# Patent

## Title
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.

## Background
This application is a continuation of U.S. application Ser. No. 10/149,835, filed Jun. 14, 2002, which is a national stage entry under 35 U.S.C. §371 of International Patent Application No. PCT/US2000/35325, the entire disclosures of which are hereby incorporated by reference.


FIELD OF THE INVENTION
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
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 I interleukin 1 (IL-1) receptor blocking the binding of IL-α (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).
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-α (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 αVβ3 or αVβ5 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 α5β1 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. Struct. 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).
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.
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.
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).
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
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.
Such compounds preferably bind a desired plasma protein with an affinity characterized by a dissociation constant, Kd, that is less than about 100 μM, 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.
Preferred peptide ligands bind IgG-Fc and include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following core formula:
Xaai-Cys-Xaaj-Cys-Xaak, wherein Xaai is absent or is a peptide of between 1 and 4 amino acids, preferably 4 amino acids; Xj 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 Xaa1-Xaa2-Xaa3-Xaa4-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 139), wherein Xaa1 preferably is Ala, Ser, or Thr; Xaa2 preferably is Trp or Tyr; Xaa3 preferably is His, or Trp; Xaa4 preferably is Leu or Met, and Xaak 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.
Preferred among this group of compounds are compounds that bind IgG-Fc comprising the sequence:
Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Val-Trp-Cys-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO: 11);
Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Gly-Glu-Leu-Val-Trp-Cys-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO: 12);
Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Gly-Glu-Leu-Val-Trp-Cys-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13 (SEQ ID NO: 13), wherein Xaa5 is Ala, Ser, or Thr; Xaa6 is Trp or Tyr; Xaa7 is His or Trp; and Xaa8 is Leu or Met; and
Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Gly-Glu-Leu-Val-Trp-Cys-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13 (SEQ ID NO: 14) wherein Xaa4 is Ser, Arg, or Asp; Xaa5 is Ala, Ser, or Thr; Xaa6 is Trp or Tyr; Xaa7 is His or Trp; Xaa8 is Leu or Met; and Xaa9 is Glu, Ser, Thr or Val.
Preferred peptide ligands that bind serum albumin include linear and cyclic peptides, preferably cyclic peptide compounds comprising the following formulae:







Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa


 







(SEQ ID NO: 113)







Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys


 







(SEQ ID NO: 114)







Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe


 







(SEQ ID NO: 115)







Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys


and







(SEQ ID NO: 116)







Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp






wherein the general formulae further comprises the sequence (Xaa)x at the N-terminus and (Xaa)z at the C-terminus.
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 Xaa1 is selected from the group consisting of Ile, Phe, Tyr and Val.
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 a few.
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 peptideligand 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.
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.
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.
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
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).
FIG. 2. BIAcoreTM 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.
FIG. 3. Group average serum concentration vs. time data (±SD) are presented in the figure for Fab-Y0317-570 and Fab-Y0317.
FIG. 4. The peptide sequences (SEQ ID NOs: 119, 130 and 140-150) 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.
FIGS. 5A and 5B. Sequences identified following soft randomization (SEQ ID NOs: 120, 130, 140 and 148-269) are shown in FIG. 5 along with their species specificity as determined by phage ELISA.
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.
FIG. 7. Clones that bind to multiple species of albumin (multi-species binders) are listed in FIG. 7 (SEQ ID NOs: 123, 150, 207, 208 and 259-268).
FIGS. 8A, 8B and 8C. Sequences from libraries selected against rat, rabbit and human albumin are shown in FIGS. 8A, 8B, and 8C, respectively (SEQ ID NOs: 270-490 and 492).
FIG. 9. Peptides corresponding to identified phage sequences (SEQ ID NOs: 119, 121-124, 126-128, 269 and 491) were synthesized and their affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay.
FIG. 10A 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.
FIG. 11. 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, identical constructs were made with the intra-chain disulfide replaced by alanines (D3H44-Ls and D3H44-Hs, respectively) as depicted in FIG. 11.
FIG. 12. Purified D3H44. fusions retained their ability to bind TF as measured using a FX activation assay.
FIG. 13. Purified D3H44 fusions retained their ability to bind TF as measured using a protbrombin time assay that measures prolongation of tissue factor dependent clotting.
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.
FIG. 15 Both D3H44 albumin-binding fusions are capable of binding TF and albumin simultaneously as judged by a biotin-TF binding assay.
FIG. 16 Fusion of the albumin binding peptide to D3H44 results in a protein having improved phannacokinetic parameters.
FIG. 17. Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetie parameters.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
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 β turn or β 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.
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, Kd, of less than about 1 μM, 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 μM 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.
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)).
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 α-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.
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)-hydoxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr; allo-isoleucine (AIle) for Ile, Leu, and Val; p-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.
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.
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 peptideligand 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.
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.
“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.
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 Fe region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fe region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
Pepsin treatment yields an F(ab′)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.
“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.
“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.
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.
“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, t1/2 the time required for 50% completion of the process. The units of these two constants are time−1 and time, respectively. A first-order rate constant and the half-time of the reaction are simply related (k×t1/2=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.
“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, CaPO4 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
“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.
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.”
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.
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
A. Peptide Ligands
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:







Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa


 







(SEQ ID NO: 113)







Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys


 







(SEQ ID NO: 114)







Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe


 







(SEQ ID NO: 115)







Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys


and







(SEQ ID NO: 116)







Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp






wherein the general formulae further comprises the sequence (Xaa)x at the N-terminus and the sequence (Xaa)z at the C-terminus.
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 Xaa1 is selected from the group consisting of Ile, Phe, Tyr and Val.
Further preferred peptide ligands that bind a serum albumin are identified as described herein in the context of the following general formulae Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys (SEQ ID NO: 117) and Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp (SEQ ID NO: 118), wherein the general formulae further comprises the sequence (Xaa)x at the N-terminus and the sequence (Xaa)z at the C-terminus and 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.
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.
Preferred compounds according to this aspect of the invention include:








(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-Thr-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







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:
Xaai-Cys-Xaaj-Cys-Xaak, wherein Xaai is absent or is a peptide of between 1 and 4 amino acids, preferably 4 amino acids; Xj 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 Xaa1-Xaa2-Xaa3-Xaa4-Gly-Glu-Leu-Val-Trp (SEQ ID NO: 139), wherein Xaa1 is Ala, Ser, or Thr; Xaa2 is Trp or Tyr; Xaa3 is His, or Trp; Xaa4 is Leu or Met, and Xaak 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.
Preferred among this group of compounds are compounds comprising the sequence:








(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;






Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Gly-Glu-Leu-Val-Trp-Cys-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13 (SEQ ID NO: 13), wherein Xaa5 is Ala, Ser, or Thr; Xaa6 is Trp or Tyr; Xaa7 is His, or Trp; and Xaa8 is Leu or Met; and
Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Gly-Glu-Leu-Val-Trp-Cys-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13 (SEQ ID NO: 14) wherein Xaa4 is Ser, Arg, or Asp;.Xaa5 is Ala, Ser, or Thr; Xaa6 is Trp, Tyr; Xaa7 is His, or Trp; Xaa8 is Leu or Met; and Xaa9 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.
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 shown below:







(SEQ ID NO: 116)







Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp


 







(SEQ ID NO: 113)







Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys


 







(SEQ ID NO: 114)







Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe


or







(SEQ ID NO: 115)







Cys-Tyr-Xaa1-Pro-Gly-Xaa-Cys






wherein the general formulae further comprises the sequence (Xaa)x at the N-terminus and the sequence (Xaa)z at the C-terminus.
wherein Xaa is an amino acid, x and z are preferably 4 or 5 and Xaa1 is selected from the group consisting of Ile, Phe, Tyr and Val.
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.
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 μM, preferably when present at 1 μM, 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 μM, 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.
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 bercomes bound to the ligand.
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.
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:
(a) Radioisotopes, such as 35S, 14C, 125I, 3H, and 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.
(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.
(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 emitlight 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, β-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.
Examples of enzyme-substrate combinations include, for example:
(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));
(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and
(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.
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 themaximally-bound tracer is displaced is referred to as the “IC50” and reflects the IgG binding affinity of the candidate compound. Therefore a candidate compound with an IC50 of 1 mM displays a substantially weaker interaction with the target than a candidate compound with an IC50 of 1 μM.
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 EC50. Assays were performed under conditions where EC50(con)/EC50(mut) will approximate Kd(con)/Kd(mut).
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 IC50 for the target such as IgG or human serum albumin of less than 1 μM. Preferred among these compound are compounds having an IC50 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 IC50 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.
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.
B. Peptide Ligand Combinations
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.
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 (i.e., 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.
C. Linker Domains
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.
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.
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.
D. Recombinant Synthesis
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.
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.
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.
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.
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, kanr) (U.S. Pat. No. 5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium, Serratia marcesans, and Pseudomonas species.
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. 
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/-DHR (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).
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, lpp, 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).
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.
Prokaryotic host cells used to produce the present peptides can be cultured as described generally in Sambrook et al., supra.
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 GentamycinTM 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.
The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.
E. Chemical Synthesis
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).
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 cain be used to prepare the hybrid molecule compositions of the invention if the active domain is or comprises a polypeptide.
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 α-amino group of the blocked amino acids in peptide synthesis. If a base-labile α-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 α-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.
After the initial amino acid is coupled to an inert solid support, the α-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 α-amino group, the next α-amino and side chain protected amino acid in the synthesis is added. The remaining α-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.
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.
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 α-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 α-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.
α- and ε-amino side chains can be protected with benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2Cl)], p-nitrobenzyloxycarbonyl [Z(NO2)], 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.
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.
Many of the blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif.), Bachem CA (Torrence, Calif.) or Peninsula Labs (Belmont, Calif.).
Stewart and Young, supra, provides detailed information regarding procedures for preparing peptides. Protection of α-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.
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 (), 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 thiocresol 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.
F. Chemical Conjugation of Hybrids
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.
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).
G. Disulfide-Linked Peptides
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 μM, and preferably 500 μM to 1 μM, and more preferably 25 μM to 1 μM, 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.
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, αβ-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.
H. Pharmaceutical Compositions
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.
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.
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).
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.
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).
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.
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.
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.
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.
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 chloroflourocarbon, a hydrofluorocarbon, a hydrochlorofluorocarbon, or a hydrocarbon, including triflouromethane, dichlorodiflouromethane, dichlorotetrafloroethanol, and 1,1,1,2-tetraflouroethane, or combinations thereof.
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 a on. 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.
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.
I. Liquid Aerosol Formulations
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. No. 4,624,251 issued Nov. 25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No. 3,561,444 issued Feb. 9, 1971 and U.S. Pat. No. 4,635,627 issued Jan. 13, 1971.
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.
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.
J. Aerosol Dry Powder Formulations
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.
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.
K. Research, Manufacturing, and Diagnostic Compositions
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, pH 5.0, that will release the immunoglobulin protein from the peptide ligand.
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.
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.
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.
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.
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 “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.
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
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 4×109 different peptides of the form Xaai-Cys-Xaaj-Cys-Xaak 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., Xaai-Cys-Xaaj-Cys-Xaak 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.
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 μg/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-IgG1 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.
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-Gly-Glu-Leu-Val-Trp-Cys-Val-Ala-Gly-Val-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.
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 μg/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 μM 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, “IC50”, was extrapolated to zero biotin-IgG-Fc concentration in order to obtain a Ki. 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 (Ki) of about 5 μM. The results imply that these peptides bind to an overlapping site on IgG-Fc that coincides with the Protein A binding site.
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-Val-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.
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-Val-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 1×108. 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 OWA 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.
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 IC50 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 KdS 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.
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 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 (Ki)



ETQRCTWHMGELVWCEREHN
SEQ ID NO: 2
5000 nM (Ki)


DLADCSWHMGELVWCSRVEG
SEQ ID NO: 15
  50 nM (Kd)


WEADCAWHLGELVWCTPMEF
SEQ ID NO: 16
  30 nM (IC50)


   DCAWHLGELVWCT
SEQ ID NO: 8
 100 nM (IC50)


 


Phage Clones

All phage


(M13/gIII Display)

affinities




are EC50S


 


N/A = Not individ-


ually assayed. Since


they were selected


for binding, EC50


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


KEASGSWHLGELVWCVAGVE
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


KEASGSYWLGELVWCDNGVE
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


NQVSGSYSRGELVWCSKQSQ
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


DLADCSWHLGELVWCSRVEG
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


EVADCSWHLGELVWGTEGEF
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
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 1 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).
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.
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
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.
An irrelevant humanized IgG, 4D5-IgG, also known as Herceptin®, 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® in solution, equilibrated for 20 min in a non-adsorbent plate (Nunc F96), then transferred to the Herceptin®-oated 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 IC50 values of about 100-300 nM for each of the constructs, pY0192-569, pY0192-570, pY0317-569, and pY0317-570.
EXAMPLE 4
BIAcoreTM Analysis of IgG Binding to Anti-VEGF Fab Tagged with an IgG-Fc Peptide Ligand
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®, to Fab that previously had been bound to an immobilized VEGF biosensor chip.
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 Iigand 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 BIAcoreTM 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® was injected, and the dissociation off-rate (koff) following injection was observed.
The results (FIG. 2) show that Herceptin® 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 koff of 2.8×10−3, sec−1, and a corresponding dissociation half-life (t1/2) of 8.5 min were calculated for Y0317-570. Limitations of material prevented reliable on-rate determinations. However, the observed koff suggests an equilibrium binding affinity, Kd, of 30 M to 300 nM (assuming kon of 104-105 M−1 sec−1), consistent with peptide binding and phage-ELISA results (above). Importantly, the BIAcoreTM 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
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.
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-αt+Be-βt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-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 β-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((α−k21)/(α−β), B=D/V (β−k21)/(α−β), and α and β (for α>β) are roots of the quadratic equation: r2+(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.
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.
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.


















