Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention provides peptides and compounds that bind the interleukin 5 receptors (IL-5R), methods for assaying interleukin 5 (IL-5), and methods for inhibiting the binding of IL-5 to the IL-5R.
• IL-5R interleukin 5 receptors
• the invention has application in the fields of biochemistry and medicinal chemistry and particularly provides IL-5 antagonists for use in the treatment of human disease.
• Interleukin-5 is a lymphokine secreted by T cells and mast cells having biological activities on B cells and eosinophils.
• IL-5 is a selective signal for the proliferation and differentiation of the eosinophilic lineage.
• IL-5 function shows analogies with colony- stimulating factors for other myeloid lineages.
• human (h) IL-5 is very potent in the activation of human eosinophils. See Lopez et al. (1988) T. Exp. Med i 16Z:219-224 and Saito et al. (1988) Proc. Natl. Acad. Sci. USA 85:2288-2292.
• IL-5 mediates its activity through a cell membrane receptor- complex. This complex has been characterized physicochemically in both the murine and human system.
• Mouse pre-B cell lines depending on IL-5 for their growth have been developed from bone marrow and are used for IL-5 receptor analysis. See Rolink et al. (1989) T. Exp. Med. 169:1693-1701.
• the human IL-5 receptor can be studied on a subclone of the promyelocytic cell line HL60 induced towards eosinophil differentiation. See Plaetinck et al. (1990) T. Exp. Med. 172:683-691. Eosinophilic differentiation is initiated using sodium butyrate.
• a soluble human IL-5R-0C chain can be used as an IL-5 antagonist in chronic asthma or other disease states with demonstrated eosinophilia.
• Eosinophils are white blood cells of the granulocytic lineage. Their normal function appears to be combating parasitic infections, particularly helminthis infections. However, their accumulation in tissues, a condition referred to as eosinophilia, is also associated with several disease states, most notably asthma. It is believed that the damage to the epithelial lining of the bronchial passages in severe asthmatic attacks is largely caused by the compounds released by degranulating eosinophils.
• Fab fragments The use of antibody fragments, e.g., Fab fragments, was also reported.
• glucocorticoid steroids are the most effective drugs for treating the acute effects of allergic diseases, such as asthma.
• the long term use of steroids is associated with certain side effects.
• the steroids apparently do not affect the production or accumulation of granulocytic cells, such as eosinophils, in the afflicted tissues.
• the availability of alternative or complementary approaches to the treatment of disorders associated with eosinophilia would have important clinical utility.
• IL-5R IL-5R
• soluble IL- 5R derivative IL-5R
• the availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems. These systems include the "peptides on plasmids" system described in U.S. Patent No. 5,270,170, the "peptides on phage” system described in U.S. patent application Serial No. 718,577, filed June 20, 1991, and in Cwirla et al, (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382.
• This invention is directed, in part, to the novel and unexpected discovery that defined low molecular weight peptides and peptide mimetics have strong binding properties to the IL-5 R. Accordingly, such peptides and peptide mimetics are useful for therapeutic purposes in treating conditions mediated by IL-5 or involving improper production of or response to IL-5 and can be used to inhibit production and accumulation of eosinophils. These compounds will find particular use in the treatment of asthma.
• the present invention also provides a method for treating a patient having a disorder that is susceptible to treatment with a IL-5 inhibitor, wherein the patient receives, or is administered, a therapeutically effective dose or amount of a compound of the present invention.
• Peptides and peptide mimetics suitable for therapeutic and/or diagnostic purposes have an IC50 of about 2 mM or less, as determined by the binding aff nity assay set forth in Example 2 below wherein a lower IC50 correlates to a stronger binding affinity to IL-5R.
• the peptides and peptidomimetics preferably have an IC50 of no more than about 100 ⁇ m.
• the molecular weight of the peptide or peptide mimetic is from about 250 to about 5000 daltons.
• the peptides and peptide mimetics When used for diagnostic purposes, the peptides and peptide mimetics preferably are labeled with a detectable label and, accordingly, the peptides and peptide mimetics without such a label serve as intermediates in the preparation of labeled peptides and peptide mimetics.
• Peptides meeting the defined criteria for molecular weight and binding affinity for IL-5R comprise 12 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids.
• Peptide mimetics include peptides having one or more of the following modifications: peptides wherein one or more of the peptidyl [-C(0)NR-] linkages (bonds) have been replaced by a non-peptidyl linkage such as a -CH 2 - carbamate linkage [-CH 2 -OC(0)NR-]; a phosphonate linkage; a -CH 2 - sulfonamide [-CH 2 -S(0) 2 NR-] linkage; a urea [-NHC(0)NH-] linkage; a -CH 2 - secondary amine linkage; or an alky la ted peptidyl linkage [-C(0)NR - where
• R 6 is lower alkyl]; peptides wherein the N-terminus is derivatized to a -NRR 1 group; to a -NRC(0)R group; to a -NRC(0)OR group; to a -NRS(0) 2 R group; to a
• R and R are hydrogen or lower alkyl with the proviso that R and R 1 are not both hydrogen; to a succinimide group; to a benzyloxycarbonyl-NH- (CBZ-NH-) group; or to a benzyloxycarbonyl-NH- group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo; or peptides wherein the C terminus is derivatized to -C(0)R where
• R is selected from the group consisting of lower alkoxy, and -NR R where R and R are independently selected from the group consisting of hydrogen and lower alkyl.
