug Design pp. 378-422 (ACS, Washington D.C. 1995)).

[0040] Therefore, the term "amino acid" within the scope of the present invention is used in its broadest sense and is meant to include naturally occurring L .alpha.-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein (Lehninger, A. L., Biochemistry, 2d ed., pp. 71-92, (1975), Worth Publishers, New York). The correspondence between the standard single letter codes and the standard three letter codes is well known to the skilled artisan; and is reproduced here: A=Ala; C=Cys; D=Asp; E=Glu; F Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr, V=Val; W=Trp; Y=Tyr. The term includes D-amino acids as well as chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. For example, analogs or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as natural Phe or Pro are included within the definition of amino acid. Such analogs and mimetics are referred to herein as "functional equivalents" of an amino acid. Other examples of amino acids are listed by Roberts and Vellaccio The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983, which is incorporated herein by reference.

[0041] Peptide ligands synthesized by, for example, standard solid phase synthesis techniques, are not limited to amino acids encoded by genes. Commonly encountered amino acids which are not encoded by the genetic code, include, for example, those described in International Publication No. WO 90/01940 such as, for example, 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminoisobutyric acid (Aib) for Gly; cyclohexylalanine (Cha) for Val, and Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparigine (EtAsn) for Asn, and Gln; Hydroxyllysine (Hyl) for Lys; allohydroxyllysine (AHyl) for Lys; 3-(and 4)-hydroxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr; allo-isoleucine (AIle) for Ile, Leu, and Val; .rho.-amidinophenylalanine for Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala; N-methylisoleucine (MeIle) for Ile; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (Nle) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln; N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br, and I) phenylalanine, trifluorylphenylalanine, for Phe.

[0042] Peptide ligands within the context of the present invention may be "engineered", i.e., they are non-native or non-naturally occurring peptide ligands. By "non-native" or "non-naturally occurring" is meant that the amino acid sequence of the particular peptide ligand is not found in nature. That is to say, amino acid sequences of non-native or non-naturally occurring peptide ligands do not correspond to an amino acid sequence of a naturally occurring protein or polypeptide. Peptide ligands of this variety may be produced or selected using a variety of techniques well known to the skilled artisan. For example, constrained or unconstrained peptide libraries may be randomly generated and displayed on phage utilizing art standard techniques, for example, Lowman et al., (1998) Biochemistry 37: 8870-8878.

[0043] Peptide ligands, when used within the context of the present invention, may be "conjugated" to a therapeutic or diagnostic substance. The term "conjugated" is used in its broadest sense to encompass all methods of attachment or joining that are known in the art. For example, in a typical embodiment, the therapeutic or diagnostic substance is a protein (referred to herein as a "protein therapeutic"), and the peptide ligand will be an amino acid extension of the C- or N-terminus of the protein therapeutic. In addition, a short amino acid linker sequence may lie between the protein therapeutic and the peptide ligand. In this scenario, the peptide ligand, optional linker and protein therapeutic will be coded for by a nucleic acid comprising a sequence encoding protein therapeutic operably linked to (in the sense that the DNA sequences are contiguous and in reading frame) an optional linker sequence encoding a short polypeptide as described below, and a sequence encoding the peptide ligand. In this typical scenario, the peptide ligand is considered to be "conjugated" to the protein therapeutic optionally via a linker sequence. In a related embodiment, the peptide ligand amino acid sequence may interrupt or replace a section of the protein therapeutic amino acid sequence, provided, of course, that the insertion of the peptide ligand amino acid sequence does not interfere with the function of the protein therapeutic. In this embodiment, the "conjugate" may be coded for by a nucleic acid comprising a sequence encoding protein therapeutic interrupted by and operably linked to a sequence encoding the peptide ligand. In a further typical embodiment, the peptide will be linked, e.g., by chemical conjugation to the protein therapeutic or other therapeutic optionally via a linker sequence. Typically, according to this embodiment, the peptide ligand will be linked to the protein therapeutic via a side chain of an amino acid somewhere in the middle of the protein therapeutic that doesn't interfere with the therapeutic's activity. Here again, the peptide is considered to be "conjugated" to the therapeutic.

[0044] As used within the context of the present invention the term "target molecule" includes, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, and the like. Target molecules include, for example, extracellular molecules such as various serum factors including but not limited to plasma proteins such as serum albumin, immunoglobulins, apolipoproteins or transferrin, or proteins found on the surface of erythrocytes or lymphocytes, provided, of course, that binding of the peptide ligand to the cell surface protein does not substantially interfere with the normal function of the cell.

[0045] "Antibodies" and "immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains.

[0046] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments or regions, each with a single antigen-binding site, and a residual "Fc" fragment or region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.

[0047] Pepsin treatment yields an F(ab').sub.2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. The Fab' fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.

[0048] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0049] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

[0050] A "disorder" is any condition that would benefit from treatment with the compositions comprising the peptide ligands of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.

[0051] "Elimination half-time" is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, t 1/2, the time required for 50% completion of the process. The units of these two constants are time.sup.-1 and time, respectively. A first-order rate constant and the half-time of the reaction are simply related (k.times.t 1/2=0.693) and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e. after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

[0052] "Transfection" refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO.sub.4 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.

[0053] "Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et al., Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, NY (1989), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., (1983) Gene, 23:315 and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method described in sections 16.30-16.37 of Sambrook et al., supra, is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued 16 Aug. 1983. Transformations into yeast are typically carried out according to the method of Van Solingen et al., (1977) J. Bact., 130:946 and Hsiao et al., (1979) Proc. Natl. Acad. Sci. (USA), 76:3829. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or by protoplast fusion may also be used.

[0054] As used herein, the term "pulmonary administration" refers to administration of a formulation of the invention through the lungs by inhalation. As used herein, the term "inhalation" refers to intake of air to the alveoli. In specific examples, intake can occur by self-administration of a formulation of the invention while inhaling, or by administration via a respirator, e.g., to an patient on a respirator. The term "inhalation" used with respect to a formulation of the invention is synonymous with "pulmonary administration."

[0055] As used herein, the term "parenteral" refers to introduction of a compound of the invention into the body by other than the intestines, and in particular, intravenous (i.v.), intraarterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), intraventricular, and subcutaneous (s.c.) routes.

[0056] As used herein, the term "aerosol" refers to suspension in the air. In particular, aerosol refers to the particlization of a formulation of the invention and its suspension in the air. According to the present invention, an aerosol formulation is a formulation comprising a compound of the present invention that is suitable for aerosolization, i.e. particlization and suspension in the air, for inhalation or pulmonary administration.

II. Modes for Carrying out the Invention

[0057] A. Peptide Ligands

[0058] Peptide ligands within the context of the present invention bind a target, preferably a serum protein such as serum albumin or an immunoglobulin, and can be identified in a direct binding assay, or by their ability to compete for target binding with a known ligand for the target. Preferred peptide ligands that bind serum albumin include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following formulae or are peptides that compete for binding serum albumin of a particular mammalian species with peptides of the following formulae:

TABLE-US-00003 (Xaa).sub.x-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa- Xaa-(Xaa).sub.z (SEQ ID NO: 113) (Xaa).sub.x-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser- Cys-(Xaa).sub.z (SEQ ID NO: 114) (Xaa).sub.x-Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe-(Xaa).sub.z (SEQ ID NO: 115) (Xaa).sub.x-Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys-(Xaa).sub.z and (SEQ ID NO: 116) (Xaa).sub.x-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu- Trp-(Xaa).sub.z

Preferred are peptide compounds of the foregoing general formulae wherein Xaa is an amino acid and x and z are a whole number greater or equal to 0 (zero), generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5 and more preferably 4 or 5 and wherein Xaa.sub.1 is selected from the group consisting of Ile, Phe, Tyr and Val.

[0059] Further preferred peptide ligands that bind a serum albumin are identified as described herein in the context of the following general formulae (Xaa).sub.x-Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys-(Xaa- ).sub.z (SEQ ID NO: 117) and (Xaa).sub.x-Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp-(Xaa).sub.z (SEQ ID NO: 118) wherein Xaa is an amino acid and x and z are a whole number greater or equal to zero, generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5 and more preferably 4 or 5.

[0060] According to this aspect of the invention reference is made to the Figures and especially FIGS. 5A and 5B, 8A, 8B and 8C and FIG. 9 for exemplary peptides and appropriate amino acids for selecting peptides ligands that bind a mammalian serum albumin. In a preferred aspect, reference is made to FIG. 9 for selecting peptide ligands that bind across several species of serum albumin.

[0061] Preferred compounds according to this aspect of the invention include:

TABLE-US-00004 (SEQ ID NO: 119) Asp-Leu-Cys-Leu-Arg-Asp-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 120) Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 121) Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu- Trp-Glu-Asp (SEQ ID NO: 122) Gln-Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp- Gly-Cys-Leu-Trp-Glu-Asp-Asp-Phe (SEQ ID NO: 123) Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp- Gly-Cys-Leu-Trp-Gly-Asp-Ser-Val (SEQ ID NO: 124) Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp- Gly-Cys-Leu-Trp-Gly-Asp-Ser-Val-Lys (SEQ ID NO: 125) Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp- Glu-Asp-Asp (SEQ ID NO: 126) Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly- Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 127) Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu- Trp-Glu-Asp-Asp (SEQ ID NO: 121) Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu- Trp-Glu-Asp (SEQ ID NO: 128) Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Ala-Arg-Trp-Gly- Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 129) Glu-Val-Arg-Ser-Phe-Cys-Thr-Asp-Trp-Pro-Ala-Glu- Lys-Ser-Cys-Lys-Pro-Leu-Arg-Gly (SEQ ID NO: 130) Arg-Ala-Pro-Glu-Ser-Phe-Val-Cys-Tyr-Trp-Glu-Thr- Ile-Cys-Phe-Glu-Arg-Ser-Glu-Gln (SEQ ID NO: 131) Glu-Met-Cys-Tyr-Phe-Pro-Gly-Ile-Cys-Trp-Met

[0062] In a preferred embodiment, peptide ligands of the present invention bind IgG-Fc and can be identified by their ability to compete for binding of IgG-Fc in an in vitro assay with a peptide ligand having the general formula:

[0063] Xaa.sub.i-Cys-Xaa.sub.j-Cys-Xaa.sub.k, wherein Xaa.sub.i is absent or is a peptide of between 1 and 4 amino acids, preferably 4 amino acids; X.sub.j is preferably 9 amino acids having a preferred sequence Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp (SEQ ID NO: 9); or Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10); or Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 10), wherein Xaa.sub.1 is Ala, Ser, or Thr; Xaa.sub.2 is Trp or Tyr; Xaa.sub.3 is His, or Trp; Xaa.sub.4 is Leu or Met, and Xaa.sub.k is absent or between 1 and 5 amino acids, preferably 5 amino acids, so long as the cyclic peptide or analog thereof retains the qualitative biological activity of binding IgG-Fc described above.

[0064] Preferred among this group of compounds are compounds comprising the sequence:

[0065] Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp-Cys-Xaa-Xaa- -Xaa-Xaa-Xaa (SEQ ID NO: 11);

[0066] Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp-Cys-Xaa-Xaa- -Xaa-Xaa-Xaa (SEQ ID NO: 12);

[0067] Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Cys-Xaa.sub.5-Xaa.sub.6-Xaa- .sub.7-Xaa.sub.8-Gly-Glu-Leu-Val-Trp-Cys-Xaa.sub.9-Xaa.sub.10-Xaa.sub.11-X- aa.sub.12-Xaa.sub.13 (SEQ ID NO: 13), wherein Xaa.sub.5 is Ala, Ser, or Thr; Xaa.sub.6 is Trp or Tyr; Xaa.sub.7 is His, or Trp; and Xaa.sub.8 is Lou or Met; and

[0068] Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Cys-Xaa.sub.5-Xaa.sub.6-Xaa- .sub.7-Xaa.sub.8-Gly-Glu-Leu-Val-Trp-Cys-Xaa.sub.9-Xaa.sub.10-Xaa.sub.11-X- aa.sub.12-Xaa.sub.13 (SEQ ID NO: 14) wherein Xaa.sub.4 is Ser, Arg, or Asp; Xaa.sub.5 is Ala, Ser, or Thr; Xaa.sub.6 is Trp, Tyr; Xaa.sub.7 is His, or Trp; Xaa.sub.8 is Leu or Met; and Xaa.sub.9 is Glu, Ser, Thr or Val. In particular embodiments, the IgG-Fc binding peptide ligands of the present invention will compete with any of the peptide ligands represented in SEQ ID NO: 2-SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 11-SEQ ID NO: 111 described herein and preferably will compete with SEQ ID NO: 8 for binding IgG-Fc.

[0069] In another preferred embodiment, peptide ligands of the present invention bind human serum albumin and can be identified by their ability to compete for binding of human serum albumin in an in vitro assay with peptide ligands having the general formulae:

TABLE-US-00005 (SEQ ID NO: 116) (Xaa).sub.x-Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu- Trp-(Xaa).sub.z (SEQ ID NO: 113) (Xaa).sub.x-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser- Cys-(Xaa).sub.z (SEQ ID NO: 114) (Xaa).sub.x-Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe-(Xaa).sub.z or (SEQ ID NO: 115) (Xaa).sub.x-Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys-(Xaa).sub.z

wherein Xaa is an amino acid, x and z are preferably 4 or 5 and Xaa.sub.1 is selected from the group consisting of Ile, Phe, Tyr and Val.

[0070] In particular embodiments, the human serum albumin binding peptide ligands of the present invention will compete with any of the peptide ligands represented in SEQ ID NO: 120-131 described herein above and preferably will compete with SEQ ID NO: 122 for binding human serum albumin.

[0071] As will be appreciated from the foregoing, the term "compete" and "ability to compete" are relative terms. Thus the terms, when used to describe the peptide ligands of the present invention, refer to peptide ligands that produce a 50% inhibition of binding of, for example SEQ ID NO: 8 or SEQ ID NO: 122, when present at 50 .mu.M, preferably when present at 1 .mu.M, more preferably 100 nM, and preferably when present at 1 nM or less in a standard competition assay as described herein. Such peptide ligands generally will bind IgG-Fc with an affinity of less than 1 .mu.M, preferably less than about 100 nM and more preferably less than about 10 nM as determined by a standard competition assay such as the one described in the Example sections. However, peptide ligands having an affinity for a serum protein such as serum albumin or IgG-Fc of less than about 1 nM and preferably between about 1 pM and 1 nM are equally likely to be peptide ligands within the context of the present invention.

[0072] For in vitro assay systems to determine whether a peptide or other compound has the "ability" to compete with a peptide ligand for binding to an IgG-Fc (or other plasma protein such as, e.g., serum albumin) as noted herein, the skilled artisan can employ any of a number of standard competition assays. Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of ligand. The amount of analyte in the test sample is inversely proportional to the amount of standard that becomes bound to the ligand.

[0073] Thus, the skilled artisan may determine whether a peptide or other compound has the ability to compete with a peptide ligand for binding to an IgG-Fc (or other target such as a plasma protein) employing procedures which include but are not limited to competitive assay systems using techniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA), preferably the enzyme linked immunosorbent assay (ELISA), "sandwich" immunoassays, immunoradiometric assays, fluorescent immunoassays, and immunoelectrophoresis assays, to name but a few.

[0074] For these purposes the selected peptide ligand will be labeled with a detectable moiety (the detectably labeled peptide ligand hereafter called the "tracer") and used in a competition assay with a candidate compound for binding IgG-Fc domain or other target. Numerous detectable labels are available which can be preferably grouped into the following categories:

[0075] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I, .sup.3H, and .sup.131I. The peptide compound can be labeled with the radioisotope using the techniques described in Coligen et al., eds., Current Protocols in Immunology, Volumes 1 and 2 (1991), Wiley-Interscience, New York, N.Y., for example and radioactivity can be measured using scintillation counting.

[0076] (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated to the peptide compounds using the techniques disclosed in Current Protocols in Immunology, supra, for, example. Fluorescence can be quantified using a fluorimeter.

[0077] (c) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme preferably catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase, .theta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0078] Examples of enzyme-substrate combinations include, for example:

[0079] (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. ABTS, orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));

[0080] (ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and

[0081] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.

[0082] According to a particular assay, the tracer is incubated with immobilized target in the presence of varying concentrations of unlabeled candidate compound. Increasing concentrations of successful candidate compound effectively compete with binding of the tracer to immobilized target. The concentration of unlabeled candidate compound at which 50% of the maximally-bound tracer is displaced is referred to as the "IC.sub.50" and reflects the IgG binding affinity of the candidate compound. Therefore a candidate compound with an IC.sub.50 of 1 mM displays a substantially weaker interaction with the target than a candidate compound with an IC.sub.50 of 1 .mu.M.

[0083] In some phage display ELISA assays, binding affinity of a mutated ("mut") sequence was directly compared of a control ("con") peptide using methods described in B. C. Cunningham, D. G. Lowe, B. Li, B. D. Bennett, and J. A. Wells, EMBO J. 13:2508 (1994) and characterized by the parameter EC.sub.50. Assays were performed under conditions where EC.sub.50(con)/EC.sub.50(mut) will approximate K.sub.d(con)/K.sub.d(mut).

[0084] Accordingly, the invention provides compounds "having the ability to compete" for target molecules such as IgG or human serum albumin binding in an in vitro assay as described. Preferably the compound has an IC.sub.50 for the target such as IgG or human serum albumin of less than 1 .mu.M. Preferred among these compound are compounds having an IC.sub.50 of less than about 100 nM and preferably less than about 10 nM or less than about 1 nM. In further preferred embodiments according to this aspect of the invention the compounds display an IC.sub.50 for the target molecule such as IgG or human serum albumin of less than about 100 pM and more preferably less than about 10 pM.

[0085] A preferred in vitro assay for the determination of a candidate compound's ability to compete with a peptide ligand described herein is as follows and is described more fully in the Examples. In preferred embodiments the candidate compound is a peptide. The ability of a candidate compound to compete with a labeled peptide ligand tracer for binding to IgG or human serum albumin is monitored using an ELISA. Dilutions of a candidate compound in buffer are added to microtiter plates coated with IgG or human serum albumin (as described in the Example Sections) along with tracer for 1 hr. The microtiter plate is washed with wash buffer and the amount of tracer bound to IgG or human serum albumin measured.