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-Y0317-570 1

3
0.93


2
3.0
Fab-Y0317-570 1

3
1.00


2
2.9
Fab-Y0317-570 1

3
0.97









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 ( ̃30 min) at room temperature, and centrifuged. Serum was harvested and immediately stored at −70C until analyzed.
ELISA plates were coated with 0.5 microg/ml VEGF in 50 mM carbonate buffer, pH 9.6, at 4° C. overnight and blocked with 0.5% bovine serum albumin, 10 ppm Proclin 300 (Supelco, Bellefonte, Pa.) in PBS (8 mM Na2HPO4, 1.5 mM KH2PO4, 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 lmmunoResearch, 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.
Data analysis: Graphs of concentration versus time profiles were made using KaleidaGraph (KaleidaGraphTM 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® Professional V. 3.1 WinNonlinTM 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).
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.










Pharmacokinetic Parameter Summary


(IV bolus; 1 mg/kg)












Group 1
Group 2



Parameter
Control Fab-Y0317
Fab-Y0317-570







AUC (h*μg/mL)
13.6 ± 1.2
215 ± 56



Cmax (μg/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










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/h/d). The overall exposure (AUC) of Fab-Y0317-570 was  ̃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 μg/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.
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
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 CX2GPX4C (SEQ ID NO: 133), X4CX2GPX4CX4 (SEQ ID NO: 134) and XiCXjCXk where j=8-10; Pool B contained X20 and XiCXjCXk where j=4-7; Pool C contained X8 and X2CXjCX2 where j=4-6; Pool D contained X2CXjCX2 where j=7-10; Pool E contained CX6CX6CCX3CX6C (SEQ ID NO: 135), CCX3CX6C (SEQ ID NO: 136), CCX5CX4CX4CC (SEQ ID NO: 137), CXCX7CX3CX6 (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.
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 μg/ml in PBS, overnight at 4° 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.
Phage ELISA-Phage clones ( ̃1011 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/H2O2 substrate and monitoring the change at 405 nm.
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.
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.
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.
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 X5DXCLPXWGCLWX4 (SEQ ID NO: 366), 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, 8B, and 8C respectively.
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.
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-QGLIGDICLPRWGCLWGDSVKb (SEQ ID NO: 124)-n where Kb refers to lysine-biotin).
SA08b Binding Assay—Rabbit, rat or mouse albumin was immobilized directly oil maxisorp plates at 10 μg/ml in PBS, overnight at 4° C. Plates were blocked using Blocker Casein (Pierce Chemical, Rockford, Ill.) for 1 hr, at 25° 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° 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/H2O2 substrate and monitoring the change at 405 nm.
Peptides corresponding to identified phage sequences were synthesized and their affinity for rat, rabbit or mouse albumin measured using the SA08b binding assay (FIG. 9 and 10).
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.
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° C. Cell debris was removed by centrigation 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)).
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 TF. Wild-type D3H44 (WT) is unable to bind albumin and thus does not generate a signal upon addition of biotinylated TF.
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 ELSA.
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-αt+Be-βt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-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 β-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(α−k21)/(α−β), B=D/V (β−k21)/(α−β), and α and β (for α>62 ) are roots of the quadratic equation: r2+(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.
Data analysis: Graphs of concentration versus time profiles were made. using KaleidaGraph KaleidaGraphTM 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® Professional V. 3.1 WinNonlinTM 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).
Fusion of the albumin binding peptide to D3H44 results in a protein having improved pharmacokinetic parameters (FIG. 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).
All publications cited herein are expressly incorporated by reference in their entirety.