• preferred preferred peptides and peptide mimetics comprise a compound having:
• a molecular weight of less than about 5000 daltons and (2) a binding affinity to IL5-R as expressed by an IC50 of no more than about 100 ⁇ m, wherein from zero to all of the -C(0)NH- linkages of the peptide have been replaced by a linkage selected from the group consisting of a -CH 2 OC(0)NR- linkage; a phosphonate linkage; a -CH 2 S(0) 2 NR- linkage; a - CH 2 NR- linkage; and a -C(0)NR 6 - linkage; and a -NHC(0)NH- linkage where R is hydrogen or lower alkyl and R 6 is lower alkyl, further wherein the N-terminus of said peptide or peptide mimetic is selected from the group consisting of a -NRR group; a -NRC(0)R group; a -NRC(0)OR group; a -NRS(0) 2 R group; a -NHC(0)NHR group;
• the invention is directed to a labeled peptide or peptide mimetic comprising a peptide or peptide mimetic described as above having covalently attached thereto a label capable of detection. More preferably, these peptides are twelve to forty or more amino acid residues in length, preferably twelve to twenty-five amino acid residues in length, and comprise a core sequence of amino acids: C X1 R X2 X7 X8 3 X4 X5 W X6 C where Xi is D, E, I, S, T, W, or Y; X2 is D, F, G, I, L, S, V, W, or Y; X3 is D, E, G, L, N, S, T, or W; X4 is H or R; X5 is A, K, R, S, T, V, or W; X6 is D, E, F, L, M, P, Q, or V; X7 is I or V; and X is
• the core peptide comprises a sequence of amino acids:
• the core peptide comprises either a sequence of amino acids:
• X9 XlO Xll C Xi R X 2 X7 X ⁇ X3 X4 X5 W X 6 C
• X9 is D, E, F, G, L, Q, or V
• X10 is D, E, G, H, K, N, or V
• Xi 1 is D, E, G, S, V, or W (SEQ ID NO:4)
• CX1RX2X7X8X3X4X5WX6CX12X13X14 where X12 is D, E, G, M, S, or T; X13 is D, E, I, L, K, M, Q, T, or V; and X1 is D,
• E, F, G, L, Q, T, V, or W (SEQ ID NO:5). More preferably, X9 is D, E, F, G, or V; XlO is D, E, G, or V; and X11 is D, G, or V (SEQ ID NO:6); or X12 is D or G; X13 is D, E, L, M, or V; and X14 is D, E, or G (SEQ ID NO:7). More preferably, the core peptide comprises a sequence of amino acids:
• X9 is D, E, F, G, L, Q, or V
• Xi 0 is D, E, G, H, K, N, or V
• Xn is D, E, G, S, V, or W
• X12 is D, E, G, M, S, or T
• Xi 3 is D, E, I, L, K, M, Q, T, or V
• X14 is D, E, F, G, L, Q, T, V, or W (SEQ ID NO:8).
• X9 is D, E, F, G, or V
• X10 is D, E, G, or V
• X ⁇ is D, G, or V
• X12 is D or G
• X13 is D, E, L, M, or V
• X14 is D, E, or G (SEQ ID NO:9).
• Particularly preferred peptides include: GEVCTRDVANH R W M C G V D (SEQ ID NO:10), GEDCIRIVRTHSWDCGVD (SEQ ID NO:ll), V V D C W R S V A T H T W F C G E E (SEQ ID NO:12), and F D G C T R I V A T R S W D C D L D (SEQ ID NO:13).
• compositions comprising one or more of the compounds described herein and a physiologically acceptable carrier.
• These pharmaceutical compositions can be in a variety of forms including oral dosage forms, as well as inhalable powders and solutions and injectable and infusible solutions.
• Figures 1A-C illustrates the construction of peptides-on-plasmids libraries in vector pJS142.
• Figure 1A shows a restriction map and position of the genes.
• the library plasmid includes the rrnB transcriptional terminator, the bla gene to permit selection on ampicillin, the M13 phage intragenic region (M23 JG) to permit rescue of single-stranded DNA, a plasmid replication origin (on), two lacO s ssequence, and the araC gene to permit positive and negative regulation of the araB promoter driving expression of the lac fusion gene.
• Figure IB shows the sequence of the cloning region at the 3' end of the lac I gene, including the Sfil and EagI sites used during library construction.
• Figure IC shows the ligation of annealed library oligonucleotides, ON-829 and ON-830, to Sfil sites of pJS142 to produce a library. Singles spaces in the sequence indicate sites of lig
• Figures 2A-B illustrate cloning into the pELM3 and pELM15 MBP vectors.
• Figure 2A shows the sequence at the 3' end of the malE fusion gene, including the MBP coding sequence, the poly asparagine linker, the factor Xa protease cleavagge site, and the available cloning sites. The remaining portions of the vectors are derived from pMALc2 (pELM3) and pMALp2 (pELM15), available from New England Biolabs.
• Figure 2B shows the sequence of the vectors after transfer of the BspEII-Scal library fragment into Agel-Scal digested pELM3/pELM15. The transferred sequence includes the sequence encoding the GGG peptide linker from the pJS142 library.
• Figure 3A depicts a restriction map and position of the genes for the construction of headpiece dimer libraries in vector pCMG14.
• the library plasmid includes: the rrnB transcriptional terminator, the bla gene to permit selection on ampicillin, the M13 phage intragenic region (M13 IG) to permit rescue of single-stranded DNA, a plasmid replication origin (on), one lacO s sequence, and the araC gene to permit positive and negative regulation of the araB promoter driving expression of the headpiece dimer fusion gene.
• Figure 3B depicts the sequence of the cloning region at the 3' end of the headpiece dimer gene, including the Sfil and EagI sites used during library construction.
• Figure 3C shows the ligation of annealed ON-1679, ON-829, and ON-830 to Sfil sites of pCMG14 to produce a library. Single spaces in the sequence indicate sites of ligation.
• “Pharmaceutically acceptable salts” refer to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art.
• the term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid.
• Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, and the like.
• “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, menthanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
• organic acids such as acetic acid, propionic acid, glycolic acid,
• “Pharmaceutically acceptable ester” refers to those esters which retain, upon hydrolysis of the ester bond, the biological effectiveness and properties of the carboxylic acid or alcohol and are not biologically or otherwise undesirable.
• esters are typically formed from the corresponding carboxylic acid and an alcohol.
• ester formation can be accomplished via conventional synthetic techniques. (See, e.g., March Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York (1985) p. 1157 and references cited therein, and Mark et al.
• the alcohol component of the ester will generally comprise (i) a C2-C 2 aliphatic alcohol that can or can not contain one or more double bonds and can or can not contain branched carbon chains or (ii) a C7-C12 aromatic or heteroaromatic alcohols.
• compositions which are both esters as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.