[0086] B. Peptide Ligand Combinations

[0087] According to the present invention, the peptide ligand is optionally linked to a bioactive compound to form a hybrid molecule that comprises a peptide ligand domain and an active domain The bioactive compounds of the invention include any compound useful as a therapeutic or diagnostic agent. Non-limiting examples of bioactive compounds include polypeptides such as enzymes, hormones, cytokines, antibodies or antibody fragments, as well as organic compounds such as analgesics, antipyretics, antiinflammatory agents, antibiotics, antiviral agents, anti-fungal drugs, cardiovascular drugs, drugs that affect renal function and electrolyte metabolism, drugs that act on the central nervous system, chemotherapeutic drugs, etc. According to the present invention the peptide ligand domain is joined to an active domain, optionally via a flexible linker domain.

[0088] The hybrid molecules of the present invention are constructed by combining a peptide ligand domain with a suitable active domain. Depending on the type of linkage and its method of production, the peptide ligand domain may be joined via its N- or C-terminus to the N- or C-terminus of the active domain. For example, when preparing the hybrid molecules of the present invention via recombinant techniques, nucleic acid encoding a peptide ligand will be operably linked to nucleic acid encoding the active domain sequence, optionally via a linker domain Typically the construct encodes a fusion protein wherein the C-terminus of the peptide ligand is joined to the N-terminus of the active domain. However, especially when synthetic techniques are employed, fusions where, for example, the N-terminus of the peptide ligand is joined to the N- or C-terminus of the active domain also are possible. In some instances, the peptide ligand domain may be inserted within the active domain molecule rather than being joined to the active domain at its N- or C-terminus. This configuration may be used to practice the invention so long as the functions of the peptide ligand domain and the active domain are preserved. For example, a peptide ligand may be inserted into a non-binding light chain CDR of an immunoglobulin without interfering with the ability of the immunoglobulin to bind to its target. Regions of active domain molecules that can accommodate peptide ligand domain insertions may be identified empirically by selecting an insertion site, randomly, and assaying the resulting conjugate for the function of the active domain), or by sequence comparisons amongst a family of related active domain molecules (e.g., for active domains that are proteins) to locate regions of low sequence homology. Low sequence homology regions are more likely to tolerate insertions of peptide ligands domains than are regions that are well-conserved. For active domain molecules whose three-dimensional structures are known (e.g. from X-ray crystallographic or NMR studies), the three-dimensional structure may provide guidance as to peptide ligand insertion sites. For example, loops or regions with high mobility (i.e., large temperature or "B" factors) are more likely to accommodate peptide ligand domain insertions than are highly ordered regions of the structure, or regions involved in ligand binding or catalysis.

[0089] C. Linker Domains

[0090] According to the present invention, the peptide ligand domain is optionally linked to the active domain via a linker. The linker component of the hybrid molecule of the invention does not necessarily participate in but may contribute to the function of the hybrid molecule. Therefore, according to the present invention, the linker domain, is any group of molecules that provides a spatial bridge between the active domain and the peptide ligand domain.

[0091] The linker domain can be of variable length and makeup, however, according to the present invention, it is the length of the linker domain and not its structure that is important. The linker domain preferably allows for the peptide ligand domain of the hybrid molecule to bind, substantially free of steric and/or conformational restrictions to the target molecule. Therefore, the length of the linker domain is dependent upon the character of the two "functional" domains of the hybrid molecule, i.e., the peptide ligand domain and the active domain.

[0092] One skilled in the art will recognize that various combinations of atoms provide for variable length molecules based upon known distances between various bonds (Morrison, and Boyd, Organic Chemistry, 3rd Ed, Allyn and Bacon, Inc., Boston, Mass. (1977)). For example, the linker domain may be a polypeptide of variable length. The amino acid composition of the polypeptide determines the character and length of the linker. In a preferred embodiment, the linker molecule comprises a flexible, hydrophilic polypeptide chain. Exemplary, linker domains comprises one or more Gly and or Ser residues, such as those described in the Example sections herein.

[0093] D. Recombinant Synthesis

[0094] The present invention encompasses a composition of matter comprising an isolated nucleic acid, preferably DNA, encoding a peptide ligand or a hybrid molecule comprising a peptide ligand domain and a polypeptide active domain as described herein. DNAs encoding the peptides of the invention can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al. (1989), Agnew. Chem. Int. Ed. Engl. 28:716-734, the entire disclosure of which is incorporated herein by reference, such as the triester, phosphite, phosphoramidite and H-phosphonate methods. In one embodiment, codons preferred by the expression host cell are used in the design of the encoding DNA. Alternatively, DNA encoding the peptides of the invention can be altered to encode one or more variants by using recombinant DNA techniques, such as site specific mutagenesis (Kunkel et al. (1991), Methods Enzymol., 204:125-139; Carter et al. (1986), Nucl. Acids Res. 13:4331; Zoller et al. (1982), Nucl. Acids Res. 10:6487), cassette mutagenesis (Wells et al. (1985), Gene 34:315), restriction selection mutagenesis (Carter, Directed Mutagenesis: A Practical Approach (M. J. McPherson, ed.) IRL Press, Oxford, 1991), and the like.

[0095] According to preferred aspects described above, the nucleic acid encodes a peptide ligand capable of binding a target molecule. Target molecules include, for example, extracellular molecules such as various serum factors including but not limited to plasma proteins such as serum albumin, immunoglobulins, apolipoproteins or transferrin, or proteins found on the surface of erythrocytes or lymphocytes, provided, of course, that binding of the peptide ligand to the cell surface protein does not substantially interfere with the normal function of the cell.

[0096] According to another preferred aspect of the invention, the nucleic acid encodes a hybrid molecule comprising a peptide ligand domain sequence and an active domain. In this aspect of the invention, the active domain may comprise any polypeptide compound useful as a therapeutic or diagnostic agent, e.g., enzymes, hormones, cytokines, antibodies or antibody fragments. The nucleic acid molecule according to this aspect of the present invention encodes a hybrid Molecule and the nucleic acid encoding the peptide ligand domain sequence is operably linked to (in the sense that the DNA sequences are contiguous and in reading frame) the nucleic acid encoding the biologically active agent. Optionally these DNA sequences may be linked through a nucleic acid sequence encoding a linker domain amino acid sequence.

[0097] According to this aspect, the invention further comprises an expression control sequence operably linked to the DNA molecule encoding a peptide of the invention, an expression vector, such as a plasmid, comprising the DNA molecule, wherein the control sequence is recognized by a host cell transformed with the vector, and a host cell transformed with the vector. In general, plasmid vectors contain replication and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells.

[0098] For expression in prokaryotic hosts, suitable vectors include pBR322 (ATCC No. 37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, any vector in the pRIT20 or pRIT30 series (Nilsson and Abrahmsen (1990), Meth. Enzymol. 185:144-161), pRIT2T, pKK233-2, pDR540 and pPL-lambda. Prokaryotic host cells containing the expression vectors of the present invention include E. coli K12 strain 294 (ATCC NO. 31,446), E. coli strain JM101 (Messing et al.(1981), Nucl. Acid Res. 9:309), E. coli strain B, E. coli strain 1776 (ATCC No. 31537), E. coli c600, E. coli W3110 (F-, gamma-, prototrophic, ATCC No. 27,325), E. coli strain 27C7 (W3110, tonA, phoA E15, (argF-lac)169, ptr3, degP41, ompT, kan.sup.r) (U.S. Pat. No. 5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhinzuriumn, Serratia marcesans, and Pseudomonas species.

[0099] In addition to prokaryotes, eukaryotic organisms, such as yeasts, or cells derived from multicellular organisms can be used as host cells. For expression in yeast host cells, such as common baker's yeast or Saccharomyces cerevisiae, suitable vectors include episomally-replicating vectors based on the 2-micron plasmid, integration vectors, and yeast artificial chromosome (YAC) vectors. For expression in insect host cells, such as Sf9 cells, suitable vectors include baculoviral vectors. For expression in plant host cells, particularly dicotyledonous plant hosts, such as tobacco, suitable expression vectors include vectors derived from the Ti plasmid of Agrobacterium tumefaciens.

[0100] Examples of useful mammalian host cells include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. (1977), J. Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA 77:4216); mouse sertoli cells (TM4, Mather (1980), Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al. (1982), Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). For expression in mammalian host cells, useful vectors include vectors derived from SV40, vectors derived from cytomegalovirus such as the pRK vectors, including pRK5 and pRK7 (Suva et al. (1987), Science 237:893-896; EP 307,247 (Mar. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived from vaccinia viruses or other pox viruses, and retroviral vectors such as vectors derived from Moloney's murine leukemia virus (MoMLV).

[0101] Optionally, the DNA encoding the peptide of interest is operably linked to a secretory leader sequence resulting in secretion of the expression product by the host cell into the culture medium. Examples of secretory leader sequences include STII, ecotin, lamB, herpes GD, 1 pp, alkaline phosphatase, invertase, and alpha factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (Abrahmsen et al. (1985), EMBO J. 4:3901).

[0102] Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0103] Prokaryotic host cells used to produce the present peptides can be cultured as described generally in Sambrook et al., supra.

[0104] The mammalian host cells used to produce peptides of the invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace (1979), Meth. in Enz. 58:44, Barnes and Sato (1980), Anal. Biochem. 102:255, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of which are incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin.TM. drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0105] The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.

[0106] E. Chemical Synthesis

[0107] Another method of producing the compounds of the invention involves chemical synthesis. This can be accomplished by using methodologies well known in the art (see Kelley, R. F. & Winkler, M. E. in Genetic Engineering Principles and Methods, Setlow, J. K., ed., Plenum Press, N.Y., Vol. 12, pp 1-19 (1990); Stewart, J. M. Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill. (1984); see also U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

[0108] Peptide ligands of the invention can be prepared conveniently using solid-phase peptide synthesis. Merrifield (1964), J. Am. Chem. Soc. 85:2149; Houghten (1985), Proc. Natl. Acad. Sci. USA 82:5132. Solid-phase peptide synthesis also can be used to prepare the hybrid molecule compositions of the invention if the active domain is or comprises a polypeptide.

[0109] Solid-phase synthesis begins at the carboxy terminus of the nascent peptide by coupling a protected amino acid to an inert solid support. The inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid. Typically, the macromolecular support is a cross-linked polymeric resin (e.g., a polyamide or polystyrene resin) as shown in FIGS. 1-1 and 1-2, on pages 2 and 4 of Stewart and Young, supra. In one embodiment, the C-terminal amino acid is coupled to a polystyrene resin to form a benzyl ester. A macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the .alpha.-amino group of the blocked amino acids in peptide synthesis. If a base-labile .alpha.-protecting group is used, then it is desirable to use an acid-labile link between the peptide and the solid support. For example, an acid-labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis as described on page 16 of Stewart and Young, supra. Alternatively, a peptide anchor link and .alpha.-protecting group that are differentially labile to acidolysis can be used. For example, an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis as described on pages 11-12 of Stewart and Young, supra.

[0110] After the initial amino acid is coupled to an inert solid support, the .alpha.-amino protecting group of the initial amino acid is removed with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralized in, for example, triethylamine (TEA). Following deprotection of the initial amino acid's .alpha.-amino group, the next .alpha.-amino and side chain protected amino acid in the synthesis is added. The remaining .alpha.-amino and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support. Alternatively, some amino acids may be coupled to one another to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain.

[0111] The condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the axide method, mixed acid anhydride method, DCC (N,N'-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy-tris [dimethylamino] phosphonium hexafluorophosphate) method, N-hydroxysuccinic acid imido ester method, etc., and Woodward reagent K method.

[0112] It is common in the chemical synthesis of peptides to protect any reactive side chain groups of the amino acids with suitable protecting groups. Ultimately, these protecting groups are removed after the desired polypeptide chain has been sequentially assembled. Also common is the protection of the .alpha.-amino group on an amino acid or peptide fragment while the C-terminal carboxy group of the amino acid or peptide fragment reacts with the free N-terminal amino group of the growing solid phase polypeptide chain, followed by the selective removal of the .alpha.-amino group to permit the addition of the next amino acid or peptide fragment to the solid phase polypeptide chain. Accordingly, it is common in polypeptide synthesis that an intermediate compound is produced which contains each of the amino acid residues located in the desired sequence in the peptide chain wherein individual residues still carry side-chain protecting groups. These protecting groups can be removed substantially at the same time to produce the desired polypeptide product following removal from the solid phase.

[0113] .alpha.- and .epsilon.-amino side chains can be protected with benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2Cl)], p-nitrobenzyloxycarbonyl [Z(NO.sub.2)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl (Mpt) groups, and the like.

[0114] Protective groups for the carboxy functional group are exemplified by benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group. For example, the guanidino group of arginine may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group of cysteine can be protected with p-methoxybenzyl, trityl, and the like.

[0115] Many of the blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif.), Bachem Calif. (Torrence, Calif.) or Peninsula Labs (Belmont, Calif.).

[0116] Stewart and Young, supra, provides detailed information regarding procedures for preparing peptides. Protection of .alpha.-amino groups is described on pages 14-18, and side chain blockage is described on pages 18-28. A table of protecting groups for amine, hydroxyl and sulfhydryl functions is provided on pages 149-151.

[0117] After the desired amino acid sequence has been completed, the peptide can be cleaved away from the solid support, recovered and purified. The peptide is removed from the solid support by a reagent capable of disrupting the peptide-solid phase link, and optionally deprotects blocked side chain functional groups on the peptide. In one embodiment, the peptide is cleaved away from the solid phase by acidolysis with liquid hydrofluoric acid (HF), which also removes any remaining side chain protective groups. Preferably, in order to avoid alkylation of residues in the peptide (for example, alkylation of methionine, cysteine, and tyrosine residues), the acidolysis reaction mixture contains thio-cresol and cresol scavengers. Following HF cleavage, the resin is washed with ether, and the free peptide is extracted from the solid phase with sequential washes of acetic acid solutions. The combined washes are lyophilized, and the peptide is purified.

[0118] F. Chemical Conjugation of Hybrids

[0119] In certain embodiments of the present invention, the hybrid molecules may comprise active domains that are organic compounds having diagnostic or therapeutic utility, or alternatively, fusions between a peptide ligand domain and a polypeptide active domain in configurations that cannot be encoded in a single nucleic acid. Examples of the latter embodiment include fusions between the amino terminus of a peptide ligand and the amino terminus of the active domain, or fusions between the carboxy-terminus of a peptide ligand and the carboxy-terminus of the active domain.

[0120] Chemical conjugation may be employed to prepare these embodiments of the hybrid molecule, using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene, 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0121] G. Disulfide-Linked Peptides

[0122] As described above; some embodiments of the invention include cyclized peptide ligands. Peptide ligands may be cyclized by formation of a disulfide bond between cysteine residues. Such peptides can be made by chemical synthesis as described above and then cyclized by any convenient method used in the formation of disulfide linkages. For example, peptides can be recovered from solid phase synthesis with sulfhydryls in reduced form, dissolved in a dilute solution wherein the intramolecular cysteine concentration exceeds the intermolecular cysteine concentration in order to optimize intramolecular disulfide bond formation, such as a peptide concentration of 25 mM to 1 .mu.M, and preferably 500 .mu.M to 1 .mu.M, and more preferably 25 .mu.M to 1 .mu.M, and then oxidized by exposing the free sulfhydryl groups to a mild oxidizing agent that is sufficient to generate intramolecular disulfide bonds, e.g., molecular oxygen with or without catalysts such as metal cations, potassium ferricyanide, sodium tetrathionate, etc. Alternatively, the peptides can be cyclized as described in Pelton et al. (1986), J. Med. Chem. 29:2370-2375.

[0123] Cyclization can be achieved by the formation, for example, of a disulfide bond or a lactam bond between a first Cys and a second Cys. Residues capable of forming a disulfide bond include, for example, Cys, Pen, Mpr, and Mpp and its 2-amino group-containing equivalents. Residues capable of forming a lactam bridge include, for example, Asp Glu, Lys, Orn, .alpha..beta.-diaminobutyric acid, diaminoacetic acid, aminobenzoic acid and mercaptobenzoic acid. The compounds herein can be cyclized for example via a lactam bond which can utilize the side chain group of a non-adjacent residue to form a covalent attachment to the N-terminus amino group of Cys or other amino acid. Alternative bridge structures also can be used to cyclize the compounds of the invention, including for example, peptides and peptidomimetics, which can cyclize via S--S, CH2-S, CH2-O--CH2, lactam ester or other linkages.

[0124] H. Pharmaceutical Compositions

[0125] Pharmaceutical compositions which comprise the hybrid molecules of the invention may be administered in any suitable manner, including parental, topical, oral, or local (such as aerosol or transdermal) or any combination thereof.

[0126] Other suitable compositions of the present invention comprise any of the above-noted compositions with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, in oral administration, usually using a solid carrier and ini.v. administration, a liquid salt solution carrier.

[0127] The compositions of the present invention include pharmaceutically acceptable components that are compatible with the subject and the protein of the invention. These generally include suspensions, solutions and elixirs, and most especially biological buffers, such as phosphate buffered saline, saline, Dulbecco's Media, and the like. Aerosols may also be used, or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like (in the case of oral solid preparations, such as powders, capsules, and tablets).

[0128] As used herein, the term "pharmaceutically acceptable" generally means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0129] The formulation of choice can be accomplished using a variety of the aforementioned buffers, or even excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate, and the like. "PEGylation" of the compositions may be achieved using techniques known to the art (see for example International Patent Publication No. WO92/16555, U.S. Pat. No. 5,122,614 to Enzon, and international Patent Publication No. WO92/00748).

[0130] A preferred route of administration of the present invention is in the aerosol or inhaled form. The compounds of the present invention, combined with a dispersing agent, or dispersant, can be administered in an aerosol formulation as a dry powder or in a solution or suspension with a diluent.

[0131] As used herein, the term "dispersant" refers to a agent that assists aerosolization of the compound or absorption of the protein in lung tissue, or both. Preferably the dispersant is pharmaceutically acceptable. Suitable dispersing agents are well known in the art, and include but are not limited to surfactants and the like. For example, surfactants that are generally used in the art to reduce surface induced aggregation of a compound, especially a peptide compound, caused by atomization of the solution forming the liquid aerosol, may be used. Nonlimiting examples of such surfactants are surfactants such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts of surfactants used will vary, being generally within the range of from about 0.001% to about 4% by weight of the formulation. In a specific aspect, the surfactant is polyoxyethylene sorbitan monooleate or sorbitan trioleate. Suitable surfactants are well known in the art, and can be selected on the basis of desired properties, depending on the specific formulation, concentration of the compound, diluent (in a liquid formulation) or form of powder (in a dry powder formulation), etc.