Table of Sequence Identification Numbers










Location in



SEQ ID
Specification/Figures



No:
(Page Nos./FIG.Nos.)
SEQUENCE*












1
30
GGGSGGG


2
30, 32
ETQRCTWHMGELVWCEREHN


3
30, 31, 32
KEASCSYWLGELVWCVAGVE


4
31
GGGPGGG


5
31
DRG GWA GMA RRC TGC KCT TRS CAC




MTG GGC GAG CTG GTC TGG TGC RVC




RVM BKC GAS KDW


6
31
DRS VWG SVG RRC TGC KCC TRS YRS




MTG GGC GAG CTG GTC TGG TGC RNC




VVS NBS GWS KDM


7
31
DNS NNS NNS VNS TGC BVG TDS HRS




MDS GGC GAG STC KKG WRG TGC RNM




NNS NNS NNS NNM


8
31, 32, 36
DCAWHLGELVWCT


9
3, 13
XXXXXXLVW


10
3, 13
XXXXGELVW


11
3, 13
XXXXCXXXXXXLVWCXXXXX


12
3, 13
XXXXCXXXXGELVWCXXXXX


13
3, 13
X1X2X3X4CX5X6X7X8GELVWCX9X10X11X12X13


14
3, 13
X1X2X3X4CX5X6X7X8GELVWCX9X10X11X12X13


15
32
DLADCSWHMGELVWCSRVEG


16
32
WEADCAWHLGELVWCTPMEF


17
32
KEASCSYWLGELVWCDTLTE


18
32, 33
KEASCSYWLGELVWCSPGVE


19
32
KEASCSYWLGELVWCSGVEG


20
32
KEASCSYWLGELVWCSAGVE


21
33
ESEDCSYWLGELVWCVAGVE


22
33
EKEDCSYWLGELVWCVAGVE


23
33
EDPDCSYWLGELVWCVAGVE


24
33
EEADCSYWLGELVWCVAGVE


25
33
NADDCSYWLGELVWCVAGVE


26
33
SETTCSYWLGELVWCVAGVE


27
33
AWKTCQWLGELVWCVAGVE


28
33
DLADCSYWLGELVWCSRVEG


29
33
KEADCAWHLGELVWCVAGVE


30
33
KEAECSYHLGELVWCVAGVE


31
33
KEARCWYWHGELVWCSDPEE


32
33
KEASCSYHLGELVWCVAGVE


33
33
KEASCSWHLGELVWCVAGVE


34
33
KEASCSYWLGELVWCTEGVE


35
33
KEASCSYWLGELVWCDDGVE


36
33
KEASCSYWLGELVWCSEGVE


37
33
KEASCSYWLGEVWKCKSGVE


38
33
KEASCSYWLGELVWCDNGVE


39
33
KEASCSYWLGELVWCDTFDE


40
33
KEASCSYWLGELVWCDGLDE


41
33
KEASCSYWLGELVWCVGLDE


42
33
KEASCSYWLGELVWCEDTLE


43
33
KEASCSYWLGELVWCEDTME


44
33
KEASCSYWLGELVWCEDMME


45
33
WVEDCSWHMGELVWCDGGEF


46
33
KEASCSYWLGELVWCDWMNG


47
33
KEASCSYWLGELVWCDDTPV


48
33
KEASCSYWLGELVWCDDYGE


49
33
KEASCSYWLGELVWCSDLWE


50
33
WRGGCSWHMGELVWCEHDME


51
33
AVSKCSFHMGELVWCSDVMN


52
33
NQVSCSYSRGELVWCSKQSQ


53
33
GRMECAWHQGELVWCTPTLE


54
33
GTMECSWHQGELVWCTPTLA


55
33
EMRDCSWHLGELVWCAHMEG


56
33
GSWECAYHLGELVWCETGSG


57
34
VAEPCAYHLGELVWCEVLKG


58
34
KEAMCSYWLGELVWCESDMP


59
34
DLADCSWHLGELVWCSRVEG


60
34
DLADCSWHLGELVWCVGLDE


61
34
WVEDCSWHLGELVWCVGLDF


62
34
KVADCAWHMGELVWCTEVEG


63
34
GEEDCSYHLGELVMCTELDD


64
34
GVADCAWHLGELVWCTERED


65
34
GEEDCAWHLGELVWCSGGDF


66
34
WEADCAWHLGELVWCTKVEE


67
34
GEADCSYHLGELVWCNDFEE


68
34
WVDCAYHLGELVWCSTFEE


69
34
WVEDCAWHMGELVWCTKVDE


70
34
READCAWHLGELVWCSERDL


71
34
EEASCAYHLGELVWCDAFDV


72
34
RVASCAWHLGELVWCDGLDG


73
34
GEADCAWHLGELVWCTKVEE


74
34
GEASCAYHLGELVWCDEGEG


75
34
RVEDCAYHLGELVWCTEGDE


76
34
EEPDCSWHLGELVMCTPMEV


77
34
KEADCAWHMGELVWCSEMEG


78
34
EQADCAWHLGELVWCTPMVF


79
34
EEPDCSWHLGELVWCTPIEV


80
34
GEPDCAWHLGELVWCTPMVF


81
34
GEQDCSYHMGELVWCTTVDG


82
34
GVRNCAYHLGELVWCTPMEF


83
34
RVADCAWHMGELVWCSELEV


84
34
GEADCAWHLGELVWCTPMDL


85
34
GEQDCSWHLGELVWCTPMEV


86
34
GMRDCSYHLGELVWCSDMEL


87
35
EVADCSWHLGELVWCTEGEF


88
35
GEEDCAWHLGELVWCTDVED


89
35
EVEDCAYHLGELVWCSDLEG


90
35
WEEDCAWHLGELVWCAEFDE


91
35
KEASCAWHLGELVWCSEVEE


92
35
AEADCAWHLGELVWCTKVEE


93
35
WAADCAWHLGELVWCTKVEE


94
35
WEPDCAWHLGELVWCTKVEE


95
35
WEAACAWHLGELVWCTKVEE


96
35
WEAACSWHLGELVWCTKVEE


97
35
WEADCAAHLGELVWCTKVEE


98
35
WEADCAWALGELVWCTKVEE


99
35
WEADCAWHAGELVWCTKVEE


100
35
WEADCAWHLAELVWCTKVEE


101
35
WEADCAWHLGALVWCTKVEE


102
35
WEADCAWHLGEAVWCTKVEE


103
35
WEADCAWHLGELAWCTKVEE


104
35
WEADCAWHLGELVACTKVEE


105
35
WEADCAWHLGELVWCAKVEE


106
35
WEADCAWHLGELVWCTAVEE


107
35
WEADCAWHLGELVWCTKAEE


108
35
WEADCAWHLGELVWCTKVAE


109
36
DCAWHLGELVWCTGGG


110
36
GGGWEADCAWHLGELVWCT


111
36
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


112
36
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


113
4, 11, 14
FCXDWPXXXSC


114
4, 11, 14
VCYXXXICF


115
4, 12, 14
CYX1PGXC


116
4, 12, 13
DXCLPXWGCLW


117
12
WCDXXLXAXDLC


118
12
DLVXLGLECW


119
12, FIGS. 4, 9
DLCLRDWGCLW


120
12, FIG. 5
DICLPRWGCLW