• “Pharmaceutically acceptable amide” refers to those amides which retain, upon hydrolysis of the amide bond, the biological effectiveness and properties of the carboxylic acid or amine and are not biologically or otherwise undesirable.
• pharmaceutically acceptable amides as prodrugs, see Bundgaard, H., ed., (1985) Design of Prodrugs. Elsevier Science Publishers, Amsterdam. These amides are typically formed from the corresponding carboxylic acid and an amine. Generally, amide formation can be accomplished via conventional synthetic techniques. [See, e.g., March
• compositions which are both amides as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.
• “Pharmaceutically or therapeutically acceptable carrier” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
• “Stereoisomer” refers to a chemical compound having the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical moieties are at different orientations in space and, therefore, when pure, has the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so slight that it is undetectable with present instrumentation.
• the compounds of the instant invention may have one or more asymmetrical carbon atoms and therefore include various stereoisomers. All stereoisomers are included within the scope of the invention.
• “Therapeutically- or pharmaceutically-effective amount” as applied to the compositions of the instant invention refers to the amount of composition sufficient to induce a desired biological result. That result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present invention, the result will typically involve a decrease in the immunological and/or inflammatory responses to infection or tissue injury.
• Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is lie or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y;
• Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is
• peptide mimetics In addition to peptides consisting only of naturally-occurring amino acids, pep idomimetics or peptide analogs are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or
• peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad- spectrum of biological activities), reduced antigenicity, and others.
• Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure- activity data and /or molecular modeling.
• a spacer e.g., an amide group
• non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) (e.g., immunoglobulin superfamily molecules) to which the peptidomimetic binds to produce the therapeutic effect.
• peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
• peptidomimetics of receptor-binding peptides bind to the receptor with high affinity and possess detectable biological activity (i.e., are agonistic or antagonistic to one or more receptor-mediated phenotypic changes).
• Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used to generate more stable peptides.
• constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
• the present invention provides compounds that bind to the IL-
• compositions comprising an effective IL-5R binding, IL-5 blocking compound, and more particularly a compound, that is useful for treating disorders associated with the overexpression of IL-5 or with the production and accumulation of eosinophils.
• random peptide diversity generating systems include the "peptides on plasmids" system described in U.S. Patent
• random peptides were generally designed to have a defined number of amino acid residues in length
• the codon motif (NNK)x was used to specify any one of the 32 possible codons resulting from the NNK motif: 1 for each of 12 amino acids, 2 for each of 5 amino acids, 3 for each of 3 amino acids, and only one of the three stop codons.
• NNK motif encodes all of the amino acids, encodes only one stop codon, and reduces codon bias.
• the random peptides were presented either on the surface of a phage particle, as part of a fusion protein comprising either the pill or the pVIII coat protein of a phage fd derivative (peptides on phage) or as a fusion protein with the Lad peptide fusion protein bound to a plasmid (peptides on plasmids).
• the phage or plasmids including the DNA encoding the peptides, were identified and isolated by an affinity enrichment process using immobilized IL-5R.
• the affinity enrichment process sometimes called
• panning involves multiple rounds of incubating the phage or plasmids with the immobilized receptor, collecting the phage or plasmids that bind to the receptor (along with the accompanying DNA), and producing more of the phage or plasmids (along with the accompanying Lacl-peptide fusion protein) collected.
• the extracellular domain (ECD) of the ⁇ -chain of the IL-5R, the ECD of the ⁇ -chain of the IL-5R, the ⁇ -chain of the IL-5R, ⁇ -chain of the IL-5R, or the ⁇ / ⁇ -heterodimer can be used during panning.
• the phage or plasmids and accompanying peptides were examined by ELISA to determine if the peptides bind specifically to IL-5 R.
• This assay was carried out similarly to the procedures used in the affinity enrichment process, except that after removing unbound phage, the wells were typically treated with rabbit anti- phage antibody, then with alkaline phosphatase (AP)-conjugated goat anti- rabbit antibody. The amount of alkaline phosphatase in each well was determined by standard methods. A similar ELISA procedure for use in the pepides on plasmids system is described in detail below.
• phage pools found to bind to IL-5R were screened in a colony lift probing format using bivalent IL-5R probes. This probe was constructed by pre- incubating radiolabeled Ab 179 with the receptor. The complex was subsequently purified over an immobilized Ab 179 affinity column to separate the complex from free receptor. Peptides found to bind specifically to the receptor were then tested using an ELISA format to see if the binding was competitive with IL-5.
• IL-5 The competition assay was carried out in similar fashion to the ELISA , except that the peptide was added to the wells before the IL-5. Peptides which compete with IL-5 for binding to the receptor are preferred compounds of the invention.
• the immobilized ⁇ chain, ⁇ chain, and heterodimer, as well as the extracellular domains of the single chains of the IL-5 receptors were produced in recombinant host cells.
• the DNA encoding IL-5R was obtained by PCR of cDNA from TF-1 cells using primers obtained from the published receptor sequences. See Murata (1992) T. Exp. Med. 175:341-351 and Hayashida
• IL-5R IL-5R
• a signal peptide for protein secretion and for glycophospholipid membrane anchor attachment This form of anchor attachment is called "PIG-tailing". See Caras and Wendell (1989) Science 243:1196-1198 and Lin et al. (1990) Science 249:677- 679.
• the receptor e.g., transformed CHO cells selected for high level expression of receptor with a cell sorter
• the cleaved receptor still comprises a carboxy terminal sequence of amino acids, called the "HPAP tail", from the signal protein for membrane attachment and can be immobilized without further purification.
• the recombinant receptor protein can be immobilized by coating the wells of microtiter plates with an anti-HP AP tail antibody (Ab 179), blocking non- specific binding with bovine serum albumin (BSA) in PBS, and then binding cleaved recombinant receptor to the antibody.
• Ab 179 anti-HP AP tail antibody
• BSA bovine serum albumin
• a monovalent receptor probe frequently is used.
• This probe can be produced using protein kinase A to phosphorylate a kemptide sequence fused to the C-terminus of the soluble receptor.