[0132] Moreover, depending on the choice of the peptide ligand, the desired therapeutic effect, the quality of the lung tissue (e.g., diseased or healthy lungs), and numerous other factors, the liquid or dry formulations can comprise additional components, as discussed further below.

[0133] The liquid aerosol formulations generally contain the peptide ligand/active domain hybrid and a dispersing agent in a physiologically acceptable diluent. The dry powder aerosol formulations of the present invention consist of a finely divided solid form of the peptide ligand/active domain hybrid and a dispersing agent. With either the liquid or dry powder aerosol formulation, the formulation must be aerosolized. That is, it must be broken down into liquid or solid particles in order to ensure that the aerosolized dose actually reaches the alveoli. In general the mass median dynamic diameter will be 5 micrometers or less in order to ensure that the drug particles reach the lung alveoli (Wearley, L. L. (1991), Crit. Rev. in Ther. Drug Carrier Systems 8:333). The term "aerosol particle" is used herein to describe the liquid or solid particle suitable for pulmonary administration, i.e., that will reach the alveoli. Other considerations such as construction of the delivery device, additional components in the formulation and particle characteristics are important. These aspects of pulmonary administration of a drug are well known in the art, and manipulation of formulations, aerosolization means and construction of a delivery device require at most routine experimentation by one of ordinary skill in the art.

[0134] With regard to construction of the delivery device, any form of aerosolization known in the art, including but not limited to nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used in the practice of the invention. A delivery device that is uniquely designed for administration of solid formulations is envisioned. Often, the aerosolization of a liquid or a dry powder formulation will require a propellant. The propellant may be any propellant generally used in the art. Specific nonlimiting examples of such useful propellants are a chlorofluorocarbon, a hydrofluorocarbon, a hydrochlorofluorocarbon, or a hydrocarbon, including trifluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.

[0135] In a preferred aspect of the invention, the device for aerosolization is a metered dose inhaler. A metered dose inhaler provides a specific dosage when administered, rather than a variable dose depending on administration. Such a metered dose inhaler can be used with either a liquid or a dry powder aerosol formulation. Metered dose inhalers are well known in the art.

[0136] Once the peptide ligand/active domain hybrid reaches the lung, a number of formulation-dependent factors affect the drug absorption. It will be appreciated that in treating a disease or disorder that requires circulatory levels of the compound, such factors as aerosol particle size, aerosol particle shape, the presence or absence of infection, lung disease or emboli may affect the absorption of the compounds. For each of the formulations described herein, certain lubricators, absorption enhancers, protein stabilizers or suspending agents may be appropriate. The choice of these additional agents will vary depending on the goal. It will be appreciated that in instances where local delivery of the compounds is desired or sought, such variables as absorption enhancement will be less critical.

[0137] I. Liquid Aerosol Formulations

[0138] The liquid aerosol formulations of the present invention will typically be used with a nebulizer. The nebulizer can be either compressed air driven or ultrasonic. Any nebulizer known in the art can be used in conjunction with the present invention such as but not limited to: Ultravent, Mallinckrodt, Inc. (St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood Colo.). Other nebulizers useful in conjunction with the present invention are described in U.S. Pat. Nos. 4,624,251 issued Nov. 25, 1986; 3,703,173 issued Nov. 21, 1972; 3,561,444 issued Feb. 9, 1971 and 4,635,627 issued Jan. 13, 1971.

[0139] The formulation may include a carrier. The carrier is a macromolecule which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those of skill in the art would accept injection of said carrier into a patient as part of a therapeutic regime. The carrier preferably is relatively stable in the circulatory system with an acceptable elimination half-time. Such macromolecules include but are not limited to soya lecithin, oleic acid and sorbetan trioleate, with sorbitan trioleate preferred.

[0140] The formulations of the present embodiment may also include other agents useful for protein stabilization or for the regulation of osmotic pressure. Examples of the agents include but are not limited to salts, such as sodium chloride, or potassium chloride, and carbohydrates, such as glucose, galactose or mannose, and the like.

[0141] J. Aerosol Dry Powder Formulations

[0142] It is also contemplated that the present pharmaceutical formulation will be used as a dry powder inhaler formulation comprising a finely divided powder form of the peptide ligand and a dispersant. The form of the compound will generally be a lyophilized powder. Lyophilized forms of peptide ligand/active domain hybrid compounds can be obtained through standard techniques.

[0143] In another embodiment, the dry powder formulation will comprise a finely divided dry powder containing one or more compounds of the present invention, a dispersing agent and also a bulking agent. Bulking agents useful in conjunction with the present formulation include such agents as lactose, sorbitol, sucrose, or mannitol, in amounts that facilitate the dispersal of the powder from the device.

[0144] K. Research, Manufacturing, and Diagnostic Compositions

[0145] In a preferred embodiment, the peptide ligands or the hybrid molecules of the invention are non-covalently adsorbed or covalently bound to a macromolecule, such as a solid support. It will be appreciated that the invention encompasses macromolecules complexed with the peptide ligands or hybrid molecules. In a preferred embodiment, the peptide ligands of the invention are directed against an immunoglobulin, such as, e.g., the IgG-Fc peptide ligands disclosed infra. Such peptide ligands may be used as affinity purification agents. In this process, the peptide ligands are immobilized on a solid phase support such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized peptide ligand is contacted with a sample containing the immunoglobulin protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the immunoglobulin protein, which is bound to the immobilized peptide ligand. Finally, the support is washed with another suitable solvent, such as glycine buffer, 5.0, that will release the immunoglobulin protein from the peptide ligand.

[0146] In general, the solid support is an inert matrix, such as a polymeric gel, comprising a three-dimensional structure, lattice or network of a material. Almost any macromolecule, synthetic or natural, can form a gel in a suitable liquid when suitably cross-linked with a bifunctional reagent. Preferably, the macromolecule selected is convenient for use in affinity chromatography. Most chromatographic matrices used for affinity chromatography are xerogels. Such gels shrink on drying to a compact solid comprising only the gel matrix. When the dried xerogel is resuspended in the liquid, the gel matrix imbibes liquid, swells and returns to the gel state. Xerogels suitable for use herein include polymeric gels, such as cellulose, cross-linked dextrans (e.g. Sepharose), agarose, cross-linked agarose, polyacrylamide gels, and polyacrylamide-agarose gels.

[0147] Alternatively, aerogels can be used for affinity chromatography. These gels do not shrink on drying but merely allow penetration of the surrounding air. When the dry gel is exposed to liquid, the latter displaces the air in the gel. Aerogels suitable for use herein include porous glass and ceramic gels.

[0148] Also encompassed herein are the peptide ligands or hybrid molecules of the invention coupled to derivatized gels wherein the derivative moieties facilitate the coupling of the hybrid molecules to the gel matrix and avoid steric hindrance of the peptide ligand-target molecule interaction in affinity chromatography. Alternatively, spacer arms can be interposed between the gel matrix and the hybrid molecules for similar benefits.

[0149] A variation on the above contemplates the use of gene fusions and the use of the peptide ligands as purification reagents. According to this aspect of the invention the gene encoding a peptide ligand is associated, in a vector, with a gene encoding another protein or a fragment of another protein. This results in the peptide ligand being produced by the host cell as a fusion with another protein or peptide. The "other" protein or peptide is often a protein or peptide which can be secreted by the cell, making it possible to isolate and purify the other protein from the culture medium and eliminating the necessity of destroying the host cells which arises when the other protein remains inside the cell. Alternatively, the fusion protein can be expressed intracellularly. It is useful to use fusion proteins that are highly expressed.

[0150] The use of gene fusions is analogous to the use of Protein A fusions which are often used because the binding of protein A, or more specifically the Z domain of protein A binds to IgG and provides an "affinity handle" for the purification of the fused protein. According to a preferred aspect of the invention, peptide ligands which bind serum albumin are use as "affinity handles" for the purification of fused proteins on a solid serum albumin support. For example, a DNA sequence encoding the desired peptide ligand can be fused by site directed mutagenesis to the gene for protein. After expression and secretion, the fusion protein can be purified on a matix of serum albumin to which the peptide ligand will bind. After purification the peptide ligand can be enzymatically or chemically cleaved to yield free protein or left intact to aid in increasing the elimination half life of the fused protein. Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly residue. Using standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5' end of the gene encoding the desired peptide. Alternatively, one can employ proteolytic cleavage of fusion protein. Carter, in Protein Purification From Molecular Mechanisms to Large-Scale Processes, Ladisch et al., eds. (American Chemical Society Symposium Series No. 427, 1990), Ch 13, pages 181-193.

[0151] The following examples are offered by way of illustration and not by way of limitation. The disclosures of all citations in the specification are expressly incorporated herein by reference.

Example 1

IgG-Fc Peptide Ligands

[0152] An in vitro selection designed to identify peptide ligands which bind the IgG-Fc surface without the constraint that the peptides function in vivo was performed. The selection was accomplished using a combination of polyvalent and monovalent phage display which has recently been applied to generate peptides that bind a variety of cellular hormones and receptors. N. C. Wrighton, et al, (1996), Science 273:458, O. Livnah, et al. (1996), Science 273:464. A single disulfide-constrained peptide library was constructed that consisted of 4.times.10.sup.9 different peptides of the form Xaa.sub.i-Cys-Xaa.sub.j-Cys-Xaa.sub.k wherein Xaa is a random amino acid from an NNS codon, i+j+k=18, and j=4 through 10. This library was expressed on the surface of M13 bacteriophage as an N-terminal fusion to the gene VIII protein with a short linker consisting of glycine and serine residues. H. B. Lowman et al. (1998). Biochemistry 37: 8870-8878. More particularly, the library construct contained an STII secretion signal peptide, the peptide library of twenty amino acid length, i.e., Xaa.sub.i-Cys-Xaa.sub.j-Cys-Xaa.sub.k wherein Xaa is a random amino acid from an NNS codon, i+j+k=18, and j=4 through 10, a Gly-Gly-Gly-Ser-Gly-Gly-Gly linker (SEQ ID NO: 1), and the M13 gene VIII starting at the first residue of the mature protein.

[0153] In principle, peptides could be selected that bind to potentially any region of the IgG-Fc due to the unbiased nature of this library. However, after several rounds of selection, the library became dominated by a single peptide, Fc-I (Glu-Thr-Gln-Arg-Cys-Thr-Trp-His-Met-Gly-Glu-Leu-Val-Trp-Cys-Glu-Arg-Glu-- His-Asn) (SEQ ID NO: 2). Selections were performed as described in H. B. Lowman, et al., supra, with the following modifications: microtiter wells were coated using 5 .mu.g/ml IgG-Fc; Casein Blocker Buffer (Pierce) was used in place of 0.1% BSA to better prevent non-specific binding; elution of phage was effected with either 75 mM DTT or 0.2 mM glycine pH 2.0 with equivalent results. IgG-Fc was obtained by papain cleavage of CD4-IgG.sub.1 immunoadhesin protein, Capon et al. (1989), Nature, 337: 525. Cleaved material was purified over Protein A Sepharose followed by Superdex-75 (Pharmacia) and then quantified by absorbance at 280 nm.

[0154] Repetition of the selection experiment again gave Fc-I and also a related peptide, Fc-II (Lys-Glu-Ala-Ser-Cys-Ser-Tyr-Trp-Leu-G-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-Va- l-Glu) (SEQ ID NO: 3). The Fc-II peptide shared the cysteine spacing and the internal Gly-Glu-Leu-Val-Trp (SEQ ID NO: 132) sequence seen in Fc-I. Apparently, these two peptides bound IgG-Fc with an affinity high enough to be selected over any of the other IgG-Fc binding peptides present in the starting pool. Both peptides were synthesized on solid phase using standard 9-fluorenylmethoxycarbonyl protocols and purified by reversed-phase HPLC. Masses were confirmed by electrospray mass spectrometry, and purified peptides were quantified by UV absorbance at 280 nm.

[0155] Competition ELISAs were performed in a manner similar to the method described in H. B. Lowman, et al., supra. Briefly, Protein A Z-domain was immobilized on microtiter wells at a concentration of 5 .mu.g/ml, blocked, and washed as described. A matrix of mixtures of biotinylated-IgG-Fc at concentrations from 312 nM to 0.3 nM and peptide at concentrations from 215 .mu.M to 0.8 nM was prepared. These mixtures were incubated with immobilized Protein A Z-domain for 1 hour. Plates were then washed and developed as described using avidin/HRP conjugate. Inhibition curves were then computed for each concentration of biotin-IgG-Fc, and then the curve of half-maximal inhibition, "IC.sub.50", was extrapolated to zero biotin-IgG-Fc concentration in order to obtain a K.sub.i. The Fc-I and Fc-II peptides both were found to compete with Protein A (Z-domain) (B. Nilsson et al. (1987), Protein Eng. 1:107) for binding to IgG-Fc with inhibition constants (K.sub.i) of about 5 .mu.M. The results imply that these peptides bind to an overlapping site on IgG-Fc that coincides with the Protein A binding site.

[0156] The DNA sequence of the Fc-II peptide was moved to a monovalent phage display format by cassette mutagenesis to give a construct with the STII signal sequence, the Fc-II peptide Lys-Glu-Ala-Ser-Cys-Ser-Tyr-Trp-Leu-Gly-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-V- al-Glu (SEQ ID NO: 3), a Gly-Gly-Gly-Pro-Gly-Gly-Gly linker (SEQ ID NO: 4), and the M13 gene III protein starting at residue 253. The Fc-II sequence was affinity-matured by monovalent phage display. Five residue blocks were randomly mutated in six separate libraries to exhaustively cover the non-cysteine positions in the peptide sequence and then screened against IgG-Fc.

[0157] A series of second generation monovalent phage display libraries were constructed based on the Fc-II sequence Lys-Glu-Ala-Ser-Cys-Ser-Tyr-Trp-Leu-Gly-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-V- al-Glu (SEQ ID NO: 3) in which five sequential residues were randomized using NNS codons in each library starting at positions 1, 4, 7, 10, 12, and 16, excluding the two cysteines. Each library had a diversity of approximately 1.times.10.sup.8. These libraries were independently screened for binding to IgG-Fc for six rounds and then sequenced. Preferred residues from this selection were then recombined using three additional libraries that spanned the entire peptide sequence. The three additional libraries were constructed using the degeneracy of the genetic code to recombine the preferred amino acids at each position into one peptide. The DNA sequences for these libraries contained the following mixtures of bases (IUPAC codes): DRG GWA GMA RRC TGC KCT TRS CAC MTG GGC GAG CTG GTC TGG TGC RVC RVM BKC GAS KDW (SEQ ID NO: 5), DRS VWG SVG RRC TGC KCC TRS YRS MTG GGC GAG CTG GTC TGG TGC RNC VVS NBS GWS KDM (SEQ ID NO: 6), and DNS NNS NNS VNS TGC BVG TDS HRS MDS GGC GAG STC KKG WRG TGC RNM NNS NNS NNS NNM (SEQ ID NO: 7). These libraries also were sorted against IgG-Fc for six rounds and then sequenced.

[0158] After screening against IgG-Fc, the consensus patterns from these libraries suggested a highly conserved 13-residue core sequence (Asp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr) (SEQ ID NO: 8). The corresponding peptide (Fc-III) was synthesized and found to inhibit binding of Protein A (Z-domain) to Fc with an IC.sub.50 of 100 nM. Thus, although Fc-III is seven residues shorter than Fc-II, it binds 50-times more tightly. Despite its smaller size, the binding affinity of Fc-III to Fc was only ten-fold weaker than that of the domains from Protein A and Protein G, which are each about four times larger and bind with K.sub.dS around 10 nM. S. R. Fahnestock, et al. in Bacterial Immunoglobulin-Binding Proteins (Academic Press, Inc. 1990) Vol. 1, chap. 11. R. Karlsson, L. Jendeberg, B. Nilsson, J. Nilsson, P. Nygren (1995), J. Immuno. Methods 183:43.

[0159] Table I lists the amino acid sequences and IgG-Fc binding affinities of exemplary IgG-Fc peptide ligands that were identified using the procedures described above.