121
12, FIG. 9
MEDICLPRWGCLWED


122
12, FIG. 9
QRLMEDICLPRWGCLWEDDF


123
12, FIGS. 7, 9
QGLIGDICLPRWGCLWGDSV


124
12, 42, FIG. 9
QGLIGDICLPRWGCLWGDSVK


125
12
EDICLPRWGCLWEDD


126
12, FIG. 9
RLMEDICLPRWGCLWEDD


127
12, FIG. 9
MEDICLPRWGCLWEDD


128
12, FIG. 9
RLMEDICLARWGCLWEDD


129
13
EVRSFCTDWPAEKSCKPLRG


130
13, FIGS. 4, 5
RAPESFVCYWETICFERSEQ


131
13
EMCYFPGICWM


132
30
GELVW


133
40
CXXGPXXXXC


134
40
XXXXCXXGPXXXXCXXXX


135
41
CXXXXXXCXXXXXXCCXXXCXXXXXXC


136
41
CCXXXCXXXXXXC


137
41
CCXXXXXCXXXXCXXXXCC


138
41
CXCXXXXXXXCXXXCXXXXXX


139
3, 13
X1X2X3X4GELVW


140
FIGS. 4, 5
EMCYFPGICWM


141
FIG. 4
CEVALDACRGGESGCCRHICELIRQLC


142
FIG. 4
CGCVDVSDWDCWSECLWSHGA


143
FIG. 4
DLCDVDFCWF


144
FIG. 4
DTCVDLVRLGLECWG


145
FIG. 4
KSCSELHWLLVEECLF


146
FIG. 4
MDELAFYCGPVVECLMHQEQK


147
FIG. 4
RNEDPCVVLLEMGLECWEGV


148
FIGS. 4, 5
EVRSFCTDWPAEKSCKPLRG


149
FIGS. 4, 5
GENWCDSTLMAYDLCGQVNM


150
FIGS. 4, 5, 7
QRQMVDFCLPQWGCLWGDGF


151
FIG. 5
ALCYFPGICWM


152
FIG. 5
ASEICYFPGICWMVE


153
FIG. 5
DICYIPGICWM


154
FIG. 5
DLCYFPGICWM


155
FIG. 5
DMCYFPGICFN


156
FIG. 5
DMCYFPGICWL


157
FIG. 5
DSEVCYFPGICWSGT


158
FIG. 5
DVCYFPGICWM


159
FIG. 5
EHDMCYFPGICWIAD


160
FIG. 5
EICYFPGICWI


161
FIG. 5
EICYFPGICWM


162
FIG. 5
ELCYFPGICWM


163
FIG. 5
ELCYFPGICWP


164
FIG. 5
ELCYFPGICWT


165
FIG. 5
EMCYFPGICWS


166
FIG. 5
EMCYFPGICWT


167
FIG. 5
ETCYFPGICWL


168
FIG. 5
EVCYFPGICWE


169
FIG. 5
EVCYFPGICWF


170
FIG. 5
EVCYFPGICWM


171
FIG. 5
EVRSFCTDWPAHYSCTSLQG


172
FIG. 5
GEDWCDSTLLAFDLCGEGAR


173
FIG. 5
GENWCDWVLLAYDLCGEDNT


174
FIG. 5
GGEICYFPGICRVLP


175
FIG. 5
GMCYFPGICWA


176
FIG. 5
G-RSFCMDWPAHKSCTPLML


177
FIG. 5
GTEVCYFPGICWGGG


178
FIG. 5
GVRTFCQDWPAHNSCKLLRG


179
FIG. 5
HAEICYFPGICWTER


180
FIG. 5
IVEMCYYPGICWISP


181
FIG. 5
KLCYFPGICWS


182
FIG. 5
KTCYFPGICWM


183
FIG. 5
KTEICYFPGICWMSG


184
FIG. 5
KVCYFPGICWM


185
FIG. 5
LAEMCYFPGICWMSA


186
FIG. 5
LVPERIVCYFESICYERSEL


187
FIG. 5
MELWCDSTLMAYDLCGDFNM


188
FIG. 5
MEPSRSVCYAEGICFDRGEQ


189
FIG. 5
NDEICYFPGVCWKSG


190
FIG. 5
QCFPGWVK


191
FIG. 5
QTELCYFPGICWNES


192
FIG. 5
QTRSFCADWPRHESCKPLRG


193
FIG. 5
RAAESSVCYWPGICFDRTEQ


194
FIG. 5
RAPERWVCYWEGICFDRYEQ


195
FIG. 5
RDTVCYFPGICWMAS


196
FIG. 5
REPASLVCYFEDICFVRAEA


197
FIG. 5
RGPDVCYWPSICFERSMP


198
FIG. 5
RMPASLPCYWETICYESSEQ


199
FIG. 5
RRTCDWPHNSCKLRG


200
FIG. 5
RTAESLVCYWPGICFAQSER


201
FIG. 5
SGAICYVPGICWTHA


202
FIG. 5
SREVCYYPGICWNGA


203
FIG. 6
SYAPCYFPGICWMGN


204
FIG. 5
TTEMCYFPGICWKTE


205
FIG. 5
VQEVCYFPGICWMQE


206
FIG. 5
VRDMCYFPGICWKSE


207
FIGS. 5, 7
NRQMEDICLPQWGCLWGDDF


208
FIGS. 5, 7
QRHPEDICLPRWGCLWGDDD


209
FIG. 5
DFDLCLPDWGCLWDD


210
FIG. 5
DICLERWGCLW


211
FIG. 5
DICLPAWGCLW


212
FIG. 5
DICLPDWGCLW


213
FIG. 5
DICLPEWGCLW


214
FIG. 5
DICLPVWGCLW


215
FIG. 5
DLCLPEWGCLW


216
FIG. 5
DLCLPKWGCLW


217
FIG. 5
DLCLPVWGCLW


218
FIG. 5
DSCGDLLRLGLECWA


219
FIG. 5
DTCADLVRLGLECWA


220
FIG. 5
DTCDDLVQLGLECWA


221
FIG. 5
DTCEDLVRLGLECWA


222
FIG. 5
DTCSDLVGLGLECWA


223
FIG. 5
EEDLCLPVWGCLWGA


224
FIG. 5
EEDVCLPVWGCLWEG


225
FIG. 5
EFDLCLPTWGCLWED


226
FIG. 5
ERQMEDFCLPQWGCLWGDGV


227
FIG. 5
ERQMVDFCLPKWGCLWGDGF


228
FIG. 5
EWDVCFPAWGCLWDQ


229
FIG. 5
EWDVCLPHWGCLWDG


230
FIG. 5
FEDFCLPNWGCLWGS


231
FIG. 5
FEDLCVVRWGCLWGD


232
FIG. 5
GRQVVDFCLPKWGCLWEEGL


233
FIG. 5
KMGRVDFCLPKWGCLWGDEL


234
FIG. 5
KSRMGDFCLPEWGCLWGDEL


235
FIG. 5
LRIFEDICLPKWGCLWGEGF


236
FIG. 5
MDDICLHHWGCLWDE


237
FIG. 5
MDDLCLPNWGCLWGD


238
FIG. 5
MFDLCLPKWGCLWGN


239
FIG. 5
MWDVCFPDWGCLWDV


240
FIG. 5
NTCADLVRLGLECWA


241
FIG. 5
NWDLCFPDWGCLWDD


242
FIG. 5
QGDFWDICLPRWGCLSGEGY


243
FIG. 5
QGGMGDFCLPQWGCLWGEDL


244
FIG. 5
QGYMVDFCLPRWGCLWGDAN


245
FIG. 5
QMHMMDICLPKWGCLWGDTS


246
FIG. 5
QMQMSDFCLPQWGCLWGDGY


247
FIG. 5
QRHMMDFCLPKWGCLWGDGY


248
FIG. 5
QRLMWEICLPLWGCLWGDGL


249
FIG. 5
QRPIMDFCLPKWGCLWEDGF


250