• the "engineered" form of the IL-5 receptor ⁇ and ⁇ chains are then expressed in host cells, typically CHO cells. Following PI-PLC harvest of the receptors, the receptor is labeled to high specific activity with 33p or 32p f or use as a monovalent probe to identify high affinity ligands using colony lifts.
• Preferred screening methods to facilitate identification of peptides which bind IL-5R involve first identifying lead peptides which bind the receptor and then making other peptides which resemble the lead peptides. Specifically, using a pill or pVIII-based peptides on phage system, a random library can be screened to discover a phage that presents a peptide that binds to IL-5R. The phage DNAs are sequenced to determine the sequences of the peptides displayed on the surface of the phages.
• Examples of primary libraries which were produced and panned against immobilized IL-5R ⁇ ECD include: GGC(X) ⁇ oC(G4S)3 (SEQ ID NO:14); GGC(X)l2C(G4S)3 (SEQ ID NO:15); XXXC(X)5CXXX(G4S)3 (SEQ ID NO:16); XXXC(X)6CXXX(G4S)3 (SEQ ID NO:17); XXXC(X)7CXXX(G4S)3 (SEQ ID NO:18); XXXC(X) ⁇ CXXX(G4S)3 (SEQ ID NO:19); XXC(X)9CXXX(G4S)3 (SEQ ID NO:20); and XXC(X)ioCXXX(G4S)3 (SEQ ID NO:21) wherein X represents random residue encoded by NNK; and (G4S)3
• SEQ ID NO:22 is a peptide linker. ⁇ -Chain specific clones and clones capable of specific binding to the ⁇ -chain of IL-5R were identified. The sequences of these peptides serve as the basis for the construction of other peptide libraries designed to contain a high frequency of derivatives of the initially identified peptides. These libraries can be synthesized so as to favor the production of peptides that differ from the binding peptide in only a few residues.
• This approach involves the synthesis of an oligonucleotide with the binding peptide coding sequence, except that rather than using pure preparations of each of the four nucleoside triphosphates in the synthesis, one uses mixtures of the four nucleoside triphosphates (i.e., 55% of the "correct" nucleotide, and 15% each of the other three nucleotides is one preferred mixture for this purpose and 70% of the "correct" nucleotide and 10% of each of the other three nucleotides is another preferred mixture for this purpose) so as to generate derivatives of the binding peptide coding sequence.
• mutagenesis strategies were used to derivatize the lead peptides by making "mutagenesis on a theme" libraries, which included mutagenesis of the original coding sequences at 70:10:10:10 frequencies with 3 NNK codons on each terminus (panned for 3 rounds against the immobilized extracellular domain (ECD) of the IL-5 receptor ⁇ chain (IL-5R ⁇ ) in the presence of known binding peptide; alternatively, known binding peptide was included during the wash step); fixed-sliding libraries, such as X X X X X X X W R V P C G G (SEQ ID NO:23); and G G C W S G E X X X X X X X (SEQ ID NO:24) (panned for 3 rounds against the immobilized extracellular domain (ECD) of the IL-5 receptor ⁇ chain (IL-5R ⁇ ) in the presence of known binding peptide; alternatively, known binding peptide was included during the wash step;
• the various phage libraries can be rescreened against IL-5R ⁇ in the presence of known binding peptide or non-IL-5 competitive peptide during the final wash or at room temperature or 4°C to identify peptides of higher affinity. Pools of phage are then probed by colony lift using P or 33 P labeled IL-5R ⁇ (as discussed above).
• MBP maltose binding protein
• This MBP ELISA identified one of the previously identified clones as well as a new clone, which was also competed by IL-5 for receptor binding.
• a Lad ELISA was then performed in which the receptor was preincubated with 250 ⁇ M of a known IL-5 binding peptide prior to the addition of the Lacl-peptide fusions. Binding of all but one of the clones tested was blocked by the peptide.
• Three of the libraries were 70:10:10:10 codon mutagenesis libraries and the fourth library was based on a common sequence motif seen in a number of plasmid derived clones. All of the libraries contained > 10 ⁇ members. These libraries were panned against the IL-5R ⁇ ECD for four rounds, optionally with the addition of IL-5 during the washes. Pools of clones showing binding to the receptor were transferred from the headpiece dimer expression plasmid into a maltose binding protein (MBP) fusion expression system in order to probe for high affinity clones using 32p labeled monovalent receptor probe in a colony lift assay.
• MBP maltose binding protein
• IC50 values for some additional representative peptides are given in the table below.
• a variety of methods can be used to evaluate IC50 values. For example, an [125-1] IL-5 binding assay was used to determine whether the peptides inhibit the binding of IL-5 to the extracellular domain of the IL-5 receptor ⁇ -chain. Alternatively, for some peptides, a microphysiometer assay was used to determine whether the peptide blocked the response of TF-1 cells to IL-5 (5 ng/ml).
• the IC50 value were determined using the free peptide.
• the IC50 value can be determined using the free peptide, which optionally can be C-terminally amidated, or can be prepared as an ester or other carboxy amide.
• the IC50 values were typically evaluated on both the parent MBP- fusion and the corresponding synthetic peptide. To recreate the exact sequence displayed by the phage, the N-terminal and C-terminal amino acids of the synthetic peptides are often preceded by one or two glycine residues. These glycines are not believed to be necessary for binding or activity. IC50 values are indicated symbolically by the symbols "-", "+”, and "++”.
• those peptides which showed IC50 values in excess of 100 ⁇ M are indicated with a "-”.
• Those peptides which gave IC50 values of less than or equal to 100 ⁇ M are given a "+”, while those which gave IC50 values of 500 nm or less are indicated with a "++”.
• Those peptides which gave IC50 values at or near the cutoff point for a particular symbol are indicated with a hybrid designator, e.g., "+/-”.
• Those peptides for which IC50 values were not determined are listed as "N.D.”.
• Xl is D, E, I, S, T, W, or Y
• X2 is D, F, G, I, L, S, V, W, or Y
• X3 is D, E, G, L, N, S, T, or W
• X4 is H or R
• X5 is A, K, R, S, T, V, or W
• X6 is D, E, F, L, M, P, Q, or V
• X7 is I or V
• X ⁇ is A or R (SEQ ID NO:l), and dimers and oligomers thereof.