TABLE-US-00006 TABLE I IgG-Fc Peptide Ligand Sequences and Affinities Binding Sequence Sequence ID NO Affinity Peptides *All peptides have an N-terminal amine and a C-terminal amide KEASCSYWLGELVWCVAGVE SEQ ID NO: 3 5000 nM (K.sub.i) ETQRCTWHMGELVWCEREHN SEQ ID NO: 2 5000 nM (K.sub.i) DLADCSWHMGELVWCSRVEG SEQ ID NO: 15 50 nM (K.sub.d) WEADCAWHLGELVWCTPMEF SEQ ID NO: 16 30 nM (IC.sub.50) DCAWHLGELVWCT SEQ ID NO: 8 100 nM (IC.sub.50) Phage Clones All phage af- (M13/gIII Display) finities are EC.sub.50S N/A = Not individu- ally assayed. Since they were selected for binding, EC.sub.50 likely to be <1 uM or better. All of the peptides listed bind IgG-Fc. Focused Libraries KEASCSYWLGELVWCDTLTE SEQ ID NO: 17 N/A KEASCSYWLGELVWCSPGVE SEQ ID NO: 18 734 nM KEASCSYWLGELVWCSGVEG SEQ ID NO: 19 N/A KEASCSYWLGELVWCSAGVE SEQ ID NO: 20 N/A ESEDCSYWLGELVWCVAGVE SEQ ID NO: 21 N/A EKEDCSYWLGELVWCVAGVE SEQ ID NO: 22 N/A EDPDCSYWLGELVWCVAGVE SEQ ID NO: 23 N/A EEADCSYWLGELVWCVAGVE SEQ ID NO: 24 N/A NADDCSYWLGELVWCVAGVE SEQ ID NO: 25 N/A SETTCSYWLGELVWCVAGVE SEQ ID NO: 26 N/A AWKTCQWLGELVWCVAGVE SEQ ID NO: 27 N/A DLADCSYWLGELVWCSRVEG SEQ ID NO: 28 776 nM KEADCAWHLGELVWCVAGVE SEQ ID NO: 29 138 nM KEAECSYHLGELVWCVAGVE SEQ ID NO: 30 N/A KEARCWYWHGELVWCSDPEE SEQ ID NO: 31 809 nM KEASCSYHLGELVWCVAGVE SEQ ID NO: 32 416 nM KEASCSWHLGELVWCVAGVE SEQ ID NO: 33 225 nM KEASCSYWLGELVWCTEGVE SEQ ID NO: 34 818 nM KEASCSYWLGELVWCDDGVE SEQ ID NO: 35 N/A KEASCSYWLGELVWCSEGVE SEQ ID NO: 36 N/A KEASCSYWLGELVWCSPGVE SEQ ID NO: 18 N/A KEASCSYWLGEVWKCKSGVE SEQ ID NO: 37 N/A KEASCSYWLGELVWCDNGVE SEQ ID NO: 38 N/A KEASCSYWLGELVWCDTFDE SEQ ID NO: 39 301 nM KEASCSYWLGELVWCDGLDE SEQ ID NO: 40 326 nM KEASCSYWLGELVWCVGLDE SEQ ID NO: 41 278 nM KEASCSYWLGELVWCEDTLE SEQ ID NO: 42 N/A KEASCSYWLGELVWCEDTME SEQ ID NO: 43 N/A KEASCSYWLGELVWCEDMME SEQ ID NO: 44 N/A WVEDCSWHMGELVWCDGGEF SEQ ID NO: 45 139 nM KEASCSYWLGELVWCDWMNG SEQ ID NO: 46 N/A KEASCSYWLGELVWCDDTPV SEQ ID NO: 47 N/A KEASCSYWLGELVWCDDYGE SEQ ID NO: 48 N/A KEASCSYWLGELVWCSDLWE SEQ ID NO: 49 N/A WRGGCSWHMGELVWCEHDME SEQ ID NO: 50 N/A AVSKCSFHMGELVWCSDVMN SEQ ID NO: 51 N/A NQVSCSYSRGELVWCSKQSQ SEQ ID NO: 52 N/A GRMECAWHQGELVWCTPTLE SEQ ID NO: 53 N/A GTMECSWHQGELVWCTPTLA SEQ ID NO: 54 N/A EMRDCSWHLGELVWCAHMEG SEQ ID NO: 55 N/A GSWECAYHLGELVWCETGSG SEQ ID NO: 56 N/A VAEPCAYHLGELVWCEVLKG SEQ ID NO: 57 N/A KEAMCSYWLGELVWCESDMP SEQ ID NO: 58 N/A Designed Clones DLADCSWHLGELVWGSRVEG SEQ ID NO: 59 9 nM DLADCSWHLGELVWCVGLDE SEQ ID NO: 60 28 nM WVEDCSWHLGELVWCVGLDF SEQ ID NO: 61 31 nM Secondary Optimization KVADCAWHMGELVWCTEVEG SEQ ID NO: 62 23 nM GEEDCSYHLGELVMCTELDD SEQ ID NO: 63 69 nM GVADCAWHLGELVWCTERED SEQ ID NO: 64 N/A GEEDCAWHLGELVWCSGGDF SEQ ID NO: 65 100 nM WEADCAWHLGELVWCTKVEE SEQ ID NO: 66 7 nM GEADCSYHLGELVWCNDFEE SEQ ID NO: 67 156 nM WVDCAYHLGELVWCSTFEE SEQ ID NO: 68 9 nM WVEDCAWHMGELVWCTKVDE SEQ ID NO: 69 70 nM READCAWHLGELVWCSERDL SEQ ID NO: 70 47 nM EEASCAYHLGELVWCDAFDV SEQ ID NO: 71 77 nM RVASCAWHLGELVWCDGLDG SEQ ID NO: 72 N/A GEADCAWHLGELVWCTKVEE SEQ ID NO: 73 38 nM GEASCAYHLGELVWCDEGEG SEQ ID NO: 74 386 nM RVEDCAYHLGELVWCTEGDE SEQ ID NO: 75 63 nM EEPDCSWHLGELVMCTPMEV SEQ ID NO: 76 14 nM KEADCAWHMGELVWCSEMEG SEQ ID NO: 77 66 nM EQADCAWHLGELVWCTPMVF SEQ ID NO: 78 8 nM EEPDCSWHLGELVWCTPIEV SEQ ID NO: 79 15 nM GEPDCAWHLGELVWCTPMVF SEQ ID NO: 80 7 nM GEQDCSYHMGELVWCTTVDG SEQ ID NO: 81 210 nM GVRNCAYHLGELVWCTPMEF SEQ ID NO: 82 10 nM RVADCAWHMGELVWCSELEV SEQ ID NO: 83 44 nM GEADCAWHLGELVWCTPMDL SEQ ID NO: 84 N/A GEQDCSWHLGELVWCTPMEV SEQ ID NO: 85 N/A GMRDCSYHLGELVWCSDMEL SEQ ID NO: 86 N/A EVADCSWHLGELVWCTEGEF SEQ ID NO: 87 54 nM GEEDCAWHLGELVWCTDVED SEQ ID NO: 88 52 nM EVEDCAYHLGELVWCSDLEG SEQ ID NO: 89 82 nM WEEDCAWHLGELVWCAEFDE SEQ ID NO: 90 44 nM KEASCAWHLGELVWCSEVEE SEQ ID NO: 91 130 nM ALA Scan on Phage AEADCAWHLGELVWCTKVEE SEQ ID NO: 92 20 nM WAADCAWHLGELVWCTKVEE SEQ ID NO: 93 34 nM WEPDCAWHLGELVWCTKVEE SEQ ID NO: 94 36 nM WEAACAWHLGELVWCTKVEE SEQ ID NO: 95 55 nM WEAACSWHLGELVWCTKVEE SEQ ID NO: 96 10 nM WEADCAAHLGELVWCTKVEE SEQ ID NO: 97 798 nM WEADCAWALGELVWCTKVEE SEQ ID NO: 98 139 nM WEADCAWHAGELVWCTKVEE SEQ ID NO: 99 56 nM WEADCAWHLAELVWCTKVEE SEQ ID NO: 100 12 nM WEADCAWHLGALVWCTKVEE SEQ ID NO: 101 11 nM WEADCAWHLGEAVWCTKVEE SEQ ID NO: 102 1890 nM WEADCAWHLGELAWCTKVEE SEQ ID NO: 103 4670 nM WEADCAWHLGELVACTKVEE SEQ ID NO: 104 3380 nM WEADCAWHLGELVWCAKVEE SEQ ID NO: 105 101 nM WEADCAWHLGELVWCTAVEE SEQ ID NO: 106 10 nM WEADCAWHLGELVWCTKAEE SEQ ID NO: 107 8 nM WEADCAWHLGELVWCTKVAE SEQ ID NO: 108 4 nM

Example 2

Construction of Anti-VEGF Fabs Tagged with IgG-Fc Peptide Ligands

[0160] IgG-Fc peptide ligands may be combined with a bioactive compound to form a hybrid molecule that comprises a peptide ligand domain and an active domain. In this Example, IgG-Fc peptide ligands are combined with a Fab fragment that recognizes human VEGF. A neutralizing antibody to human VEGF has been previously identified from murine hybridoma, humanized, and optimized by phage display. See Muller et al. (1998), Structure 6:1153-1167; Chen et al. (1999), J. Mol. Biol. 293:865-881; and International Patent Publication No. WO 98/45331. Two humanized Fab forms of this antibody were chosen to test whether binding affinity to an irrelevant IgG could be added to the Fabs without disrupting their antigen-binding affinity. An IgG-Fc peptide ligand, DCAWHLGELVWCT (SEQ ID NO: 8), identified and optimized by the peptide-phage display method described in Example I was used, along with a short peptide linker (Gly-Gly-Gly) to provide flexibility between the peptide and the Fab. The light chain of the Fab was chosen for fusions because in the case of this antibody, the light chain is known to have little contribution to antigen binding (Muller et al., 1998, supra). In principle the peptide ligand domain could function to introduce IgG-binding whether introduced at the N-terminus, C-terminus, or inserted within the original Fab sequence. Described here are N-terminal fusions DCAWHLGELVWCTGGG-(light chain) (SEQ ID NO: 109) as well as C-terminal fusions (light chain)-GGGWEADCAWHLGELVWCT (SEQ ID NO: 110).

[0161] An oligodeoxynucleotide, HL-569, was designed and synthesized for mutation of anti-VEGF plasmids to create fusions of the IgG-Fc peptide ligand at the N-terminus of the antibody light chain. The sequence of HL-569 (with added peptide sequence underlined) is: 5'-ACA AAC GCG TAC GCT GAC TGC GCT TGG CAC CTG GGC GAG CTG GTC TGG TGC ACC GGA GGA GGA GAT ATC CAG TTG ACC-3' (SEQ ID NO: 111). The GAC codon follows the STII secretion-signal sequence at the N-terminus of the light chain, and the GAT codon corresponds to the first residue of the mature (wild-type) light chain.

[0162] Another oligodeoxynucleotide, HL-570, was designed and synthesized for construction of peptide ligand fusions to the C-terminus of the antibody light chain. The sequence of HL-570 (with added peptide sequence underlined) is: 5'-AAC AGG GGA GAG TGT GGA GGA GGA TGG GAA GCA GAC TGC GCT TGG CAC CTG GGC GAG CTG GTC TGG TGC ACC TAA GCT GAT CCT CTA C-3' (SEQ ID NO: 112). The TGT codon preceding the underscored GGA codon corresponds to residue Cys-214 of the light chain, and the TAA "stop codon" marks the end of the translated peptide sequence. Phagemids pY0192 and pY0317 (described Muller et al., 1998, supra; Chen et al., 1999; and International Patent Publication No. WO 98/45331, encoding low-affinity and high-affinity forms of a humanized anti-VEGF antibody, respectively, were mutated with each of the two IgG-peptide oligos to yield constructs pY0192-569, pY0192-570, pY0317-569, and pY0317-570.

Example 3

Phage-ELISA Analysis of Hybrid Molecules Comprising Peptide-Ligand Tagged Anti-VEGF Fabs

[0163] A phage-ELISA competitive binding assay (Lowman (1998), Methods Mol. Biol. 87:249-264) was used to compare the apparent binding affinities of anti-VEGF antibody variants tagged with an IgG-Fc peptide ligand at their N-terminus or C-terminus and displayed monovalently on bacteriophage M13 particles as fusions to the C-terminal domain of the gene III protein.

[0164] An irrelevant humanized IgG, 4D5-IgG, also known as Herceptin.RTM., was coated onto Nunc Maxisorp immunosorbant plates at 2 microg/mL in phosphate buffered saline solution (PBS). Phagemid particles from overnight cultures of XL-1 Blue E. coli (Stratagene) were diluted in PBS containing 0.5% bovine serum albumin and 0.05% Tween-20. The phagemid particles were mixed with serial dilutions of Herceptin.RTM. in solution, equilibrated for 20 min in a non-adsorbent plate (Nunc F96), then transferred to the Herceptin.RTM.-coated Maxisorp plate for detection of unbound phage. After 20 min, the plate was washed with PBS/Tween, and developed with an anti-phage monoclonal antibody-HRP conjugate (Pharmacia) and OPD substrate (Sigma). Displacement curves (FIG. 1) showed IC.sub.50 values of about 100-300 nM for each of the construct, pY0192-569, pY0192-570, pY0317-569, and pY0317-570.

Example 4

BIAcore.TM. Analysis of IgG Binding to Anti-VEGF Fab Tagged with an IgG-Fc Peptide Ligand

[0165] A surface plasmon resonance instrument (BIAcore, Inc., Piscataway, N.J.) was used to measure binding of an irrelevant IgG, 4D5-IgG, also known as Herceptin.RTM., to Fab that previously had been bound to an immobilized VEGF biosensor chip.

[0166] Fab variants encoded by pY0317 and pY0317-570 (control anti-VEGF high affinity, humanized Fab, and anti-VEGF high affinity, humanized Fab tagged with an IgG-Fc peptide ligand domain, respectively; see Example 2, supra, and WO 98/45331) were expressed in E. coli and purified by protein-G (Pharmacia) affinity chromatography. Recombinant human VEGF was immobilized onto BIAcore.TM. CM-5 biosensor chips (BIAcore, Inc.) as described (Muller et al., 1998, supra). After VEGF immobilization, the chip was blocked with ethanolamine, and the peptide-ligand tagged Y0317-570 Fab, or Y0317 control, was injected in PBS buffer containing 0.05% Tween-20 and 0.01% sodium azide. Following Fab injection, Herceptin.RTM. was injected, and the dissociation off-rate (k.sub.off) following injection was observed.

[0167] The results (FIG. 2) show that Herceptin.RTM. bound to the tagged but not to the control Y0317 Fab. Using a 1:1 Langmuir binding model (Karlsson et al. (1991), J. Immunol. Methods 145:229-240 (1991)), a k.sub.off of 2.8.times.10.sup.-3, sec.sup.-1, and a corresponding dissociation half-life (t.sub.1/2) of 8.5 min were calculated for Y0317-570. Limitations of material prevented reliable on-rate determinations. However, the observed k.sub.off suggests an equilibrium binding affinity, K.sub.d, of 30 nM to 300 nM (assuming k.sub.on of 10.sup.4-10.sup.5 M.sup.-1 sec.sup.-1), consistent with peptide binding and phage-ELISA results (above). Importantly, the BIAcore.TM. results (FIG. 2) also show that the tagged Fab can simultaneously binding both antigen (immobilized VEGF) and an irrelevant IgG.

Example 5

IgG-Fc Peptide Ligand Tagged Anti-VEGF Fabs Have Prolonged Elimination Half Times

[0168] The blood clearance rates and tissue distribution of the IgG-Fc peptide ligand-tagged anti-VEGF Fab (Fab-Y0317-570) are compared to those of the untagged control anti-VEGF Fab Y0317. Determinations of the elimination half time and volume of distribution are made in New Zealand White Rabbits of 2.8 to 3 kg weight. The amount of test article present in the plasma samples is determined using any method known in the art, such as, e.g., ELISA, or RIA.

[0169] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t) Ae.sup.-.alpha.t+Be.sup.-.beta.t, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and a and 13 are the apparent first-order rate constants for the distribution and elimination phases, respectively. The .alpha.-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or .beta.-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(.alpha.-k21)/(.beta.-8), B=D/V (.beta.-k21)/(.alpha.-.beta.), and .alpha. and .beta. (for .alpha.>.beta.) are roots of the quadratic equation: r.sup.2+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[0170] On the morning of the study six New Zealand White rabbits (body weight 2.8-3.0 kg) were placed in restrainers. Catheters were installed in an ear artery for blood sample collection and in a contralateral ear vein for dosing.

[0171] Rabbits were divided into two groups (n=3/group). Group 1 animals received and IV bolus of control anti-VEGF Fab-Y0317. Rabbits in Group 2 received Fab-Y0317-570. A summary of group assignment and dosing information is presented in the table below.

TABLE-US-00007 Nominal Dose Dose Weight Dose Conc. Vol. Group (kg) Dose Group (mg/kg) (mg/mL) (mL) 1 2.9 Control-Fab-Y0317 1 3 0.97 1 3.0 Control-Fab-Y0317 1 3 1.00 1 2.9 Control-Fab-Y0317 1 3 0.97 2 2.8 Fab-Y0317-5701 3 0.93 2 3.0 Fab-Y0317-5701 3 1.00 2 2.9 Fab-Y0317-5701 3 0.97

[0172] Serial blood samples (0.5 mL) were collected just prior to dosing and at 10, 20 40 min, 1, 2, 3, 4, 6, 8, 24 and 48 hr after dose administration. Blood was collected in serum separator tubes, allowed to clot (.about.30 min) at room temperature, and centrifuged. Serum was harvested and immediately stored at -70C until analyzed.

[0173] ELISA plates were coated with 0.5 microg/ml VEGF in 50 mM carbonate buffer, pH 9.6, at 4.degree. C. overnight and blocked with 0.5% bovine serum albumin, 10 ppm Proclin 300 (Supelco, Bellefonte, Pa.) in PBS (8 mM Na.sub.2HPO.sub.4, 1.5 mM KH.sub.2PO.sub.4, 2.7 mM KCl and 137 mM NaCl, pH 7.2) at room temperature for 1 hour. Standards (0.41-100 ng/ml) and twofold serial dilutions, of samples (minimum dilution 1:100) in PBS containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 0.25% CHAPS, 0.2% bovine gamma globulins (Sigma, St. Louis, Mo.) and 5 mM EDTA were incubated on the plates for 2 hours. Bound antibody was detected using peroxidase labeled goat F(ab')2 anti-human IgG F(ab')2 (Jackson ImmunoResearch, West Grove, Pa.), followed by 3,3',5,5'-tetramethyl benzidine (Kirkegaard & Perry Laboratories) as the substrate. Plates were washed between steps. Absorbance was read at 450 nm on a Titerek stacker reader (ICN, Costa Mesa, Calif.). The standard curve was fitted using a four-parameter regression curve-fitting program (Kaleidagraph, Synergy Software, Reading, Pa.). Data points which fell in the range of the standard curve were used for calculating the Fab concentrations in samples.

[0174] Data analysis: Graphs of concentration versus time profiles were made using KaleidaGraph (KaleidaGraph.TM. V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) were not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters were determined by compartmental analysis using WinNonlin software (WinNonlin.RTM. Professional V. 3.1 WinNonlin.TM. Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters were computed as described elsewhere (Ritschel W A and Kearns G L. Handbook of basic pharmacokinetics including clinical applications, 5th edition. American Pharmaceutical Assoc., Washington, D.C. Copyright 1999).

[0175] The results are reported in FIG. 3. A two-compartment model with bolus input and first-order output (WinNonlin) was used to fit observed serum concentration vs. time data. Calculated pharmacokinetic parameters ware presented in the table below.

TABLE-US-00008 Pharmacokinetic Parameter Summary (IV bolus; 1 mg/kg) Group 1 Group 2 Parameter Control Fab-Y0317 Fab-Y0317-570 AUC (h * .mu.g/mL) 13.6 .+-. 1.2 215 .+-. 56 Cmax (.mu.g/mL) 15.6 .+-. 0.6 13 .+-. 0.7 CL (mL/h/kg) 74.2 .+-. 6.7 4.8 .+-. 1.1 K10 half-life (hr) 0.6 .+-. 0.02 11.3 .+-. 3.6 alpha half-life (hr) 0.39 .+-. 0.03 1.15 .+-. 0.31 beta half-life (hr) 1.93 .+-. 0.27 37.6 .+-. 19 V1 (mL/kg) 64.1 .+-. 2.37 75.2 .+-. 4.23 Vss (mL/kg) 112 .+-. 7.7 225 .+-. 54

[0176] The initial volume of distribution (V1) for both agents was approximately equal to serum volume. The estimated steady state volume of distribution (Vss) for Fab-Y0317-570 (225 mL/kg) was approximately 2 fold higher than estimated for the control Fab (112 mL/kg) suggesting a significant amount of binding to endogenous IgG. Control Fab-Y0317 was eliminated approximately 15-fold faster from the serum (clearance=74 mL/h/kg) compared to Fab-Y0317-570 (4.8 mL/b/d). The overall exposure (AUC) of Fab-Y0317-570 was .about.16 times higher than for Fab-Y0317. Fab-Y0317 was undetectable in the serum 24 h after dosing but serum concentrations of Fab-Y0317-570 were still above 1 .mu.g/mL 48 h after dosing. Both the distribution (alpha) half-life (1.15 h) and the elimination (beta) half-life (37.6 h) were significantly longer than the control Fab.