FIG. 5
QRQIMDFCLPHWGCLWGDGF


251
FIG. 5
QRQVVDFCLPQWGCLWGDGS


252
FIG. 5
QSQLEDFCLPKWGCLWGDGF


253
FIG. 5
QSYMEDICLPRWGCLSDDAS


254
FIG. 5
RWQTEDVCLPKWGCLFGDGV


255
FIG. 5
SEDFCLPVWGCLWED


256
FIG. 5
VWDLCLPQWGCLWDE


257
FIG. 5
WDDICFRDWGCLWGS


258
FIG. 5
WEDLCLPDWGCLWED


259
FIGS. 5, 7
HRLVEDICLPRWGCLWGNDF


260
FIGS. 5, 7
HSQMEDICLPRWGCLWGDEL


261
FIGS. 5, 7
LIFMEDVCLPQWGCLWEDGV


262
FIGS. 5, 7
LRLMDNICLPRWGCLWDDGF


263
FIGS. 5, 7
LWAMEDICLPKWGCLWEDDF


264
FIGS. 5, 7
QRDMGDICLPRWGCLWEDGV


265
FIGS. 5, 7
QRLMEDICLPRWGCLWGDRF


266
FIGS. 5, 7
QWHMEDICLPQWGCLWGDVL


267
FIGS. 5, 7
QWQMENVCLPKWGCLWEELD


268
FIGS. 5, 7
QWQVMDICLPRWGCLWADEY


269
FIGS. 5, 9
DICLPRWGCLW


270
FIG. 8
AAQVGDICLPRWGCLWSEYA


271
FIG. 8
AGWAADVCLPRWGCLWEEDV


272
FIG. 8
ALFEDVCLPVWGCLWGGE


273
FIG. 8
AQAMGDICLPRWGCLWEAEI


274
FIG. 8
ASDPGDVCLPRWGCLWGESF


275
FIG. 8
ASDRGDLCLPYWGCLWGPDG


276
FIG. 8
ASEWDVCLPTWGCLWMEG


277
FIG. 8
ASNWEDVCLPRWGCLWGERN


278
FIG. 8
ASTPRDICLPRWGCLWSEDA


279
FIG. 8
ASVVDDICLPVWGCLWGEDI


280
FIG. 8
ATMEDDICLPRWGCLWGAEE


281
FIG. 8
AYSADICLPRWGCLWMSE


282
FIG. 8
DEDFEDYCLPPWGCLWGSSM


283
FIG. 8
DGEEGDLCLPRWGCLWALEH


284
FIG. 8
EDWEDICLPQWGCLWEGM


285
FIG. 8
EDWTDLCLPAWGCLWDTE


286
FIG. 8
EEDSDICLPRWGCLWNTS


287
FIG. 8
EGEEVDICLPQWGCLWGYPV


288
FIG. 8
EGTWDDFCLPRWGCLWLGER


289
FIG. 8
EGYWDLCLPRWGCLWELE


290
FIG. 8
ELGEDLCLPRWGCLWGSE


291
FIG. 8
ERWEGDVCLPRWGCLWGESG


292
FIG. 8
ETWSDVCLPRWGCLWGAS


293
FIG. 8
EVGDLDLCLPRWGCLWGNDK


294
FIG. 8
FRDGEDFCLPQWGCLWADTS


295
FIG. 8
GDMVNDFCLPRWGCLWGSEN


296
FIG. 8
GDWMHDICLPKWGCLWDEKA


297
FIG. 8
GDYVDLCLPGWGCLWEDG


298
FIG. 8
GIEWGDTCLPKWGCLWRVEG


299
FIG. 8
GQQGEDVCLPVWGCLWDTSS


300
FIG. 8
GRMGTDLCLPRWGCLWGEVE


301
FIG. 8
GRYPMDLCLPRWGCLWEDSA


302
FIG. 8
GSAGDDLCLPRWGCLWERGA


303
FIG. 8
GVLDDICLPRWGCLWGPK


304
FIG. 8
HASDWDVCLPGWGCLWEEDD


305
FIG. 8
HEWERDICLPRWGCLWRDGD


306
FIG. 8
HMMDDVCLPGWGCLWASE


307
FIG. 8
IDYTDLCLPAWGCLWELE


308
FIG. 8
IEHEDLCLPRWGCLWAVD


309
FIG. 8
ISEWDLCLPRWGCLWDRS


310
FIG. 8
ISWADVCLPKWGCLWGKD


311
FIG. 8
ISWGDLCLPRWGCLWEGS


312
FIG. 8
KKVSGDICLPIWGCLWDNDY


313
FIG. 8
KLWDDICLPRWGCLWSPL


314
FIG. 8
LAWPDVCLPRWGCLWGGM


315
FIG. 8
LGVTHDTCLPRWGCLWDEVG


316
FIG. 8
LLESDDICLPRWGCLWHEDG


317
FIG. 8
LNESDICLPTWGCLWGVD


318
FIG. 8
LPEQDVCLPVWGCLWDAN


319
FIG. 8
LVWEEDFCLPKWGCLWGAED


320
FIG. 8
MAWGDVCLPRWGCLWAGG


321
FIG. 8
MQAESDFCLPHWGCLWDEGT


322
FIG. 8
MQGPLDICLPRWGCLWGGVD


323
FIG. 8
NEEWDVCLPRWGCLWGGV


324
FIG. 8
NVGWNDICLPRWGCLWAQES


325
FIG. 8
QELQDFCLPRWGCLWGVG


326
FIG. 8
QGVEWDVCLPQWGCLWTREV


327
FIG. 8
QMPLEDICLPRWGCLWEGRE


328
FIG. 8
QREWDVCLPRWGCLWSDV


329
FIG. 8
QRFWDTCLPRWGCLWGGD


330
FIG. 8
REEWGDLCLPTWGCLWETKK


331
FIG. 8
RLDAWDICLPQWGCLWEEPS


332
FIG. 8
RVFTDVCLPRWGCLWDLG


333
FIG. 8
RVWTEDVCLPRWGCLWSEGN


334
FIG. 8
SEAPGDYCLPRWGCLWAQEK


335
FIG. 8
SGWDDVCLPVWGCLWGPS


336
FIG. 8
SIREYDVCLPKWGCLWEPSA


337
FIG. 8
SPTEWDMCLPKWGCLWGDAL


338
FIG. 8
SSASDYCLPRWGCLWGDL


339
FIG. 8
SSGLEDICLPNWGCLWADGS


340
FIG. 8
SVGWGDICLPVWGCLWGEGG


341
FIG. 8
SWQGDICLPRWGCLWGVD


342
FIG. 8
SYETDVCLPYWGCLWEDA


343
FIG. 8
SYWGDVCLPRWGCLWSEA


344
FIG. 8
TAMDEDVCLPRWGCLWGSGS


345
FIG. 8
TEENWDLCLPRWGCLWGDDW


346
FIG. 8
TEIGQDFCLPRWGCLWVPGT


347
FIG. 8
TLEWDMCLPRWGCLWTEQ


348
FIG. 8
TLGWPDFCLPKWGCLWRESD


349
FIG. 8
TLSNQDICLPGWGCLWGGIN


350
FIG. 8
TSGSDDICLPVWGCLWGEDS


351
FIG. 8
TSTGGDLCLPRWGCLWDSSE


352
FIG. 8
TWP-GDLCLPRWGCLWEAES


353
FIG. 8
VGEFDICLPRWGCLWDAE


354
FIG. 8
VSEMDDICLPLWGCLWADAP


355
FIG. 8
VSEWEDICLPSWGCLWETQD


356
FIG. 8
VTSWDVCLPRWGCLWEED


357
FIG. 8
VVGDGDFCLPKWGCLWDQAR


358
FIG. 8
VVWDDDVCLPRWGCLWEEYG


359
FIG. 8
WDHELDFCLPVWGCLWAEDV


360
FIG. 8
WLWEDLCLPKWGCLWEED


361
FIG. 8
WSDSDDVCLPRWGCLWGNVA


362
FIG. 8
WTESEDICLPGWGCLWGPEV


363
FIG. 8
WVEEGDICLPRWGCLWESVE


364
FIG. 8
WVPFEDVCLPRWGCLWSSYQ


365
FIG. 8
XXXXDXCLPXWGCLWXXX


366