• the core peptide comprises a sequence of amino acids: C Xl R X2 X7 X ⁇ X3 X4 X5 W X6 C where Xi is I, T or W; X2 is D, I, S, or V; X3 is N, or T; X5 is R, S, or T; and X6 is D, E, F, or M (SEQ ID NO:2). More preferably, the core peptide comprises a sequence of amino acids:
• the core peptide comprises either a sequence of amino acids:
• X9 XlO XllC Xl R X2 X7 X ⁇ X3 X4 X5 W X 6 C
• X9 is D, E, F, G, L, Q, or V
• Xio is D, E, G, H, K, N, or V
• Xn is D, E, G, S, V, or W (SEQ ID NO:4); or a sequence of amino acids:
• E, F, G, L, Q, T, V, or W (SEQ ID NO:5).
• X9 is D, E, F, G, or V;
• Xio is D, E, G, or V; and
• Xn is D, G, or V (SEQ ID NO:6); or
• X12 is D or G;
• Xl3 is D, E, L, M, or V; and X14 is D, E, or G (SEQ ID NO:7).
• the core peptide comprises either a sequence of amino acids: X9 XlO Xll C Xl R X2 X7 X ⁇ X3 X4 X5 W X 6 C X12 Xl3 Xl4 where X9 is D, E, F, G, L, Q, or V; Xio is D, E, G, H, K, N, or V; Xn is D, E, G, S,
• X12 is D, E, G, M, S, or T
• X13 is D, E, I, L, K, M, Q, T, or V
• X14 is D, E,
• X9 is D, E, F, G, or V
• Xio is D, E, G, or V
• Xn is D, G, or V
• X12 is D or G
• X13 is D, E, L, M, or V
• Xl4 is D, E, or G (SEQ ID NO:9).
• Preferred peptides include those listed in the tables above.
• Particularly preferred peptides include: GEVCTRDVANHRWMCGV
• Peptides and peptidomimetics having an IC50 of greater than about 100 mM lack sufficient binding to permit use in either the diagnostic or therapeutic aspects of this invention.
• the peptides and peptidomimetics have an IC50 of about 2.5 mM or less and, for pharmaceutical purposes, the peptides and peptidomimetics have an IC50 of about 2 mM or less.
• the binding peptide sequence also provides a means to determine the minimum size of an IL-5R binding compound of the invention.
• ESL encoded synthetic library
• the peptides of the invention can be prepared by classical methods known in the art, for example, by using standard solid phase techniques.
• the standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and even by recombinant DNA technology. See, e.g., Merrifield (1963) I. Am. Chem. Soc. 85:2149, incorporated herein by reference.
• the synthesis is typically commenced from the C-terminal end of the peptide using an alpha-amino protected resin.
• a suitable starting material can be prepared, for instance, by attaching the required alpha-amino acid to a chloromethylated resin, a hydroxymethyl resin, or a benzhydrylamine resin.
• a chloromethylated resin is sold under the tradename BIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, CA, and the preparation of the hydroxymethyl resin is described by Bodonszky et al, (1966) Chem. Ind. (London) 38:1597.
• the benzhydrylamine (BHA) resin has been described by Pietta and Marshall (1970) Chem. Commn. 650, and is commercially available from Beckman Instruments, Inc., Palo Alto, CA, in the hydrochloride form.
• the compounds of the invention can be prepared by coupling an alpha-amino protected amino acid to the chloromethylated resin with the aid of, for example, cesium bicarbonate catalyst, according to the method described by Gisin (1973) Helv. Chim. Acta 56:1467.
• the alpha-amino protecting group is removed by a choice of reagents including trifluoroacetic acid (TFA) or hydrochloric acid (HCI) solutions in organic solvents at room temperature.
• TFA trifluoroacetic acid
• HCI hydrochloric acid
• the alpha-amino protecting groups are those known to be useful in the art of stepwise synthesis of peptides. Included are acyl type protecting groups (e.g.
• Boc and Fmoc are preferred protecting groups.
• the side chain protecting group remains intact during coupling and is not split off during the deprotection of the amino-terminus protecting group or during coupling. The side chain protecting group must be removable upon the completion of the synthesis of the final peptide and under reaction conditions that will not alter the target peptide.
• the side chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5- dichlorobenzyl.
• the side chain protecting groups for Asp include benzyl, 2,6- dichlorobenzyl, methyl, ethyl, and cyclohexyl.
• the side chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz.
• the side chain protecting group for Thr and Ser is benzyl.
• the side chain protecting groups for Arg include nitro, Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc.
• the side chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-BrCbz), Tos, or Boc.
• the remaining protected amino acids are coupled stepwise in the desired order.
• An excess of each protected amino acid is generally used with an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH2CI2), dimethyl formamide (DMF) mixtures.
• DCC dicyclohexylcarbodiimide
• CH2CI2 methylene chloride
• DMF dimethyl formamide
• the chloromethylated resin When the chloromethylated resin is used, hydrogen fluoride treatment results in the formation of the free peptide acids. When the benzhydrylamine resin is used, hydrogen fluoride treatment results directly in the free peptide amide.
• the side chain protected peptide can be decoupled by treatment of the peptide resin with ammonia to give the desired side chain protected amide or with an alkylamine to give a side chain protected alkylamide or dialkylamide. Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.
• the resins used to prepare the peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol, i.e., methanol. Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.
• proline analogs in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed.
• Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.
• the peptides typically are synthesized as the free acid but, as noted above, could be readily prepared as the amide or ester.
• Amino terminus modifications include methylating (i.e., -NHCH3 or -NH(CH3)2), acetylating, adding a carbobenzoyl group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO-, where R is selected from the group consisting of naphthyl, acridinyl, steroidyl, and similar groups.
• Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.
• Amino terminus modifications are as recited above and include alkylating, acetylating, adding a carbobenzoyl group, forming a succinimide group, etc. Specifically, the N-terminal amino group can then be reacted as follows:
• reaction can be conducted by contacting about equimolar or excess amounts (e.g., about 5 equivalents) of an acid halide to the peptide in an inert diluent (e.g., dichloromethane) preferably containing an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge the acid generated during reaction.
• Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes). Alkylation of the terminal amino to provide for a lower alkyl N-substitution followed by reaction with an acid halide as described above will provide for N-alkyl amide group of the formula RC(0)NR-; (b) to form a succinimide group by reaction with succinic anhydride.
• an approximately equimolar amount or an excess of succinic anhydride e.g., about 5 equivalents
• the amino group is converted to the succinimide by methods well known in the art including the use of an excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane).
• a suitable inert solvent e.g., dichloromethane
• the succinic group can be substituted with, for example, C 2 -C 6 alkyl or -SR substituents which are prepared in a conventional manner to provide for substituted succinimide at the N-terminus of the peptide.
• alkyl substituents are prepared by reaction of a lower olefin (C 2 -C 6 ) with maleic anhydride in the manner described by Wollenberg, et al, supra, and -SR substituents are prepared by reaction of RSH with maleic anhydride where R is as defined above; (c) to form a benzyloxycarbonyl-NH- or a substituted benzyloxycarbonyl-NH- group by reaction with approximately an equivalent amount or an excess of CBZ-C1 (i.e., benzyloxycarbonyl chloride) or a substituted CBZ-C1 in a suitable inert diluent (e.g., dichloromethane) preferably containing a terti
• the inert diluent contains excess tertiary amine (e.g., ten equivalents) such as diisopropylethylamine, to scavenge the acid generated during reaction.
• Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); (e) to form a carbamate group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R-OC(0)Cl or R-OC(0)OC H 4 -p- N0 2 in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a carbamate where R is as defined above.
• a suitable inert diluent e.g., dichloromethane
• the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge any acid generated during reaction.
• Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); and
• the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine. Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes).
• a tertiary amine such as diisopropylethylamine. Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes).
• the resins used to prepare the peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol, e.g., methanol. Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.
• a benzhydrylamine resin is used as the solid support for peptide synthesis.
• hydrogen fluoride treatment to release the peptide from the support results directly in the free peptide amide (i.e., the C-terminus is -C(0)NH 2 ).
• the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the -OH or the ester (-OR) of the carboxyl group or ester respectively with the N-terminal amino group to form a cyclic peptide.
• the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH 2 C1 2 ), dimethyl formamide (DMF) mixtures.
• DCC dicyclohexylcarbodiimide
• the cyclic peptide is then formed by internal displacement of the activated ester with the N- terminal amine. Internal cyclization as opposed to polymerization can be enhanced by use of very dilute solutions. Such methods are well known in the art.
• C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
• peptide compounds of the invention also serve as structural models for non-peptidic compounds with similar biological activity.
• Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis. See Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243-252, incorporated herein by reference.
• C(0)NH-] have been replaced by such linkages as a ⁇ CH 2 -carbamate linkage, a phosphonate linkage, a -CH 2 -sulfonamide linkage, a urea linkage, a secondary amine (-CH 2 NH-) linkage, and an alkylated peptidyl linkage [-C(0)NR 6 - where R is lower alkyl] are prepared during conventional peptide synthesis by merely substituting a suitably protected amino acid analogue for the amino acid reagent at the appropriate point during synthesis.
• Suitable reagents include, for example, amino acid analogues wherein the carboxyl group of the amino acid has been replaced with a moiety suitable for forming one of the above linkages. For example, if one desires to replace a -C(0)NR- linkage in the peptide with a -CH 2 -carbamate linkage (- CH 2 OC(0)NR-), then the carboxyl (-COOH) group of a suitably protected amino acid is first reduced to the -CH 2 OH group which is then converted by conventional methods to a -OC(0)Cl functionality or a para-nitrocarbonate - OC(0)0-C 6 H -p-N0 2 functionality.
• Replacement of an amido linkage in the peptide with a -CH 2 - sulfonamide linkage can be achieved by reducing the carboxyl (-COOH) group of a suitably protected amino acid to the -CH 2 OH group and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods. Reaction of the tosylated derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination will provide for the -CH 2 -S(0) 2 C1 functional group which replaces the carboxyl group of the otherwise suitably protected amino acid.
• Secondary amine linkages wherein a -CH 2 NH- linkage replaces the amido linkage in the peptide can be prepared by employing, for example, a suitably protected dipeptide analogue wherein the carbonyl bond of the amido linkage has been reduced to a CH 2 group by conventional methods. For example, in the case of diglycine, reduction of the amide to the amine will yield after deprotection H 2 NCH 2 CH 2 NHCH 2 COOH which is then used in NT- protected form in the next coupling reaction. The preparation of such analogues by reduction of the carbonyl group of the amido linkage in the dipeptide is well known in the art.
• the suitably protected amino acid analogue is employed in the conventional peptide synthesis in the same manner as would the corresponding amino acid.
• typically about 3 equivalents of the protected amino acid analogue are employed in this reaction.
• An inert organic diluent such as methylene chloride or DMF is employed and, when an acid is generated as a reaction by-product, the reaction solvent will typically contain an excess amount of a tertiary amine to scavenge the acid generated during the reaction.
• One particularly preferred tertiary amine is diisopropylethylamine which is typically employed in about 10 fold excess.
• the reaction results in incorporation into the peptide mimetic of an amino acid analogue having a non-peptidyl linkage. Such substitution can be repeated as desired such that from zero to all of the amido bonds in the peptide have been replaced by non-amido bonds.
• C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
• the compounds of the present invention may exist in a cyclized form with an intramolecular disulfide bond between the thiol groups of the cysteines .
• an intermolecular disulfide bond between the thiol groups of the cysteines can be produced to yield a dimeric (or higher oligomeric) compound.
• One or more of the cysteine residues may also be substituted with a homocysteine.
• n and n are independently 1 or 2.
• the amino-terminus of the peptide can be capped with an alpha-substituted acetic acid, wherein the alpha substituent is a leaving group, such as an ⁇ -haloacetic acid, for example, ⁇ -chloroacetic acid, ⁇ -bromoacetic acid, or ⁇ -iodoacetic acid.
• the compounds of the present invention can be cyclized or dimerized via displacement of the leaving group by the sulfur of the cysteine or homocysteine residue. See, e.g., Barker et al.