[0177] These results suggest that addition of a 13 amino acid that binds to endogenous IgG to Fab-Y0317 can significantly slow Fab clearance, increase half-life and enhance overall exposure.

Example 6

Serum Albumin Peptide Ligands

[0178] Phage Libraries and Selection Conditions--Phage-displayed peptide libraries were selected against rabbit, rat and human albumin. Phage libraries expressing random peptide sequences fused to gene 8 (Lowman et al., Biochem. 37, 8870 (1998)) were pooled into 5 groups: Pool A contained CX.sub.2GPX.sub.4C (SEQ ID NO: 133), X.sub.4CX.sub.2GPX.sub.4CX.sub.4 (SEQ ID NO: 134) and X.sub.iCX.sub.jCX.sub.k where j=8-10; Pool B contained X.sub.20 and X.sub.iCX.sub.JCX.sub.k where j=4-7; Pool C contained X.sub.8 and X.sub.2CX.sub.JCX.sub.2 where j=4-6; Pool D contained X.sub.2CX.sub.jCX.sub.2 where j=7-10; Pool E contained CX.sub.6CX.sub.6CCX.sub.3CX.sub.6C (SEQ ID NO: 135), CCX.sub.3CX.sub.6C (SEQ ID NO: 136), CCX.sub.5CX.sub.4CX.sub.4CC (SEQ ID NO: 137), CXCX.sub.7CX.sub.3CX.sub.6 (SEQ ID NO: 138) where X represents any of the 20 naturally occurring L-amino acids. In each case i+j+k=18 and |i-k|<2. Each of the 10 libraries has in excess of 108 clones.

[0179] The phage library pools were suspended in binding buffer (PBS, 1% ovalbumin, 0.005% Tween 20) and sorted against rabbit, rat or human albumin immobilized directly on maxisorp plates (10 .mu.g/ml in PBS, overnight at 4.degree. C.; plates were blocked with Blocker Casein (Pierce Chemical, Rockford, Ill.)). After 2 hours, unbound phage were removed by repetitive washing (PBS, 0.05% Tween 20) and bound phage were eluted with 500 mM KCl, 10 mM HCl, pH 2. Eluted phage were propagated in XL1-Blue cells with VCSM13 helper phage (Stratagene, La Jolla, Calif.). Enrichment was monitored by titering the number of phage that bound to an albumin coated well compared to a well coated with ovalbumin or casein.

[0180] Phage ELISA--Phage clones (.about.10.sup.11 phage) were added to plates coated with rat, rabbit or human albumin. The microtiter plate was washed with wash buffer and bound phage were detected with HRP/Anti-M13 Conjugate. The amount of HRP bound was measured using ABTS/H.sub.2O.sub.2 substrate and monitoring the change at 405 nm.

[0181] The peptide sequences displayed by phage clones selected for binding to rabbit, human or rat albumin are shown in FIG. 4. Also indicated is the ability of individual phage clones to bind the 3 species of immobilized albumin. This was tested using a phage ELISA. Note that clone RB, selected for binding to rat albumin is also capable of binding human and rabbit albumin.

[0182] Sequence Maturation on Monovalent Phage--Partially randomized libraries were designed using oligonucleotides coding for each of the selected clones in FIG. 4, but synthesized with a 70-10-10-10 mixture of bases as described (Dennis et al., Nature 404, 465 (2000)). Although the potential diversity of these libraries is the same as the initial naive libraries, each `soft randomized` library maintains a bias towards the selected sequence in FIG. 4. Each library was again selected for binding to rat, rabbit or human albumin regardless of its origin. For example, the library resulting from soft randomization of clone RB was selected against rat, rabbit or human albumin even though it was originally identified for binding to rat albumin. Sequences identified following soft randomization are shown in FIG. 5 along with their species specificity as determined by phage ELISA. Most clones appear to be specific for the species of albumin for which they were selected, however, clones from the RB soft randomization library bind to all three species.

[0183] Phage clones were also tested for binding to rhesus, mouse and bovine albumin. Clones originating from the RB soft randomization library were found to bind each of these species of albumin as well and were specific for albumin based upon their lack of binding to ovalbumin and casein (FIG. 6). Clones that bind to multiple species of albumin (multi-species binders are listed in FIG. 7.

[0184] Hard randomization--Sequences from soft randomization of the RB sequence were further matured using a hard randomization strategy. A new library was designed that kept highly selected residues (underlined) constant X.sub.5DXCLPXWGCLWX.sub.4 (SEQ ID NO: 116), while fully randomizing the remaining positions. A second library, one residue shorter at both the N and C terminus was also constructed. Sequences from these libraries selected against rat, rabbit and human albumin are shown in FIGS. 8A, 88, and 8C respectively.

[0185] Peptide Synthesis--Peptides were synthesized by either manual or automated (Milligen 9050) Fmoc-based solid (phase synthesis on a 0.25 mmol scale using a PEG-polystyrene resin (Bodanszky M., (1984) Principles of Peptide Synthesis, Springer, Berlin). Side chain protecting groups were removed and the peptides were cleaved from the resin with 95% trifluoroacetic acid (TFA) and 5% triisopropylsilane. A saturated iodine solution in acetic acid was added for oxidation of disulfide bonds. Peptides were purified by reversed phase HPLC using a water/acetonitrile gradient containing 0.1% TFA. Peptides were >95% pure by analytical HPLC and its identity verified by mass spectrometry.

[0186] The carboxy terminal lysine of peptide SA08 was derivatized with NHS-LC-biotin (Pierce Chemical, Rockford, Ill.) and purified by HPLC as above yielding SA08b (Ac-QGLIGDICLPRWGCLWGDSVK.sub.b (SEQ ID NO: 124)-n where K.sub.b refers to lysine-biotin).

[0187] SA08b Binding Assay--Rabbit, rat or mouse albumin was immobilized directly on maxisorp plates at 10 .mu.g/ml in PBS, overnight at 4.degree. C. Plates were blocked using Blocker Casein (Pierce Chemical, Rockford, Ill.) for 1 hr, at 25.degree. C. Serially diluted samples were suspended in binding buffer (above) and added to the plate followed by the addition of 10 nM SA08b for 1 hr, at 25.degree. C. The microtiter plate was washed with PBS, 0.05% Tween 20 and the SA08b bound to albumin was detected with Streptavidin/HRP. The amount of HRP bound was measured using ABTS/H.sub.2O.sub.2 substrate and monitoring the change at 405 nm.

[0188] Peptides corresponding to identified phage sequences were synthesized and their affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay (FIGS. 9 and 10).

[0189] Construction, Expression and Purification of Albumin Binding Fab Fusions--In order to test whether association with albumin could increase the half-life of proteins and peptides in vivo, the sequence of SA06 was fused to a Fab fragment (D3H44) directed for binding tissue factor (TF). The SA06 sequence was added to the carboxy terminus of either the light chain (D3H44-L) or heavy chain (D3H44-Ls) of the Fab. In addition, as a precaution against folding problems, identical constructions were made but with the intra-chain disulfide replaced by alanines (D3H44 Ls and D3H44-Hs, respectively) as depicted in FIG. 11.

[0190] The fusions were expressed under control of the alkaline phosphatase promoter and secreted from E. coli using the stII secretion signal. Fab fusions were recovered from the periplasm by suspending cells in 1 mM EDTA, 10 mM Tris-HCl, pH8, for 1 hr at 4.degree. C. Cell debris was removed by centrifugation and the anti-TF Fab was selectively purified using a Hi-Trap (Amersham Pharmacia Biotech, Piscataway, N.J.) TF affinity column. Properly folded D3H44 L or D3H44-Ls was further purified using a rabbit albumin affinity column (rabbit albumin coupled to CNBr-activated Sepharose 4B, Amersham Pharmacia Biotech, Piscataway, N.J.). Both columns were washed with PBS and eluted with 50 mM HCl. Eluted fractions were neutralized with 1 M Tris pH 8. Endotoxin was further removed following extraction with triton X114 (Aida and Pabst, J. Immunol. Methods 132, 191 (1990)).

[0191] Purified D3H44 fusions retained their ability to bind TF as measured using a FX activation assay (FIG. 12), and a prothrombin time assay that measures prolongation of tissue factor dependent clotting (FIG. 13) (for methods see Dennis et al., Nature 404, 465 (2000)). Unlike D3H44 lacking the albumin binding sequence (WT), both D3H44 L and D3H44-Ls are able to bind to albumin as measured in the SA08b binding assay (FIG. 14). Further, both D3H44 albumin-binding fusions are capable of binding TF and albumin simultaneously as judged by a biotin-TF binding assay (FIG. 15). In this assay, the binding of the D3H44 fusions to immobilized albumin is detected with biotinylated IV. Wild-type D3H44 (WT) is unable to bind albumin and thus does not generate a signal upon addition of biotinylated TF.

[0192] Pharmacokinetics of D3H44 albumin-binding fusions D3H44 variants were given as a 0.5 mg/kg bolus in rabbit. Each group consisted of 3 rabbits (5 in the F(ab')2 group). Serum samples taken at the indicated time points were serially diluted and the concentration of D3H44 determined using a TF binding ELISA.

[0193] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae.sup.-.alpha.t+Be.sup.-.beta.t, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and .alpha. and .beta. are the apparent first-order rate constants for the distribution and elimination phases, respectively. The .alpha.-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or .beta.-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(.alpha.-k21)/(.alpha.-.beta.), B=D/V (.beta.-k21)/(.alpha.-.beta.), and .alpha. and .beta. (for .alpha.>.beta.) are roots of the quadratic equation: r.sup.2+(k12+k21+k 10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[0194] Data analysis: Graphs of concentration versus time profiles were made using KaleidaGraph (KaleidaGraph.TM. V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) were not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters were determined by compartmental analysis using WinNonlin software (WinNonlin.RTM. Professional V. 3.1 WinNonlin.TM. Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters were computed as described elsewhere (Ritschel W A and Kearns G L. Handbook of basic pharmacokinetics including clinical applications, 5th edition. American Pharmaceutical Assoc., Washington, D.C. Copyright 1999).

[0195] Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters (FIGS. 16 and 17). D3H44-L has a 70-fold increase in half-life (K10-HL) relative to wild-type Fab and a comparable half-life to D3H44 Fabs derivatized with 20K or 40K polyethylene glycol (PEG).

[0196] All publications cited herein are expressly incorporated by reference in their entirety.

Sequence CWU 1

1

49217PRTArtificial SequenceSynthesized 1Gly Gly Gly Ser Gly Gly Gly1 5220PRTArtificial SequenceSynthesized 2Glu Thr Gln Arg Cys Thr Trp His Met Gly Glu Leu Val Trp Cys Glu1 5 10 15Arg Glu His Asn20320PRTArtificial SequenceSynthesized 3Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu2047PRTArtificial SequenceSynthesized 4Gly Gly Gly Pro Gly Gly Gly1 5560DNAArtificial SequenceSynthesized 5drggwagmar rctgckcttr scacmtgggc gagctggtct ggtgcrvcrv mbkcgaskdw 60660DNAArtificial SequenceSynthesized 6drsvwgsvgr rctgckcctr syrsmtgggc gagctggtct ggtgcrncvv snbsgwskdm 60760DNAArtificial SequenceSynthesized 7dnsnnsnnsv nstgcbvgtd shrsmdsggc gagstckkgw rgtgcrnmnn snnsnnsnnm 60813PRTArtificial SequenceSynthesized 8Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 1099PRTArtificial SequenceSynthesized 9Xaa Xaa Xaa Xaa Xaa Xaa Leu Val Trp1 5109PRTArtificial SequenceSynthesized 10Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp1 51120PRTArtificial SequenceSynthesized 11Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Leu Val Trp Cys Xaa1 5 10 15Xaa Xaa Xaa Xaa201220PRTArtificial SequenceSynthesized 12Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp Cys Xaa1 5 10 15Xaa Xaa Xaa Xaa201320PRTArtificial SequenceSynthesized 13Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp Cys Xaa1 5 10 15Xaa Xaa Xaa Xaa201420PRTArtificial SequenceSynthesized 14Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp Cys Xaa1 5 10 15Xaa Xaa Xaa Xaa201520PRTArtificial SequenceSynthesized 15Asp Leu Ala Asp Cys Ser Trp His Met Gly Glu Leu Val Trp Cys Ser1 5 10 15Arg Val Glu Gly201620PRTArtificial SequenceSynthesized 16Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Met Glu Phe201720PRTArtificial SequenceSynthesized 17Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Thr Leu Thr Glu201820PRTArtificial SequenceSynthesized 18Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Pro Gly Val Glu201920PRTArtificial SequenceSynthesized 19Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Gly Val Glu Gly202020PRTArtificial SequenceSynthesized 20Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Ala Gly Val Glu202120PRTArtificial SequenceSynthesized 21Glu Ser Glu Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu202220PRTArtificial SequenceSynthesized 22Glu Lys Glu Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu202320PRTArtificial SequenceSynthesized 23Glu Asp Pro Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu202420PRTArtificial SequenceSynthesized 24Glu Glu Ala Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu202520PRTArtificial SequenceSynthesized 25Asn Ala Asp Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu202620PRTArtificial SequenceSynthesized 26Ser Glu Thr Thr Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu202719PRTArtificial SequenceSynthesized 27Ala Trp Lys Thr Cys Gln Trp Leu Gly Glu Leu Val Trp Cys Val Ala1 5 10 15Gly Val Glu2820PRTArtificial SequenceSynthesized 28Asp Leu Ala Asp Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Arg Val Glu Gly202920PRTArtificial SequenceSynthesized 29Lys Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu203020PRTArtificial SequenceSynthesized 30Lys Glu Ala Glu Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu203120PRTArtificial SequenceSynthesized 31Lys Glu Ala Arg Cys Trp Tyr Trp His Gly Glu Leu Val Trp Cys Ser1 5 10 15Asp Pro Glu Glu203220PRTArtificial SequenceSynthesized 32Lys Glu Ala Ser Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu203320PRTArtificial SequenceSynthesized 33Lys Glu Ala Ser Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Ala Gly Val Glu203420PRTArtificial SequenceSynthesized 34Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Glu Gly Val Glu203520PRTArtificial SequenceSynthesized 35Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Asp Gly Val Glu203620PRTArtificial SequenceSynthesized 36Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Glu Gly Val Glu203720PRTArtificial SequenceSynthesized 37Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Val Trp Lys Cys Lys1 5 10 15Ser Gly Val Glu203820PRTArtificial SequenceSynthesized 38Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Asn Gly Val Glu203920PRTArtificial SequenceSynthesized 39Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Thr Phe Asp Glu204020PRTArtificial SequenceSynthesized 40Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Gly Leu Asp Glu204120PRTArtificial SequenceSynthesized 41Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Gly Leu Asp Glu204220PRTArtificial SequenceSynthesized 42Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu1 5 10 15Asp Thr Leu Glu204320PRTArtificial SequenceSynthesized 43Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu1 5 10 15Asp Thr Met Glu204420PRTArtificial SequenceSynthesized 44Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu1 5 10 15Asp Met Met Glu204520PRTArtificial SequenceSynthesized 45Trp Val Glu Asp Cys Ser Trp His Met Gly Glu Leu Val Trp Cys Asp1 5 10 15Gly Gly Glu Phe204620PRTArtificial SequenceSynthesized 46Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Trp Met Asn Gly204720PRTArtificial SequenceSynthesized 47Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Asp Thr Pro Val204820PRTArtificial SequenceSynthesized 48Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Asp Tyr Gly Glu204920PRTArtificial SequenceSynthesized 49Lys Glu Ala Ser Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Asp Leu Trp Glu205020PRTArtificial SequenceSynthesized 50Trp Arg Gly Gly Cys Ser Trp His Met Gly Glu Leu Val Trp Cys Glu1 5 10 15His Asp Met Glu205120PRTArtificial SequenceSynthesized 51Ala Val Ser Lys Cys Ser Phe His Met Gly Glu Leu Val Trp Cys Ser1 5 10 15Asp Val Met Asn205220PRTArtificial SequenceSynthesized 52Asn Gln Val Ser Cys Ser Tyr Ser Arg Gly Glu Leu Val Trp Cys Ser1 5 10 15Lys Gln Ser Gln205320PRTArtificial SequenceSynthesized 53Gly Arg Met Glu Cys Ala Trp His Gln Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Thr Leu Glu205420PRTArtificial SequenceSynthesized 54Gly Thr Met Glu Cys Ser Trp His Gln Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Thr Leu Ala205520PRTArtificial SequenceSynthesized 55Glu Met Arg Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Ala1 5 10 15His Met Glu Gly205620PRTArtificial SequenceSynthesized 56Gly Ser Trp Glu Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Glu1 5 10 15Thr Gly Ser Gly205720PRTArtificial SequenceSynthesized 57Val Ala Glu Pro Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Glu1 5 10 15Val Leu Lys Gly205820PRTArtificial SequenceSynthesized 58Lys Glu Ala Met Cys Ser Tyr Trp Leu Gly Glu Leu Val Trp Cys Glu1 5 10 15Ser Asp Met Pro205920PRTArtificial SequenceSynthesized 59Asp Leu Ala Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Arg Val Glu Gly206020PRTArtificial SequenceSynthesized 60Asp Leu Ala Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Gly Leu Asp Glu206120PRTArtificial SequenceSynthesized 61Trp Val Glu Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Val1 5 10 15Gly Leu Asp Phe206220PRTArtificial SequenceSynthesized 62Lys Val Ala Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Thr1 5 10 15Glu Val Glu Gly206320PRTArtificial SequenceSynthesized 63Gly Glu Glu Asp Cys Ser Tyr His Leu Gly Glu Leu Val Met Cys Thr1 5 10 15Glu Leu Asp Asp206420PRTArtificial SequenceSynthesized 64Gly Val Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Glu Arg Glu Asp206520PRTArtificial SequenceSynthesized 65Gly Glu Glu Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Gly Gly Asp Phe206620PRTArtificial SequenceSynthesized 66Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu206720PRTArtificial SequenceSynthesized 67Gly Glu Ala Asp Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Asn1 5 10 15Asp Phe Glu Glu206819PRTArtificial SequenceSynthesized 68Trp Val Asp Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Ser Thr1 5 10 15Phe Glu Glu6920PRTArtificial SequenceSynthesized 69Trp Val Glu Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Asp Glu207020PRTArtificial SequenceSynthesized 70Arg Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Glu Arg Asp Leu207120PRTArtificial SequenceSynthesized 71Glu Glu Ala Ser Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Ala Phe Asp Val207220PRTArtificial SequenceSythesized 72Arg Val Ala Ser Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Gly Leu Asp Gly207320PRTArtificial SequenceSynthesized 73Gly Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu207420PRTArtificial SequenceSynthesized 74Gly Glu Ala Ser Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Asp1 5 10 15Glu Gly Glu Gly207520PRTArtificial SequenceSynthesized 75Arg Val Glu Asp Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Glu Gly Asp Glu207620PRTArtificial SequenceSynthesized 76Glu Glu Pro Asp Cys Ser Trp His Leu Gly Glu Leu Val Met Cys Thr1 5 10 15Pro Met Glu Val207720PRTArtificial SequenceSynthesized 77Lys Glu Ala Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Ser1 5 10 15Glu Met Glu Gly207820PRTArtificial SequenceSynthesized 78Glu Gln Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Met Val Phe207920PRTArtificial SequenceSynthesized 79Glu Glu Pro Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Ile Glu Val208020PRTArtificial SequenceSynthesized 80Gly Glu Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Met Val Phe208120PRTArtificial SequenceSynthesized 81Gly Glu Gln Asp Cys Ser Tyr His Met Gly Glu Leu Val Trp Cys Thr1 5 10 15Thr Val Asp Gly208220PRTArtificial SequenceSynthesized 82Gly Val Arg Asn Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Met Glu Phe208320PRTArtificial SequenceSynthesized 83Arg Val Ala Asp Cys Ala Trp His Met Gly Glu Leu Val Trp Cys Ser1 5 10 15Glu Leu Glu Val208420PRTArtificial SequenceSynthesized 84Gly Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Met Asp Leu208520PRTArtificial SequenceSynthesized 85Gly Glu Gln Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Pro Met Glu Val208620PRTArtificial SequenceSynthesized 86Gly Met Arg Asp Cys Ser Tyr His Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Asp Met Glu Leu208720PRTArtificial SequenceSynthesized 87Glu Val Ala Asp Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Glu Gly Glu Phe208820PRTArtificial SequenceSynthesized 88Gly Glu Glu Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Asp Val Glu Asp208920PRTArtificial SequenceSynthesized 89Glu Val Glu Asp Cys Ala Tyr His Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Asp Leu Glu Gly209020PRTArtificial SequenceSynthesized 90Trp Glu Glu Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ala1 5 10 15Glu Phe Asp Glu209120PRTArtificial SequenceSynthesized 91Lys Glu Ala Ser Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ser1 5 10 15Glu Val Glu Glu209220PRTArtificial SequenceSynthesized 92Ala Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209320PRTArtificial SequenceSynthesized 93Trp Ala Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209420PRTArtificial SequenceSynthesized 94Trp Glu Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209520PRTArtificial SequenceSynthesized 95Trp Glu Ala Ala Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209620PRTArtificial SequenceSynthesized 96Trp Glu Ala Ala Cys Ser Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209720PRTArtificial SequenceSynthesized 97Trp Glu Ala Asp Cys Ala Ala His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209820PRTArtificial SequenceSynthesized 98Trp Glu Ala Asp Cys Ala Trp Ala Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu209920PRTArtificial SequenceSynthesized 99Trp Glu Ala Asp Cys Ala Trp His Ala Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu2010020PRTArtificial SequenceSynthesized 100Trp Glu Ala Asp Cys Ala Trp His Leu Ala Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu2010120PRTArtificial SequenceSynthesized 101Trp Glu Ala Asp Cys Ala Trp His Leu Gly Ala Leu Val Trp Cys Thr1 5 10 15Lys Val Glu Glu2010220PRTArtificial SequenceSynthesized 102Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Ala Val Trp Cys Thr1 5 10 15Lys Val Glu Glu2010320PRTArtificial SequenceSynthesized 103Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Ala Trp Cys Thr1 5 10 15Lys Val Glu Glu2010420PRTArtificial SequenceSynthesized 104Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Ala Cys Thr1 5