42, FIG. 8
XXXXXDXCLPXWGCLWXXXX


367
FIG.8
AFWSDICLPRWGCLWEED


368
FIG. 8
AGLDEDICLPRWGCLWGKEA


369
FIG. 8
AGMMGDICLPRWGCLWQGEP


370
FIG. 8
APGDWDFCLPKWGCLWDDDA


371
FIG. 8
AQLFDDICLPRWGCLWSDGY


372
FIG. 8
ARTMGDICLPRWGCLWGASD


373
FIG. 8
AVSWADICLPRWGCLWERAD


374
FIG. 8
AWLDEDICLPKWGCLWNTGV


375
FIG. 8
AWQDFDVCLPRWGCLWEPES


376
FIG. 8
DTTWGDICLPRWGCLWSEEA


377
FIG. 8
DWGRDICLPRWGCLWDEE


378
FIG. 8
EAWGDICLPRWGCLWELE


379
FIG. 8
EGFLGDICLPRWGCLWGHQA


380
FIG. 8
EQWLHDICLPKWGCLWDDTD


381
FIG. 8
ETGWPDICLPRWGCLWEEGE


382
FIG. 8
FELGEDICLPRWGCLWEEHN


383
FIG. 8
FITQDICLPRWGCLWGEN


384
FIG. 8
FLWRDICLPRWGCLWSEG


385
FIG. 8
FSLDEDICLPKWGCLWGAEK


386
FIG. 8
FVHEDICLPRWGCLWGEG


387
FIG. 8
GASLGDICLPRWGCLWGPED


388
FIG. 8
GDLGDDICLPRWGCLWDEYP


389
FIG. 8
GEGWSDICLPRWGCLWAEDE


390
FIG. 8
GEWWEDICLPRWGCLWGSSS


391
FIG. 8
GLGDDICLPRWGCLWGRD


392
FIG. 8
GLMGEDICLPRWGCLWKGDI


393
FIG. 8
GMFDDICLPKWGCLWGLG


394
FIG. 8
GPGWDICLPRWGCLWGEE


395
FIG. 8
GPWYDICLPRWGCLWDGV


396
FIG. 8
GSLESDICLPRWGCLWGTDE


397
FIG. 8
GWDDDICLPRWGCLWGDG


398
FIG. 8
GWHDRDICLPRWGCLWEQND


399
FIG. 8
GWLEEDICLPKWGCLWGADN


400
FIG. 8
HEQWDDICLPRWGCLWGGSY


401
FIG. 8
LEYEDICLPKWGCLWGGE


402
FIG. 8
LILSDICLPRWGCLWDDT


403
FIG. 8
LKLEDICLPRWGCLWGES


404
FIG. 8
LLDEDICLPRWGCLWGVR


405
FIG. 8
LLGGHDICLPRWGCLWGGDV


406
FIG. 8
LLTRDICLPKWGCLWGSD


407
FIG. 8
LMSPDICLPKWGCLWEGD


408
FIG. 8
LRWSDICLPRWGCLWEET


409
FIG. 8
LVLGDICLPRWGCLWESD


410
FIG. 8
LYLRDICLPKWGCLWEAD


411
FIG. 8
MLSRDICLPRWGCLWEEE


412
FIG. 8
MPWTDICLPRWGCLWSES


413
FIG. 8
MRWSSDICLPKWGCLWGDEE


414
FIG. 8
NWYDDICLPRWGCLWDVE


415
FIG. 8
QDWEDICLPRWGCLWGD


416
FIG. 8
QFEWDDICLPRWGCLWEVEV


417
FIG. 8
QGWWHDICLPRWGCLWEEGE


418
FIG. 8
QRVDDDICLPRWGCLWGENS


419
FIG. 8
QSWPDICLPKWGCLWGEG


420
FIG. 8
REGWPDICLPRWGCLWSETG


421
FIG. 8
RELWGDICLPRWGCLWEHAT


422
FIG. 8
RLELMDICLPRWGCLWDPQD


423
FIG. 8
RLGSDICLPRWGCLWDYQ


424
FIG. 8
RLGSDICLPRWGCLWGAG


425
FIG. 8
SGVLGDICLPRWGCLWEEAG


426
FIG. 8
SLGLTDLCLPRWGCLWEEEQ


427
FIG. 8
SPWMDICLPRWGCLWESG


428
FIG. 8
SSLEQDICLPRWGCLWGQDA


429
FIG. 8
STFTDICLPRWGCLWELE


430
FIG. 8
SVGWGDICLPKWGCLWAESD


431
FIG. 8
SVLSDDICLPRWGCLWWDFS


432
FIG. 8
SVLSDICLPRWGCLWEES


433
FIG. 8
TLLQDICLPRWGCLWESD


434
FIG. 8
TLMSNDICLPRWGCLWDEPK


435
FIG. 8
TLVLDDICLPRWGCLWDMTD


436
FIG. 8
TSLADDICLPRWGCLWSEDG


437
FIG. 8
TSLLDDICLPRWGCLWYEEG


438
FIG. 8
TWFSDICLPRWGCLWEPG


439
FIG. 8
TWQGEDICLPRWGCLWDTEV


440
FIG. 8
VEMWHDICLPRWGCLWDSNA


441
FIG. 8
VGVFDDICLPRWGCLWEQPV


442
FIG. 8
VHQADICLPRWGCLWGDT


443
FIG. 8
VLLGDICLPLWGCLWGED


444
FIG. 8
VNWGDICLPRWGCLWGES


445
FIG. 8
VPAMGDICLPRWGCLWEARN


446
FIG. 8
VRLMDICLPRWGCLWGEE


447
FIG. 8
VRWEDICLPRWGCLWGEE


448
FIG. 8
VSLGDDICLPKWGCLWEPEA


449
FIG. 8
VVWSDICLPRWGCLWDKE


450
FIG. 8
VWTDRDICLPRWGCLWDTEN


451
FIG. 8
VWYKDICLPRWGCLWEAE


452
FIG. 8
WDLASDICLPRWGCLWEEEA


453
FIG. 8
WDVADICLPRWGCLWAED


454
FIG. 8
WDYGDICLPRWGCLWEEG


455
FIG. 8
WEVQDICLPRWGCLWGDD


456
FIG. 8
WHMGDICLPRWGCLWSEV


457
FIG. 8
WKDFDICLPRWGCLWDDH


458
FIG. 8
WLSDDICLPRWGCLWDDL


459
FIG. 8
WLSEDICLPQWGCLWEES


460
FIG. 8
WLSEDICLPRWGCLWAAD


461
FIG. 8
WRWNEDICLPRWGCLWEEEA


462
FIG. 8
YIWRDICLPRWGCLWEGE


463
FIG. 8
YRDYDICLPRWGCLWDER


464
FIG. 8
AGEWDICLPRWGCLWDVE


465
FIG. 8
EIRWDFCLPRWGCLWDED


466
FIG. 8
ESLGDICLPRWGCLWGSG


467
FIG. 8
EVREWDICLPRWGCLWENWR


468
FIG. 8
EYWGDICLPRWGCLWDWQ


469
FIG. 8
FGQEWDICLPRWGCLWGNEQ


470
FIG. 8
IWQLEDICLPRWGCLWEDGL


471
FIG. 8
KMWSDICLPRWGCLWEEE


472
FIG. 8
MGTKDICLPRWGCLWAEA


473
FIG. 8
MHEWDICLPRWGCLWESS


474
FIG. 8
NTPTYDICLPRWGCLWGDVP


475
FIG. 8
NWAENDICLPRWGCLWGDEN


476
FIG. 8
QPVWSDICLPRWGCLWGEDH


477
FIG. 8
RGLHDACLPWWGCLWAGS


478
FIG. 8
RLFGDICLPRWGCLWQGE


479
FIG. 8
SAREWDICLPTWGCLWEKDI


480
FIG. 8
SGEWDICLPRWGCLWGEG


481
FIG. 8
SMFFDHCLPMWGCLWAEQ


483
FIG. 8
TLFQDICLPRWGCLWEES


484
FIG. 8
VGEWDICLPNWGCLWERE