• the compounds of the invention can advantageously be modified with or covalently coupled to one or more of a variety of hydrophilic polymers.
• the corresponding derivative may have increased solubility and circulation half-lives and masked immunogenicity.
• Nonproteinaceous polymers suitable for use in accordance with the present invention include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol; polylactic acid; polyglycolic acid; polyoxyalkenes; polyvinylalcohol; polyvinylpyrrolidone; cellulose and cellulose derivatives; dextran and dextran derivatives; etc.
• hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, more preferably from about 2000 to about 40,000 daltons, and even more preferably, from about 5,000 to about 20,000 daltons.
• such hydrophilic polymers have an average molecular weight of about 5,000 daltons, 10,000 daltons, or 20,000 daltons.
• the compounds of the invention can be derivatized with or coupled to such polymers using any of the methods set forth in Zallipsky (1995) Bioconjugate Chem. 6:150-165: Monfardini et al. (1995) Bioconjugate Chem. 6:62-69: U.S. Patent No. 4,640,835; U.S. Patent No. 4,496,689; U.S. Patent No. 4,301,144; U.S. Patent No. 4,670,417; U.S. Patent No. 4,791,192; U.S. Patent No. 4,179,337 or WO 95/34326, all of which are incorporated by reference in their entirety herein.
• the compounds of the present invention are derivatized with polyethylene glycol (PEG).
• PEG is a linear, water-soluble polymer of ethylene oxide repeating units with two terminal hydroxyl groups.
• PEGs are classified by their molecular weights which typically range from about 500 daltons to about 40,000 daltons.
• the PEGs employed have molecular weights ranging from 5,000 daltons to about 20,000 daltons.
• PEGs coupled to the compounds of the present invention can be either branched or unbranched. See, e.g., Monfardini et al. (1995) Bioconjugate Chem. 6:62-69.
• PEGs are comemrcially availble from Shearwater Polymers Inc. (Huntsville, Alabama), Sigma Chemical Co., and other companies. Such PEGs include, but are not limited to, monomethyoxypolyethyle glycol (MePEG-OH); monomethoxy poly ethylene glycol-succinate (MePEG-S); monomethoxypoly ethylene glycol-succinimidyl succinate (MePEG-S-NHS); monomethoxypolyethylene glycol amine (MePEG-NH 2 ); monomethoxypolyethylene glycol-tresylate (MePEG-TRES); and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).
• MePEG-OH monomethyoxypolyethyle glycol
• MePEG-S monomethoxy poly ethylene glycol-succinate
• MePEG-NHS monomethoxypoly ethylene glycol amine
• the hydrophilic polymer which is employed is preferably capped at one end by an unreactive protecting group, such as a methoxy or ethoxy. Thereafter, the polymer is activated at the other end by reaction with a suitable activating agent, such as cyanuric halide (e.g., cyanuric chloride, bromide, or fluoride), diimadozle, an anhydride reagent (e.g., a dihalosuccinic anhydride, such as dibromosuccinic anhydride), acyl azide, p-diazoiumbenzyl ether), 3-(p-diazoniumphenoxy)-2- hydroxypropylether and the like.
• cyanuric halide e.g., cyanuric chloride, bromide, or fluoride
• diimadozle e.g., an anhydride reagent (e.g., a dihalosuccinic anhydride, such as dibromosucc
• the activated polymer is then reacted with a compound of the present invention to produce a compound derivatized with a polymer.
• a functional group in the compounds of the invention can be activated for reaction with the polymer, or the two groups can be joined in a concerted coupling reaction using known coupling methods. It will be readily appreciated that the compounds of the invention can be derivatized with PEG using a myriad of other reaction schemes known to and used by those of skill in the art.
• This immobilized polymer synthesis system or other peptide synthesis methods can also be used to synthesize every truncation analog and every deletion analog and every combination of truncation and deletion analog of all of the peptide compounds of the invention. IV. In Vivo and In Vitro Testing
• the activity of the compounds of the present invention can be evaluated in vivo in one of the numerous animal models of asthma. See
• An ideal animal model would duplicate the chief clinical and physiological features of human asthma, including: airway hyperresponsiveness to chemical mediators and physical stimuli; reversal of airway obstruction by drugs useful in human asthma ( ⁇ -adrenergics, methylxanthines, corticosteroids, and the like); airway inflammation with infiltration of activated leukocytes; and chronic inflammatory degenerative changes, such as basement membrane thickening, smooth muscle hypertrophy, and epithelial damage.
• Species used historically as animal models include mice, rats, guinea pigs, rabbits, dogs, and sheep. All have some limitations, and the proper choice of animal model depends upon the question which is to be addressed.
• the initial asthmatic response can be evaluated in guinea pigs, and dogs, and particularly, with a basenji-greyhound cross strain which develops nonspecific airway hyperresponsiveness to numerous nonallergenic substances, such as methacholine and citric acid.
• Certain selected sheep exhibit a dual response after antigen challenge with Ascaris proteins.
• the initial asthmatic response (IAR) is followed by a late asthmatic response (LAR) at 6-8 hours post-exposure.
• Hypersensitivity to the cholinergic agonist carbachol increases at 24 hours after antigen challenge in those animals which exhibit LAR.
• the allergic sheep model can be used to evaluate the potential antiasthmatic effects of the compounds of the present invention.
• compositions comprising aerosolized solutions of the compounds of the instant invention to allergic sheep prior to or following exposure to specific allergens will demonstrate that such compositions substantially lessen or abolish the late asthmatic response and consequent hyperresponsiveness.
• the compounds of this invention are also useful for the treatment of other immunomediated inflammatory disorders in which tryptase activity contributes to the pathological condition.
• diseases include inflammatory diseases associated with mast cells, such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, inflammatory bowel disease, peptic ulcer and various skin conditions.
• the efficacy of the compounds of the instant invention for the treatment of the vast majority of immunomediated inflammatory disorders can be evaluated by either in vitro or in vivo procedures.
• the anti- inflammatory efficacy of the compounds of the instant invention can be demonstrated by assays well known in the art, for example, the Reversed Passive Arthus Reaction (RPAR)-PAW technique (see, e.g., Ganguly et al. (1992) U.S. Patent No. 5,126,352).