10 15Lys Val Glu Glu2010520PRTArtificial SequenceSynthesized 105Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Ala1 5 10 15Lys Val Glu Glu2010620PRTArtificial SequenceSynthesized 106Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Ala Val Glu Glu2010720PRTArtificial SequenceSynthesized 107Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Ala Glu Glu2010820PRTArtificial SequenceSynthesized 108Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr1 5 10 15Lys Val Ala Glu2010916PRTArtificial SequenceSynthesized 109Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Gly Gly Gly1 5 10 1511019PRTArtificial SequenceSynthesized 110Gly Gly Gly Trp Glu Ala Asp Cys Ala Trp His Leu Gly Glu Leu Val1 5 10 15Trp Cys Thr11178DNAArtificial SequenceSynthesized 111acaaacgcgt acgctgactg cgcttggcac ctgggcgagc tggtctggtg caccggagga 60ggagatatcc agttgacc 7811288DNAArtificial SequenceSynthesized 112aacaggggag agtgtggagg aggatgggaa gcagactgcg cttggcacct gggcgagctg 60gtctggtgca cctaagctga tcctctac 8811311PRTArtificial SequenceSynthesized 113Phe Cys Xaa Asp Trp Pro Xaa Xaa Xaa Ser Cys1 5 101149PRTArtificial SequenceSynthesized 114Val Cys Tyr Xaa Xaa Xaa Ile Cys Phe1 51157PRTArtificial SequenceSynthesized 115Cys Tyr Xaa Pro Gly Xaa Cys1 511611PRTArtificial SequenceSynthesized 116Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp1 5 1011712PRTArtificial SequenceSynthesized 117Trp Cys Asp Xaa Xaa Leu Xaa Ala Xaa Asp Leu Cys1 5 1011810PRTArtificial SequenceSynthesized 118Asp Leu Val Xaa Leu Gly Leu Glu Cys Trp1 5 1011911PRTArtificial SequenceSynthesized 119Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp1 5 1012011PRTArtificial SequenceSynthesized 120Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 1012115PRTArtificial SequenceSynthesized 121Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp1 5 10 1512220PRTArtificial SequenceSynthesized 122Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Asp Asp Phe2012320PRTArtificial SequenceSynthesized 123Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Ser Val2012421PRTArtificial SequenceSynthesized 124Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Ser Val Lys2012515PRTArtificial SequenceSynthesized 125Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp1 5 10 1512618PRTArtificial SequenceSynthesized 126Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Asp Asp12716PRTArtificial SequenceSynthesized 127Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp1 5 10 1512818PRTArtificial SequenceSynthesized 128Arg Leu Met Glu Asp Ile Cys Leu Ala Arg Trp Gly Cys Leu Trp Glu1 5 10 15Asp Asp12920PRTArtificial SequenceSynthesized 129Glu Val Arg Ser Phe Cys Thr Asp Trp Pro Ala Glu Lys Ser Cys Lys1 5 10 15Pro Leu Arg Gly2013020PRTArtificial SequenceSynthesized 130Arg Ala Pro Glu Ser Phe Val Cys Tyr Trp Glu Thr Ile Cys Phe Glu1 5 10 15Arg Ser Glu Gln2013111PRTArtificial SequenceSynthesized 131Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 101325PRTArtificial SequenceSynthesized 132Gly Glu Leu Val Trp1 513310PRTArtificial SequenceSynthesized 133Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys1 5 1013418PRTArtificial SequenceSynthesized 134Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys Xaa Xaa1 5 10 15Xaa Xaa13527PRTArtificial SequenceSynthesized 135Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys1 5 10 15Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys20 2513613PRTArtificial SequenceSynthesized 136Cys Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys1 5 1013719PRTArtificial SequenceSynthesized 137Cys Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Cys Cys13821PRTArtificial SequenceSynthesized 138Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa201399PRTArtificial SequenceSynthesized 139Xaa Xaa Xaa Xaa Gly Glu Leu Val Trp1 514011PRTArtificial SequenceSynthesized 140Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1014127PRTArtificial SequenceSynthesized 141Cys Glu Val Ala Leu Asp Ala Cys Arg Gly Gly Glu Ser Gly Cys Cys1 5 10 15Arg His Ile Cys Glu Leu Ile Arg Gln Leu Cys20 2514221PRTArtificial SequenceSynthesized 142Cys Gly Cys Val Asp Val Ser Asp Trp Asp Cys Trp Ser Glu Cys Leu1 5 10 15Trp Ser His Gly Ala2014310PRTArtificial SequenceSynthesized 143Asp Leu Cys Asp Val Asp Phe Cys Trp Phe1 5 1014415PRTArtificial SequenceSynthesized 144Asp Thr Cys Val Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Gly1 5 10 1514516PRTArtificial SequenceSynthesized 145Lys Ser Cys Ser Glu Leu His Trp Leu Leu Val Glu Glu Cys Leu Phe1 5 10 1514620PRTArtificial SequenceSynthesized 146Met Asp Glu Leu Ala Phe Tyr Cys Gly Ile Trp Glu Cys Leu Met His1 5 10 15Gln Glu Gln Lys2014720PRTArtificial SequenceSynthesized 147Arg Asn Glu Asp Pro Cys Val Val Leu Leu Glu Met Gly Leu Glu Cys1 5 10 15Trp Glu Gly Val2014820PRTArtificial SequenceSynthesized 148Glu Val Arg Ser Phe Cys Thr Asp Trp Pro Ala Glu Lys Ser Cys Lys1 5 10 15Pro Leu Arg Gly2014920PRTArtificial SequenceSynthesized 149Gly Glu Asn Trp Cys Asp Ser Thr Leu Met Ala Tyr Asp Leu Cys Gly1 5 10 15Gln Val Asn Met2015020PRTArtificial SequenceSynthesized 150Gln Arg Gln Met Val Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Phe2015111PRTArtificial SequenceSynthesized 151Ala Leu Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1015215PRTArtificial SequenceSynthesized 152Ala Ser Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Met Val Glu1 5 10 1515311PRTArtificial SequenceSynthesized 153Asp Ile Cys Tyr Ile Pro Gly Ile Cys Trp Met1 5 1015411PRTArtificial SequenceSynthesized 154Asp Leu Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1015511PRTArtificial SequenceSynthesized 155Asp Met Cys Tyr Phe Pro Gly Ile Cys Phe Asn1 5 1015611PRTArtificial SequenceSynthesized 156Asp Met Cys Tyr Phe Pro Gly Ile Cys Trp Leu1 5 1015715PRTArtificial SequenceSynthesized 157Asp Ser Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Ser Gly Thr1 5 10 1515811PRTArtificial SequenceSynthesized 158Asp Val Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1015915PRTArtificial SequenceSynthesized 159Glu His Asp Met Cys Tyr Phe Pro Gly Ile Cys Trp Ile Ala Asp1 5 10 1516011PRTArtificial SequenceSynthesized 160Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Ile1 5 1016111PRTArtificial SequenceSynthesized 161Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1016211PRTArtificial SequenceSynthesized 162Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1016311PRTArtificial SequenceSynthesized 163Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Pro1 5 1016411PRTArtificial SequenceSynthesized 164Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Thr1 5 1016511PRTArtificial SequenceSynthesized 165Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Ser1 5 1016611PRTArtificial SequenceSynthesized 166Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Thr1 5 1016711PRTArtificial SequenceSynthesized 167Glu Thr Cys Tyr Phe Pro Gly Ile Cys Trp Leu1 5 1016811PRTArtificial SequenceSynthesized 168Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Glu1 5 1016911PRTArtificial SequenceSynthesized 169Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Phe1 5 1017011PRTArtificial SequenceSynthesized 170Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1017120PRTArtificial SequenceSynthesized 171Glu Val Arg Ser Phe Cys Thr Asp Trp Pro Ala His Tyr Ser Cys Thr1 5 10 15Ser Leu Gln Gly2017220PRTArtificial SequenceSynthesized 172Gly Glu Asp Trp Cys Asp Ser Thr Leu Leu Ala Phe Asp Leu Cys Gly1 5 10 15Glu Gly Ala Arg2017320PRTArtificial SequenceSynthesized 173Gly Glu Asn Trp Cys Asp Trp Val Leu Leu Ala Tyr Asp Leu Cys Gly1 5 10 15Glu Asp Asn Thr2017415PRTArtificial SequenceSynthesized 174Gly Gly Glu Ile Cys Tyr Phe Pro Gly Ile Cys Arg Val Leu Pro1 5 10 1517511PRTArtificial SequenceSynthesized 175Gly Met Cys Tyr Phe Pro Gly Ile Cys Trp Ala1 5 1017619PRTArtificial SequenceSynthesized 176Gly Arg Ser Phe Cys Met Asp Trp Pro Ala His Lys Ser Cys Thr Pro1 5 10 15Leu Met Leu17715PRTArtificial SequenceSynthesized 177Gly Thr Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Gly Gly Gly1 5 10 1517820PRTArtificial SequenceSynthesized 178Gly Val Arg Thr Phe Cys Gln Asp Trp Pro Ala His Asn Ser Cys Lys1 5 10 15Leu Leu Arg Gly2017915PRTArtificial SequenceSynthesized 179His Ala Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Thr Glu Arg1 5 10 1518015PRTArtificial SequenceSynthesized 180Ile Val Glu Met Cys Tyr Tyr Pro Gly Ile Cys Trp Ile Ser Pro1 5 10 1518111PRTArtificial SequenceSynthesized 181Lys Leu Cys Tyr Phe Pro Gly Ile Cys Trp Ser1 5 1018211PRTArtificial SequenceSynthesized 182Lys Thr Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1018315PRTArtificial SequenceSynthesized 183Lys Thr Glu Ile Cys Tyr Phe Pro Gly Ile Cys Trp Met Ser Gly1 5 10 1518411PRTArtificial SequenceSynthesized 184Lys Val Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 1018515PRTArtificial SequenceSynthesized 185Leu Ala Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met Ser Ala1 5 10 1518620PRTArtificial SequenceSynthesized 186Leu Val Pro Glu Arg Ile Val Cys Tyr Phe Glu Ser Ile Cys Tyr Glu1 5 10 15Arg Ser Glu Leu2018720PRTArtificial SequenceSynthesized 187Met Glu Leu Trp Cys Asp Ser Thr Leu Met Ala Tyr Asp Leu Cys Gly1 5 10 15Asp Phe Asn Met2018820PRTArtificial SequenceSynthesized 188Met Glu Pro Ser Arg Ser Val Cys Tyr Ala Glu Gly Ile Cys Phe Asp1 5 10 15Arg Gly Glu Gln2018915PRTArtificial SequenceSynthesized 189Asn Asp Glu Ile Cys Tyr Phe Pro Gly Val Cys Trp Lys Ser Gly1 5 10 151908PRTArtificial SequenceSynthesized 190Gln Cys Phe Pro Gly Trp Val Lys1 519115PRTArtificial SequenceSynthesized 191Gln Thr Glu Leu Cys Tyr Phe Pro Gly Ile Cys Trp Asn Glu Ser1 5 10 1519220PRTArtificial SequenceSynthesized 192Gln Thr Arg Ser Phe Cys Ala Asp Trp Pro Arg His Glu Ser Cys Lys1 5 10 15Pro Leu Arg Gly2019320PRTArtificial SequenceSynthesized 193Arg Ala Ala Glu Ser Ser Val Cys Tyr Trp Pro Gly Ile Cys Phe Asp1 5 10 15Arg Thr Glu Gln2019420PRTArtificial SequenceSynthesized 194Arg Ala Pro Glu Arg Trp Val Cys Tyr Trp Glu Gly Ile Cys Phe Asp1 5 10 15Arg Tyr Glu Gln2019515PRTArtificial SequenceSynthesized 195Arg Asp Thr Val Cys Tyr Phe Pro Gly Ile Cys Trp Met Ala Ser1 5 10 1519620PRTArtificial SequenceSynthesized 196Arg Glu Pro Ala Ser Leu Val Cys Tyr Phe Glu Asp Ile Cys Phe Val1 5 10 15Arg Ala Glu Ala2019718PRTArtificial SequenceSynthesized 197Arg Gly Pro Asp Val Cys Tyr Trp Pro Ser Ile Cys Phe Glu Arg Ser1 5 10 15Met Pro19820PRTArtificial SequenceSynthesized 198Arg Met Pro Ala Ser Leu Pro Cys Tyr Trp Glu Thr Ile Cys Tyr Glu1 5 10 15Ser Ser Glu Gln2019915PRTArtificial SequenceSynthesized 199Arg Arg Thr Cys Asp Trp Pro His Asn Ser Cys Lys Leu Arg Gly1 5 10 1520020PRTArtificial SequenceSynthesized 200Arg Thr Ala Glu Ser Leu Val Cys Tyr Trp Pro Gly Ile Cys Phe Ala1 5 10 15Gln Ser Glu Arg2020115PRTArtificial SequenceSynthesized 201Ser Gly Ala Ile Cys Tyr Val Pro Gly Ile Cys Trp Thr His Ala1 5 10 1520215PRTArtificial SequenceSynthesized 202Ser Arg Glu Val Cys Tyr Tyr Pro Gly Ile Cys Trp Asn Gly Ala1 5 10 1520315PRTArtificial SequenceSynthesized 203Ser Tyr Ala Pro Cys Tyr Phe Pro Gly Ile Cys Trp Met Gly Asn1 5 10 1520415PRTArtificial SequenceSynthesized 204Thr Thr Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Lys Thr Glu1 5 10 1520515PRTArtificial SequenceSynthesized 205Val Gln Glu Val Cys Tyr Phe Pro Gly Ile Cys Trp Met Gln Glu1 5 10 1520615PRTArtificial SequenceSynthesized 206Val Arg Asp Met Cys Tyr Phe Pro Gly Ile Cys Trp Lys Ser Glu1 5 10 1520720PRTArtificial SequenceSynthesized 207Asn Arg Gln Met Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Asp Asp Phe2020820PRTArtificial SequenceSynthesized 208Gln Arg His Pro Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Asp Asp2020915PRTArtificial SequenceSynthesized 209Asp Phe Asp Leu Cys Leu Pro Asp Trp Gly Cys Leu Trp Asp Asp1 5 10 1521011PRTArtificial SequenceSynthesized 210Asp Ile Cys Leu Glu Arg Trp Gly Cys Leu Trp1 5 1021111PRTArtificial SequenceSynthesized 211Asp Ile Cys Leu Pro Ala Trp Gly Cys Leu Trp1 5 1021211PRTArtificial SequenceSynthesized 212Asp Ile Cys Leu Pro Asp Trp Gly Cys Leu Trp1 5 1021311PRTArtificial SequenceSynthesized 213Asp Ile Cys Leu Pro Glu Trp Gly Cys Leu Trp1 5 1021411PRTArtificial SequenceSynthesized 214Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 1021511PRTArtificial SequenceSynthesized 215Asp Leu Cys Leu Pro Glu Trp Gly Cys Leu Trp1 5 1021611PRTArtificial SequenceSynthesized 216Asp Leu Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 1021711PRTArtificial SequenceSynthesized 217Asp Leu Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 1021815PRTArtificial SequenceSynthesized 218Asp Ser Cys Gly Asp Leu Leu Arg Leu Gly Leu Glu Cys Trp Ala1 5 10 1521915PRTArtificial SequenceSynthesized 219Asp Thr Cys Ala Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Ala1 5 10 1522015PRTArtificial SequenceSynthesized 220Asp Thr Cys Asp Asp Leu Val Gln Leu Gly Leu Glu Cys Trp Ala1 5 10 1522115PRTArtificial SequenceSynthesized 221Asp Thr Cys Glu

Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Ala1 5 10 1522215PRTArtificial SequenceSynthesized 222Asp Thr Cys Ser Asp Leu Val Gly Leu Gly Leu Glu Cys Trp Ala1 5 10 1522315PRTArtificial SequenceSynthesized 223Glu Glu Asp Leu Cys Leu Pro Val Trp Gly Cys Leu Trp Gly Ala1 5 10 1522415PRTArtificial SequenceSynthesized 224Glu Glu Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Glu Gly1 5 10 1522515PRTArtificial SequenceSynthesized 225Glu Phe Asp Leu Cys Leu Pro Thr Trp Gly Cys Leu Trp Glu Asp1 5 10 1522620PRTArtificial SequenceSynthesized 226Glu Arg Gln Met Glu Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Val2022720PRTArtificial SequenceSynthesized 227Glu Arg Gln Met Val Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Phe2022815PRTArtificial SequenceSynthesized 228Glu Trp Asp Val Cys Phe Pro Ala Trp Gly Cys Leu Trp Asp Gln1 5 10 1522915PRTArtificial SequenceSynthesized 229Glu Trp Asp Val Cys Leu Pro His Trp Gly Cys Leu Trp Asp Gly1 5 10 1523015PRTArtificial SequenceSynthesized 230Phe Glu Asp Phe Cys Leu Pro Asn Trp Gly Cys Leu Trp Gly Ser1 5 10 1523115PRTArtificial SequenceSynthesized 231Phe Glu Asp Leu Cys Val Val Arg Trp Gly Cys Leu Trp Gly Asp1 5 10 1523220PRTArtificial SequenceSynthesized 232Gly Arg Gln Val Val Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Glu Glu Gly Leu2023320PRTArtificial SequenceSynthesized 233Lys Met Gly Arg Val Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Glu Leu2023420PRTArtificial SequenceSynthesized 234Lys Ser Arg Met Gly Asp Phe Cys Leu Pro Glu Trp Gly Cys Leu Trp1 5 10 15Gly Asp Glu Leu2023520PRTArtificial SequenceSynthesized 235Leu Arg Ile Phe Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Glu Gly Phe2023615PRTArtificial SequenceSynthesized 236Met Asp Asp Ile Cys Leu His His Trp Gly Cys Leu Trp Asp Glu1 5 10 1523715PRTArtificial SequenceSynthesized 237Met Asp Asp Leu Cys Leu Pro Asn Trp Gly Cys Leu Trp Gly Asp1 5 10 1523815PRTArtificial SequenceSynthesized 238Met Phe Asp Leu Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly Asn1 5 10 1523915PRTArtificial SequenceSynthesized 239Met Trp Asp Val Cys Phe Pro Asp Trp Gly Cys Leu Trp Asp Val1 5 10 1524015PRTArtificial SequenceSynthesized 240Asn Thr Cys Ala Asp Leu Val Arg Leu Gly Leu Glu Cys Trp Ala1 5 10 1524115PRTArtificial SequenceSynthesized 241Asn Trp Asp Leu Cys Phe Pro Asp Trp Gly Cys Leu Trp Asp Asp1 5 10 1524220PRTArtificial SequenceSynthesized 242Gln Gly Asp Phe Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Ser1 5 10 15Gly Glu Gly Tyr2024320PRTArtificial SequenceSynthesized 243Gln Gly Gly Met Gly Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Glu Asp Leu2024420PRTArtificial SequenceSynthesized 244Gln Gly Tyr Met Val Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Ala Asn2024520PRTArtificial SequenceSynthesized 245Gln Met His Met Met Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Thr Ser2024620PRTArtificial SequenceSynthesized 246Gln Met Gln Met Ser Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Tyr2024720PRTArtificial SequenceSynthesized 247Gln Arg His Met Met Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Tyr2024820PRTArtificial SequenceSynthesized 248Gln Arg Leu Met Trp Glu Ile Cys Leu Pro Leu Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Leu2024920PRTArtificial SequenceSynthesized 249Gln Arg Pro Ile Met Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Glu Asp Gly Phe2025020PRTArtificial SequenceSynthesized 250Gln Arg Gln Ile Met Asp Phe Cys Leu Pro His Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Phe2025120PRTArtificial SequenceSynthesized 251Gln Arg Gln Val Val Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Ser2025220PRTArtificial SequenceSynthesized 252Gln Ser Gln Leu Glu Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Gly Phe2025320PRTArtificial SequenceSynthesized 253Gln Ser Tyr Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Ser1 5 10 15Asp Asp Ala Ser2025420PRTArtificial SequenceSynthesized 254Arg Trp Gln Thr Glu Asp Val Cys Leu Pro Lys Trp Gly Cys Leu Phe1 5 10 15Gly Asp Gly Val2025515PRTArtificial SequenceSynthesized 255Ser Glu Asp Phe Cys Leu Pro Val Trp Gly Cys Leu Trp Glu Asp1 5 10 1525615PRTArtificial SequenceSynthesized 256Val Trp Asp Leu Cys Leu Pro Gln Trp Gly Cys Leu Trp Asp Glu1 5 10 1525715PRTArtificial SequenceSynthesized 257Trp Asp Asp Ile Cys Phe Arg Asp Trp Gly Cys Leu Trp Gly Ser1 5 10 1525815PRTArtificial SequenceSynthesized 258Trp Glu Asp Leu Cys Leu Pro Asp Trp Gly Cys Leu Trp Glu Asp1 5 10 1525920PRTArtificial SequenceSynthesized 259His Arg Leu Val Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asn Asp Phe2026020PRTArtificial SequenceSynthesized 260His Ser Gln Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Glu Leu2026120PRTArtificial SequenceSynthesized 261Leu Ile Phe Met Glu Asp Val Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Glu Asp Gly Val2026220PRTArtificial SequenceSynthesized 262Leu Arg Leu Met Asp Asn Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Asp Gly Phe2026320PRTArtificial SequenceSynthesized 263Leu Trp Ala Met Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Glu Asp Asp Phe2026420PRTArtificial SequenceSynthesized 264Gln Arg Asp Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Asp Gly Val2026520PRTArtificial SequenceSynthesized 265Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Arg Phe2026620PRTArtificial SequenceSynthesized 266Gln Trp His Met Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Asp Val Leu2026720PRTArtificial SequenceSynthesized 267Gln Trp Gln Met Glu Asn Val Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Glu Glu Leu Asp2026820PRTArtificial SequenceSynthesized 268Gln Trp Gln Val Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ala Asp Glu Tyr2026911PRTArtificial SequenceSynthesized 269Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 1027020PRTArtificial SequenceSynthesized 270Ala Ala Gln Val Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Glu Tyr Ala2027120PRTArtificial SequenceSynthesized 271Ala Gly Trp Ala Ala Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Asp Val2027218PRTArtificial SequenceSynthesized 272Ala Leu Phe Glu Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Gly1 5 10 15Gly Glu27320PRTArtificial SequenceSynthesized 273Ala Gln Ala Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Ala Glu Ile2027420PRTArtificial SequenceSynthesized 274Ala Ser Asp Pro Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Glu Ser Phe2027520PRTArtificial SequenceSynthesized 275Ala Ser Asp Arg Gly Asp Leu Cys Leu Pro Tyr Trp Gly Cys Leu Trp1 5 10 15Gly Pro Asp Gly2027618PRTArtificial SequenceSynthesized 276Ala Ser Glu Trp Asp Val Cys Leu Pro Thr Trp Gly Cys Leu Trp Met1 5 10 15Glu Gly27720PRTArtificial SequenceSynthesized 277Ala Ser Asn Trp Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Glu Arg Asn2027820PRTArtificial SequenceSynthesized 278Ala Ser Thr Pro Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Glu Asp Ala2027920PRTArtificial SequenceSynthesized 279Ala Ser Val Val Asp Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 10 15Gly Glu Asp Ile2028020PRTArtificial SequenceSynthesized 280Ala Thr Met Glu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Ala Glu Glu2028118PRTArtificial SequenceSynthesized 281Ala Tyr Ser Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Met1 5 10 15Ser Glu28220PRTArtificial SequenceSynthesized 282Asp Glu Asp Phe Glu Asp Tyr Cys Leu Pro Pro Trp Gly Cys Leu Trp1 5 10 15Gly Ser Ser Met2028320PRTArtificial SequenceSynthesized 283Asp Gly Glu Glu Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ala Leu Glu His2028418PRTArtificial SequenceSynthesized 284Glu Asp Trp Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp Glu1 5 10 15Gly Met28518PRTArtificial SequenceSynthesized 285Glu Asp Trp Thr Asp Leu Cys Leu Pro Ala Trp Gly Cys Leu Trp Asp1 5 10 15Thr Glu28618PRTArtificial SequenceSynthesized 286Glu Glu Asp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asn1 5 10 15Thr Ser28720PRTArtificial SequenceSynthesized 287Glu Gly Glu Glu Val Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Gly Tyr Pro Val2028820PRTArtificial SequenceSynthesized 288Glu Gly Thr Trp Asp Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Leu Gly Glu Arg2028918PRTArtificial SequenceSynthesized 289Glu Gly Tyr Trp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Leu Glu29018PRTArtificial SequenceSynthesized 290Glu Leu Gly Glu Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Ser Glu29120PRTArtificial SequenceSynthesized 291Glu Arg Trp Glu Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Glu Ser Gly2029218PRTArtificial SequenceSynthesized 292Glu Thr Trp Ser Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Ala Ser29320PRTArtificial SequenceSynthesized 293Glu Val Gly Asp Leu Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asn Asp Lys2029420PRTArtificial SequenceSynthesized 294Phe Arg Asp Gly Glu Asp Phe Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Ala Asp Thr Ser2029520PRTArtificial SequenceSynthesized 295Gly Asp Met Val Asn Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Ser Glu Asn2029620PRTArtificial SequenceSynthesized 296Gly Asp Trp Met His Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Asp Glu Lys Ala2029718PRTArtificial SequenceSynthesized 297Gly Asp Tyr Val Asp Leu Cys Leu Pro Gly Trp Gly Cys Leu Trp Glu1 5 10 15Asp Gly29820PRTArtificial SequenceSynthesized 298Gly Ile Glu Trp Gly Asp Thr Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Arg Val Glu Gly2029920PRTArtificial SequenceSynthesized 299Gly Gln Gln Gly Glu Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 10 15Asp Thr Ser Ser2030020PRTArtificial SequenceSynthesized 300Gly Arg Met Gly Thr Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Glu Val Glu2030120PRTArtificial SequenceSynthesized 301Gly Arg Tyr Pro Met Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Asp Ser Ala2030220PRTArtificial SequenceSynthesized 302Gly Ser Ala Gly Asp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Arg Gly Ala2030318PRTArtificial SequenceSynthesized 303Gly Val Leu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Pro Lys30420PRTArtificial SequenceSynthesized 304His Ala Ser Asp Trp Asp Val Cys Leu Pro Gly Trp Gly Cys Leu Trp1 5 10 15Glu Glu Asp Asp2030520PRTArtificial SequenceSynthesized 305His Glu Trp Glu Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Arg Asp Gly Asp2030618PRTArtificial SequenceSynthesized 306His Met Met Asp Asp Val Cys Leu Pro Gly Trp Gly Cys Leu Trp Ala1 5 10 15Ser Glu30718PRTArtificial SequenceSynthesized 307Ile Asp Tyr Thr Asp Leu Cys Leu Pro Ala Trp Gly Cys Leu Trp Glu1 5 10 15Leu Glu30818PRTArtificial SequenceSynthesized 308Ile Glu His Glu Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala1 5 10 15Val Asp30918PRTArtificial SequenceSynthesized 309Ile Ser Glu Trp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Arg Ser31018PRTArtificial SequenceSynthesized 310Ile Ser Trp Ala Asp Val Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly1 5 10 15Lys Asp31118PRTArtificial SequenceSynthesized 311Ile Ser Trp Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Gly Ser31220PRTArtificial SequenceSynthesized 312Lys Lys Val Ser Gly Asp Ile Cys Leu Pro Ile Trp Gly Cys Leu Trp1 5 10 15Asp Asn Asp Tyr2031318PRTArtificial SequenceSynthesized 313Lys Leu Trp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser1 5 10 15Pro Leu31418PRTArtificial SequenceSynthesized 314Leu Ala Trp Pro Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Gly Met31520PRTArtificial SequenceSynthesized 315Leu Gly Val Thr His Asp Thr Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Glu Val Gly2031620PRTArtificial SequenceSynthesized 316Leu Leu Glu Ser Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15His Glu Asp Gly2031718PRTArtificial SequenceSynthesized 317Leu Asn Glu Ser Asp Ile Cys Leu Pro Thr Trp Gly Cys Leu Trp Gly1 5 10 15Val Asp31818PRTArtificial SequenceSynthesized 318Leu Pro Glu Gln Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Asp1 5 10 15Ala Asn31920PRTArtificial SequenceSynthesized 319Leu Val Trp Glu Glu Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Ala Glu Asp2032018PRTArtificial SequenceSynthesized 320Met Ala Trp Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala1 5 10 15Gly Gly32120PRTArtificial SequenceSynthesized 321Met Gln Ala Glu Ser Asp Phe Cys Leu Pro His Trp Gly Cys Leu Trp1 5 10 15Asp Glu Gly Thr2032220PRTArtificial SequenceSynthesized 322Met Gln Gly Pro Leu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Gly Val Asp2032318PRTArtificial SequenceSynthesized 323Asn Glu Glu Trp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10