485
FIG. 8
WFPKDRCLPVWGCLWERH


486
FIG. 8
WGMARDWCLPMWGCLWRGGG


487
FIG. 8
WHLTDDICLPRWGCLWGDEQ


488
FIG. 8
WWMADRCLPLWGCLWRGD


489
FIG. 8
WWVRDLCLPTWGCLWSGK


490
FIG. 8
YFDGDICLPRWGCLWGSD


491
FIG. 9
EDICLPRWGCLWEDD


492
FIG. 8
SWYGGDICLP-WGCLWSEES





*X is an amino acid


For SEQ ID NO: 10: X1 is Ala, Ser, or Thr; X2 is Trp or Tyr; X3 is His or Trp; X4 is Leu or Met


For SEQ ID NO: 13: X5 is Ala, Ser, or Thr; X6 is Trp or Tyr; X7 is His or Trp; X8 is Leu or Met; and X1-4 and 9-13 are each an amino acid.


For SEQ ID NO: 14: X4 is Ser, Arg, or Asp; X5 is Ala, Ser, or Thr; X6 is Trp or Tyr; X7 is His or Trp; X8 is Leu or Met; X9 is Glu, Ser, Thr, or Val; and X1-3 and 9-13 are each an amino acid.


For SEQ ID NO: 188: X1 is Ile, Phe, Tyr, or Val.

## Claims

1. A hybrid molecule comprising a peptide ligand domain, which comprises the amino acid sequence (Xaa)x-Xaa-Xaa1-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-(Xaa)z, wherein Xaa-Xaa1-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa is Asp-Xaa1-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp (SEQ ID NO:116), that binds serum albumin, wherein Xaa or Xaa1 is any amino acid, and x and z are 0 to 5 amino acids.

2. The hybrid molecule of claim 1 wherein x is 4 and z is 3.

3. The hybrid molecule of claim 1 wherein x is 5 and z is 4.

4. The hybrid molecule of claim 1 wherein Xaa1 is Ile, Phe, Tyr or Val.

5. The hybrid molecule of claim 1 wherein the affinity of the peptide ligand domain for serum albumin is characterized by an equilibrium dissociation constant (Kd) of less than about 1 μM.

6. The hybrid molecule of claim 1, wherein the affinity of the peptide ligand domain for serum albumin is characterized by an equilibrium dissociation constant (Kd) of less than about 500 nM.

7. The hybrid molecule of claim 1 wherein the affinity of the peptide ligand domain for serum albumin is characterized by an equilibrium dissociation constant (Kd) of less than about 50 nM.

8. The hybrid molecule of claim 1, wherein the affinity of the peptide ligand domain for serum albumin is characterized by an equilibrium dissociation constant (Kd) that is between about 1 pM and about 1 nM.

9. The hybrid molecule of claim 1 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).

10. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa is Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp (SEQ ID NO: 121).

11. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa is Gln-Arg-Leu-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Qty-Cys-Leu-Trp-Glu-Asp-Asp-Phe (SEQ ID NO: 122).

12. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa is Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-ASp-Scr-Val (SEQ ID NO: 123).

13. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa is Gln-Gly-Leu-Ile-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-ASp-Ser-Val-Lys (SEQ ID NO: 124).

14. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa is Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp (SEQ ID NO: 125).

15. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-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).

16. The hybrid molecule of claim 1 wherein Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa is Met-Gly-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-TrP-Glu-Asp-Asp (SEQ ID NO: 127).

17. The hybrid molecule of claim 1, wherein the amino acid sequence is cyclized by the presence of disulfide-bonded Cys residues.

18. The hybrid molecule of claim 1, wherein the amino acid sequence is less than about 50 amino acid residues.

19. The hybrid molecule of claim 1, wherein the amino acid sequence is less than about 40 amino acid residues.

20. The hybrid molecule of claim 1, wherein the amino acid sequence is about 20 amino acid residues.

21. The hybrid molecule of claim 1, further comprising a bioactive compound.