15Gly Val32420PRTArtificial SequenceSynthesized 324Asn Val Gly Trp Asn Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ala Gln Glu Ser2032518PRTArtificial SequenceSynthesized 325Gln Glu Leu Gln Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Val Gly32620PRTArtificial SequenceSynthesized 326Gln Gly Val Glu Trp Asp Val Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Thr Arg Glu Val2032720PRTArtificial SequenceSynthesized 327Gln Met Pro Leu Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Gly Arg Glu2032818PRTArtificial SequenceSynthesized 328Gln Arg Glu Trp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser1 5 10 15Asp Val32918PRTArtificial SequenceSynthesized 329Gln Arg Phe Trp Asp Thr Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Gly Asp33020PRTArtificial SequenceSynthesized 330Arg Glu Glu Trp Gly Asp Leu Cys Leu Pro Thr Trp Gly Cys Leu Trp1 5 10 15Glu Thr Lys Lys2033120PRTArtificial SequenceSynthesized 331Arg Leu Asp Ala Trp Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp1 5 10 15Glu Glu Pro Ser2033218PRTArtificial SequenceSynthesized 332Arg Val Phe Thr Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Leu Gly33320PRTArtificial SequenceSynthesized 333Arg Val Trp Thr Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Glu Gly Asn2033420PRTArtificial SequenceSynthesized 334Ser Glu Ala Pro Gly Asp Tyr Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ala Gln Glu Lys2033518PRTArtificial SequenceSynthesized 335Ser Gly Trp Asp Asp Val Cys Leu Pro Val Trp Gly Cys Leu Trp Gly1 5 10 15Pro Ser33620PRTArtificial SequenceSynthesized 336Ser Ile Arg Glu Tyr Asp Val Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Glu Pro Ser Ala2033720PRTArtificial SequenceSynthesized 337Ser Pro Thr Glu Trp Asp Met Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Ala Leu2033818PRTArtificial SequenceSynthesized 338Ser Ser Ala Ser Asp Tyr Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Asp Leu33920PRTArtificial SequenceSynthesized 339Ser Ser Gly Leu Glu Asp Ile Cys Leu Pro Asn Trp Gly Cys Leu Trp1 5 10 15Ala Asp Gly Ser2034020PRTArtificial SequenceSynthesized 340Ser Val Gly Trp Gly Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 10 15Gly Glu Gly Gly2034118PRTArtificial SequenceSynthesized 341Ser Trp Gln Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Val Asp34218PRTArtificial SequenceSynthesized 342Ser Tyr Glu Thr Asp Val Cys Leu Pro Tyr Trp Gly Cys Leu Trp Glu1 5 10 15Asp Ala34318PRTArtificial SequenceSynthesized 343Ser Tyr Trp Gly Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser1 5 10 15Glu Ala34420PRTArtificial SequenceSynthesized 344Thr Ala Met Asp Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Ser Gly Ser2034520PRTArtificial SequenceSynthesized 345Thr Glu Glu Asn Trp Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Asp Trp2034620PRTArtificial SequenceSynthesized 346Thr Glu Ile Gly Gln Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Val Pro Gly Thr2034718PRTArtificial SequenceSynthesized 347Thr Leu Glu Trp Asp Met Cys Leu Pro Arg Trp Gly Cys Leu Trp Thr1 5 10 15Glu Gln34820PRTArtificial SequenceSynthesized 348Thr Leu Gly Trp Pro Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Arg Glu Ser Asp2034920PRTArtificial SequenceSynthesized 349Thr Leu Ser Asn Gln Asp Ile Cys Leu Pro Gly Trp Gly Cys Leu Trp1 5 10 15Gly Gly Ile Asn2035020PRTArtificial SequenceSynthesized 350Thr Ser Gly Ser Asp Asp Ile Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 10 15Gly Glu Asp Ser2035120PRTArtificial SequenceSynthesized 351Thr Ser Thr Gly Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Ser Ser Glu2035219PRTArtificial SequenceSynthesized 352Thr Trp Pro Gly Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Ala Glu Ser35318PRTArtificial SequenceSynthesized 353Val Gly Glu Phe Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Ala Glu35420PRTArtificial SequenceSynthesized 354Val Ser Glu Met Asp Asp Ile Cys Leu Pro Leu Trp Gly Cys Leu Trp1 5 10 15Ala Asp Ala Pro2035520PRTArtificial SequenceSynthesized 355Val Ser Glu Trp Glu Asp Ile Cys Leu Pro Ser Trp Gly Cys Leu Trp1 5 10 15Glu Thr Gln Asp2035618PRTArtificial SequenceSynthesized 356Val Thr Ser Trp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Asp35720PRTArtificial SequenceSynthesized 357Val Val Gly Asp Gly Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Asp Gln Ala Arg2035820PRTArtificial SequenceSynthesized 358Val Val Trp Asp Asp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Tyr Gly2035920PRTArtificial SequenceSynthesized 359Trp Asp His Glu Leu Asp Phe Cys Leu Pro Val Trp Gly Cys Leu Trp1 5 10 15Ala Glu Asp Val2036018PRTArtificial SequenceSynthesized 360Trp Leu Trp Glu Asp Leu Cys Leu Pro Lys Trp Gly Cys Leu Trp Glu1 5 10 15Glu Asp36120PRTArtificial SequenceSynthesized 361Trp Ser Asp Ser Asp Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asn Val Ala2036220PRTArtificial SequenceSynthesized 362Trp Thr Glu Ser Glu Asp Ile Cys Leu Pro Gly Trp Gly Cys Leu Trp1 5 10 15Gly Pro Glu Val2036320PRTArtificial SequenceSynthesized 363Trp Val Glu Glu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Ser Val Glu2036420PRTArtificial SequenceSynthesized 364Trp Val Pro Phe Glu Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Ser Tyr Gln2036518PRTArtificial SequenceSynthesized 365Xaa Xaa Xaa Xaa Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp Xaa1 5 10 15Xaa Xaa36620PRTArtificial SequenceSynthesized 366Xaa Xaa Xaa Xaa Xaa Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp1 5 10 15Xaa Xaa Xaa Xaa2036718PRTArtificial SequenceSynthesized 367Ala Phe Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Asp36820PRTArtificial SequenceSynthesized 368Ala Gly Leu Asp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Lys Glu Ala2036920PRTArtificial SequenceSynthesized 369Ala Gly Met Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gln Gly Glu Pro2037020PRTArtificial SequenceSynthesized 370Ala Pro Gly Asp Trp Asp Phe Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Asp Asp Asp Ala2037120PRTArtificial SequenceSynthesized 371Ala Gln Leu Phe Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Asp Gly Tyr2037220PRTArtificial SequenceSynthesized 372Ala Arg Thr Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Ala Ser Asp2037320PRTArtificial SequenceSynthesized 373Ala Val Ser Trp Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Arg Ala Asp2037420PRTArtificial SequenceSynthesized 374Ala Trp Leu Asp Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Asn Thr Gly Val2037520PRTArtificial SequenceSynthesized 375Ala Trp Gln Asp Phe Asp Val Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Pro Glu Ser2037620PRTArtificial SequenceSynthesized 376Asp Thr Thr Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Glu Glu Ala2037718PRTArtificial SequenceSynthesized 377Asp Trp Gly Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Glu Glu37818PRTArtificial SequenceSynthesized 378Glu Ala Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Leu Glu37920PRTArtificial SequenceSynthesized 379Glu Gly Phe Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly His Gln Ala2038020PRTArtificial SequenceSynthesized 380Glu Gln Trp Leu His Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Asp Asp Thr Asp2038120PRTArtificial SequenceSynthesized 381Glu Thr Gly Trp Pro Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Gly Glu2038220PRTArtificial SequenceSynthesized 382Phe Glu Leu Gly Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu His Asn2038318PRTArtificial SequenceSynthesized 383Phe Ile Thr Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Asn38418PRTArtificial SequenceSynthesized 384Phe Leu Trp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser1 5 10 15Glu Gly38520PRTArtificial SequenceSynthesized 385Phe Ser Leu Asp Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Ala Glu Lys2038618PRTArtificial SequenceSynthesized 386Phe Val His Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Gly38720PRTArtificial SequenceSynthesized 387Gly Ala Ser Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Pro Glu Asp2038820PRTArtificial SequenceSynthesized 388Gly Asp Leu Gly Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Glu Tyr Pro2038920PRTArtificial SequenceSynthesized 389Gly Glu Gly Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ala Glu Asp Glu2039020PRTArtificial SequenceSynthesized 390Gly Glu Trp Trp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Ser Ser Ser2039118PRTArtificial SequenceSynthesized 391Gly Leu Gly Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Arg Asp39220PRTArtificial SequenceSynthesized 392Gly Leu Met Gly Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Lys Gly Asp Ile2039318PRTArtificial SequenceSynthesized 393Gly Met Phe Asp Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly1 5 10 15Leu Gly39418PRTArtificial SequenceSynthesized 394Gly Pro Gly Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Glu39518PRTArtificial SequenceSynthesized 395Gly Pro Trp Tyr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Gly Val39620PRTArtificial SequenceSynthesized 396Gly Ser Leu Glu Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Ile Asp Glu2039718PRTArtificial SequenceSynthesized 397Gly Trp Asp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Asp Gly39820PRTArtificial SequenceSynthesized 398Gly Trp His Asp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Gln Asn Asp2039920PRTArtificial SequenceSynthesized 399Gly Trp Leu Glu Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Ala Asp Asn2040020PRTArtificial SequenceSynthesized 400His Glu Gln Trp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Gly Ser Tyr2040118PRTArtificial SequenceSynthesized 401Leu Glu Tyr Glu Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly1 5 10 15Gly Glu40218PRTArtificial SequenceSynthesized 402Leu Ile Leu Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Asp Thr40318PRTArtificial SequenceSynthesized 403Leu Lys Leu Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Ser40418PRTArtificial SequenceSynthesized 404Leu Leu Asp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Val Arg40520PRTArtificial SequenceSynthesized 405Leu Leu Gly Gly His Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Gly Asp Val2040618PRTArtificial SequenceSynthesized 406Leu Leu Thr Arg Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly1 5 10 15Ser Asp40718PRTArtificial SequenceSynthesized 407Leu Met Ser Pro Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Glu1 5 10 15Gly Asp40818PRTArtificial SequenceSynthesized 408Leu Arg Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Thr40918PRTArtificial SequenceSynthesized 409Leu Val Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Ser Asp41018PRTArtificial SequenceSynthesized 410Leu Tyr Leu Arg Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Glu1 5 10 15Ala Asp41118PRTArtificial SequenceSynthesized 411Met Leu Ser Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Glu41218PRTArtificial SequenceSynthesized 412Met Pro Trp Thr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser1 5 10 15Glu Ser41320PRTArtificial SequenceSynthesized 413Met Arg Trp Ser Ser Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Gly Asp Glu Glu2041418PRTArtificial SequenceSynthesized 414Asn Trp Tyr Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Val Glu41517PRTArtificial SequenceSynthesized 415Gln Asp Trp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Asp41620PRTArtificial SequenceSynthesized 416Gln Phe Glu Trp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Val Glu Val2041720PRTArtificial SequenceSynthesized 417Gln Gly Trp Trp His Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Gly Glu2041820PRTArtificial SequenceSynthesized 418Gln Arg Val Asp Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Glu Asn Ser2041918PRTArtificial SequenceSynthesized 419Gln Ser Trp Pro Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp Gly1 5 10 15Glu Gly42020PRTArtificial SequenceSynthesized 420Arg Glu Gly Trp Pro Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Glu Thr Gly2042120PRTArtificial SequenceSynthesized 421Arg Glu Leu Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu His Ala Thr2042220PRTArtificial SequenceSynthesized 422Arg Leu Glu Leu Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Pro Gln Asp2042318PRTArtificial SequenceSynthesized 423Arg Leu Gly Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Tyr Gln42418PRTArtificial SequenceSynthesized 424Arg Leu Gly Ser Asp Ile Cys Leu Pro Arg Trp Gly

Cys Leu Trp Gly1 5 10 15Ala Gly42520PRTArtificial SequenceSynthesized 425Ser Gly Val Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Ala Gly2042620PRTArtificial SequenceSynthesized 426Ser Leu Gly Leu Thr Asp Leu Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Glu Gln2042718PRTArtificial SequenceSynthesized 427Ser Pro Trp Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Ser Gly42820PRTArtificial SequenceSynthesized 428Ser Ser Leu Glu Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Gln Asp Ala2042918PRTArtificial SequenceSynthesized 429Ser Thr Phe Thr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Leu Glu43020PRTArtificial SequenceSynthesized 430Ser Val Gly Trp Gly Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Ala Glu Ser Asp2043120PRTArtificial SequenceSynthesized 431Ser Val Leu Ser Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Trp Asp Phe Ser2043218PRTArtificial SequenceSynthesized 432Ser Val Leu Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Ser43318PRTArtificial SequenceSynthesized 433Thr Leu Leu Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Ser Asp43420PRTArtificial SequenceSynthesized 434Thr Leu Met Ser Asn Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Glu Pro Lys2043520PRTArtificial SequenceSynthesized 435Thr Leu Val Leu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Met Thr Asp2043620PRTArtificial SequenceSynthesized 436Thr Ser Leu Ala Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Ser Glu Asp Gly2043720PRTArtificial SequenceSynthesized 437Thr Ser Leu Leu Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Tyr Glu Glu Gly2043818PRTArtificial SequenceSynthesized 438Thr Trp Phe Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Pro Gly43920PRTArtificial SequenceSynthesized 439Thr Trp Gln Gly Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Thr Glu Val2044020PRTArtificial SequenceSynthesized 440Val Glu Met Trp His Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Ser Asn Ala2044120PRTArtificial SequenceSynthesized 441Val Gly Val Phe Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Gln Pro Val2044218PRTArtificial SequenceSynthesized 442Val His Gln Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Asp Thr44318PRTArtificial SequenceSynthesized 443Val Leu Leu Gly Asp Ile Cys Leu Pro Leu Trp Gly Cys Leu Trp Gly1 5 10 15Glu Asp44418PRTArtificial SequenceSynthesized 444Val Asn Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Ser44520PRTArtificial SequenceSynthesized 445Val Pro Ala Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Ala Arg Asn2044618PRTArtificial SequenceSynthesized 446Val Arg Leu Met Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Glu44718PRTArtificial SequenceSynthesized 447Val Arg Trp Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Glu44820PRTArtificial SequenceSynthesized 448Val Ser Leu Gly Asp Asp Ile Cys Leu Pro Lys Trp Gly Cys Leu Trp1 5 10 15Glu Pro Glu Ala2044918PRTArtificial SequenceSynthesized 449Val Val Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Lys Glu45020PRTArtificial SequenceSynthesized 450Val Trp Ile Asp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Asp Thr Glu Asn2045118PRTArtificial SequenceSynthesized 451Val Trp Tyr Lys Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Ala Glu45220PRTArtificial SequenceSynthesized 452Trp Asp Leu Ala Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Glu Ala2045318PRTArtificial SequenceSynthesized 453Trp Asp Val Ala Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala1 5 10 15Glu Asp45418PRTArtificial SequenceSynthesized 454Trp Asp Tyr Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Gly45518PRTArtificial SequenceSynthesized 455Trp Glu Val Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Asp Asp45618PRTArtificial SequenceSynthesized 456Trp His Met Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ser1 5 10 15Glu Val45718PRTArtificial SequenceSynthesized 457Trp Lys Asp Phe Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Asp His45818PRTArtificial SequenceSynthesized 458Trp Leu Ser Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Asp Leu45918PRTArtificial SequenceSynthesized 459Trp Leu Ser Glu Asp Ile Cys Leu Pro Gln Trp Gly Cys Leu Trp Glu1 5 10 15Glu Ser46018PRTArtificial SequenceSynthesized 460Trp Leu Ser Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala1 5 10 15Ala Asp46120PRTArtificial SequenceSynthesized 461Trp Arg Trp Asn Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Glu Glu Ala2046218PRTArtificial SequenceSynthesized 462Tyr Ile Trp Arg Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Gly Glu46318PRTArtificial SequenceSynthesized 463Tyr Arg Asp Tyr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Glu Arg46418PRTArtificial SequenceSynthesized 464Ala Gly Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Val Glu46518PRTArtificial SequenceSynthesized 465Glu Ile Arg Trp Asp Phe Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Glu Asp46618PRTArtificial SequenceSynthesized 466Glu Ser Leu Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Ser Gly46720PRTArtificial SequenceSynthesized 467Glu Val Arg Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Asn Trp Arg2046818PRTArtificial SequenceSynthesized 468Glu Tyr Trp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Asp1 5 10 15Trp Gln46920PRTArtificial SequenceSynthesized 469Phe Gly Gln Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asn Glu Gln2047020PRTArtificial SequenceSynthesized 470Ile Trp Gln Leu Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Asp Gly Leu2047118PRTArtificial SequenceSynthesized 471Lys Met Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Glu47218PRTArtificial SequenceSynthesized 472Met Gly Thr Lys Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Ala1 5 10 15Glu Ala47318PRTArtificial SequenceSynthesized 473Met His Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Ser Ser47420PRTArtificial SequenceSynthesized 474Asn Thr Pro Thr Tyr Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Val Pro2047520PRTArtificial SequenceSynthesized 475Asn Trp Ala Glu Asn Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Glu Asn2047620PRTArtificial SequenceSynthesized 476Gln Pro Val Trp Ser Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Glu Asp His2047718PRTArtificial SequenceSynthesized 477Arg Gly Leu His Asp Ala Cys Leu Pro Trp Trp Gly Cys Leu Trp Ala1 5 10 15Gly Ser47818PRTArtificial SequenceSynthesized 478Arg Leu Phe Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gln1 5 10 15Gly Glu47920PRTArtificial SequenceSynthesized 479Ser Ala Arg Glu Trp Asp Ile Cys Leu Pro Thr Trp Gly Cys Leu Trp1 5 10 15Glu Lys Asp Ile2048018PRTArtificial SequenceSynthesized 480Ser Gly Glu Trp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Glu Gly48118PRTArtificial SequenceSynthesized 481Ser Met Phe Phe Asp His Cys Leu Pro Met Trp Gly Cys Leu Trp Ala1 5 10 15Glu Gln48219PRTArtificial SequenceSynthesized 482Ser Trp Tyr Gly Gly Asp Ile Cys Leu Pro Trp Gly Cys Leu Trp Ser1 5 10 15Glu Glu Ser48318PRTArtificial SequenceSynthesized 483Thr Leu Phe Gln Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu1 5 10 15Glu Ser48418PRTArtificial SequenceSynthesized 484Val Gly Glu Trp Asp Ile Cys Leu Pro Asn Trp Gly Cys Leu Trp Glu1 5 10 15Arg Glu48518PRTArtificial SequenceSynthesized 485Trp Phe Pro Lys Asp Arg Cys Leu Pro Val Trp Gly Cys Leu Trp Glu1 5 10 15Arg His48620PRTArtificial SequenceSynthesized 486Trp Gly Met Ala Arg Asp Trp Cys Leu Pro Met Trp Gly Cys Leu Trp1 5 10 15Arg Gly Gly Gly2048720PRTArtificial SequenceSynthesized 487Trp His Leu Thr Asp Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Asp Glu Gln2048818PRTArtificial SequenceSynthesized 488Trp Trp Met Ala Asp Arg Cys Leu Pro Leu Trp Gly Cys Leu Trp Arg1 5 10 15Gly Asp48918PRTArtificial SequenceSynthesized 489Trp Trp Val Arg Asp Leu Cys Leu Pro Thr Trp Gly Cys Leu Trp Ser1 5 10 15Gly Lys49018PRTArtificial SequenceSynthesized 490Tyr Phe Asp Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly1 5 10 15Ser Asp49115PRTArtificial SequenceSynthesized 491Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp1 5 10 1549219PRTArtificial Sequencesynthesized 492Ser Trp Tyr Gly Gly Asp Ile Cys Leu Pro Trp Gly Cys Leu Trp Ser1 5 10 15Glu Glu Ser

Claims

1-60. (canceled)

61. A method of prolonging the elimination half-time of an antibody or antibody fragment comprising conjugating the antibody or antibody fragment to a serum albumin binding amino acid sequence, wherein the elimination half time of the antibody or antibody fragment exceeds that of an antibody or antibody fragment lacking a serum albumin binding amino acid sequence.

62. The method of claim 61 wherein the antibody fragment comprises a Fab.

63. The method of claim 61 wherein the antibody fragment comprises a F(ab').sub.2.

64. The method of any one of claims 61-63 wherein the affinity of the serum albumin binding amino acid sequence for albumin is characterized by an equilibrium dissociation constant (K.sub.d) that is less than about 1 .mu.M.

65. The method of any one of claims 61-63 wherein the affinity of the serum albumin binding amino acid sequence for albumin is characterized by a K.sub.d of less than about 500 nM.

66. The method of any one of claims 61-63 wherein the affinity of the serum albumin binding amino acid sequence for albumin is characterized by a K.sub.d of less than about 50 nM.

67. The method of any one of claims 61-63 wherein the affinity of the serum albumin binding amino acid sequence for albumin is characterized by a K.sub.d between about 1 .mu.M and 1 nM.

68. The method of claim 61, wherein the serum albumin binding amino acid sequence comprises the amino acid sequence (Xaa).sub.x-Xaa-Xaa.sub.1-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-(Xaa).sub.z- , wherein Xaa-Xaa.sub.1-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Asp-Xaa.sub.1-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp (SEQ ID NO:116), wherein Xaa or Xaa.sub.1 is any amino acid, and x and z are 0 to 5 amino acids.

69. The method of claim 68 wherein x is 4 and z is 3.

70. The method of claim 68 wherein x is 5 and z is 4.

71. The method of claim 68 wherein Xaa.sub.1 is Ile, Phe, Tyr or Val.

72. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 120).

73. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp (SEQ ID NO: 121).

74. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Gln-Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-A- sp-Phe (SEQ ID NO: 122).

75. The hybrid molecule of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-S- er-Val (SEQ ID NO: 123).

76. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-S- er-Val-Lys (SEQ ID NO: 124).

77. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 125).

78. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 126).

79. The method of claim 68 wherein Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 127).

80. The method of claim 61 wherein the amino acid sequence is cyclized by the presence of disulfide-bonded Cys residues.

81. The method of claim 61 wherein the amino acid sequence is less than about 50 amino acid residues.

82. The method of claim 61 wherein the amino acid sequence is less than about 40 amino acid residues.

83. The method of claim 61 wherein the amino acid sequence is about 20 amino acid residues.

85. The method of claim 61 further comprising a bioactive compound.