• Assays for determining the therapeutic value of compounds in the treatment of various skin conditions, such as hyperproliferative skin disease are well known in the art, for example, the Arachidonic Acid Mouse Ear Test (id).
• the compounds of the instant invention can be evaluated for their antiulcer activity according to the procedures described in Chiu et al. (1984) Archives Internationales de Pharmacodynamie et de Therapie 270:128-140.
• the compounds of the invention are useful in vitro as unique tools for understanding the biological role of IL-5, including the evaluation of the many factors thought to influence, and be influenced by, the production of IL-5 and the receptor binding process.
• the present compounds are also useful in the development of other compounds that bind to the IL-5R, because the present compounds provide important information on the relationship between structure and activity that should facilitate such development.
• the compounds are also useful as competitive inhibitors or tracers in assays to screen for new IL-5 receptor blockers.
• the compounds of the invention can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
• Possibilities for direct labeling include label groups such as: radiolabels such as 125 ⁇ enzymes (US Patent 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (US Patent 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
• Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
• the compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support.
• compositions and methods of the present invention also can be used in vitro for testing a patient's susceptibility to varying treatment regimens for disorders associated with the overproduction of IL-5 or an improper response to IL-5 using an in vitro diagnostic method whereby a specimen is taken from the patient and is treated with a IL-5R binding, IL-5 blocking compound of the present invention to determine the effectiveness and amount of the compound necessary to produce the desired effect.
• the blocking compound and dosage can be varied. After the blocking compounds are screened, then the appropriate treatment and dosage can be selected by the physician and administered to the patient based upon the results. Therefore, this invention also contemplates use of a blocking compound of this invention in a variety of diagnostic kits and assay methods.
• the compounds of the invention can also be administered to warm blooded animals, including humans, to block the binding of IL-5 to the IL-5R in vivo.
• the present invention encompasses methods for therapeutic treatment of IL-5 related disorders that comprise administering a compound of the invention in amounts sufficient to block or inhibit the binding of IL-5 to the IL-5R in vivo.
• the peptides and compounds of the invention can be administered to treat symptoms related to the overproduction of IL-5 or an improper response to IL-5.
• the compositions and methods described herein will find use for the treatment and/or prevention of a variety of IL-5 related disorders.
• the compositions of the present invention are useful for preventing or ameliorating asthma.
• the compounds typically will be administered prophylactically prior to exposure to allergen or other precipitating factor, or after such exposure.
• the compounds of the instant invention are particularly useful in ameliorating the late-phase tissue destruction seen in both seasonal and perennial rhinitis.
• Another aspect of the present invention is directed to the prevention and treatment of other immunomediated inflammatory disorders associated with mast cells such as urticaria and angioedema, and eczematous dermatitis
• atopic dermatitis and anaphylaxis, as well as hyperproliferative skin disease, peptic ulcers, and the like.
• the present invention also provides pharmaceutical compositions comprising, as an active ingredient, at least one of the peptides or peptide mimetics of the invention in association with a pharmaceutical carrier or diluent.
• the compounds of this invention can be administered by oral, pulmonary, parental (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (via a fine powder formulation). transdermal, nasal, vaginal, rectal, or sublingual routes of administration and can be formulated in dosage forms appropriate for each route of administration.
• aerosol when the compounds of the instant invention are to be used in the treatment of asthma, they will be formulated as aerosols.
• aerosol includes any gas-borne suspended phase of the compounds of the instant invention which is capable of being inhaled into the bronchioles or nasal passages.
• aerosol includes a gas-borne suspension of droplets of the compounds of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example.
• the preferred range of concentration of the compounds of the instant invention is 0.1-100 milligrams (mg)/ milliliter (mL), more preferably 0.1-30 mg/mL, and most preferably, 1-10 mg/mL.
• a physiologically compatible buffer such as phosphate or bicarbonate.
• the usual pH range is 5 to 9, preferably 6.5 to 7.8, and more preferably 7.0 to
• sodium chloride is added to adjust the osmolarity to the physiological range, preferably within 10% of isotonic.
• 1,1,1,2,3 ' ,3,3-heptafluoropropane optionally in the presence of a surfactant and /or cosolvent (e.g., ethanol) in a pressurized canister may also be provided with a suitable delivery device for the treatment of the above mentioned respiratory disorders, especially asthma and allergic rhinitis.
• a surfactant and /or cosolvent e.g., ethanol
• Solutions of the compounds of the instant invention may be converted into aerosols by any of the known means routinely used for making aerosol inhalant pharmaceuticals.
• such methods comprise pressurizing or providing a means of pressurizing a container of the solution, usually with an inert carrier gas, and passing the pressurized gas through a small orifice, thereby pulling droplets of the solution into the mouth and trachea of the animal to which the drug is to be administered.
• a mouthpiece is fitted to the outlet of the orifice to facilitate delivery into the mouth and trachea.
• devices of the present invention comprise solutions of the compounds of the instant invention connected to or contained within any of the conventional means for creating aerosols in asthma medication, such as metered dose inhalers, jet nebulizers, or ultrasonic nebulizers.
• such device may include a mouthpiece fitted around the orifice.
• a device may comprise a solution of a compound of the instant invention in a nasal sprayer.
• a dry powder comprising a compound of the instant invention, optionally with an excipient, is another embodiment of the present invention.
• This may be administered by a drug powder inhaler containing the above described powder.
• the compounds of the inventions can also be used in the treatment of immunomediated inflammatory skin conditions, such as urticaria and angioedema, eczematous dermatitis, and hyperproliferative skin disease, e.g., psoriasis, in mammals.
• immunomediated inflammatory skin conditions such as urticaria and angioedema, eczematous dermatitis, and hyperproliferative skin disease, e.g., psoriasis
• one affected by an immunomediated inflammatory skin condition can expect a decrease in scaling, erythema, size of the plaques, pruritus, and other symptoms associated with the skin condition.
• the dosage of medicament and the length of time required for successfully treating each individual patient may vary, but those skilled in the art will be able to recognize these variations and adjust the course of therapy accordingly.
• preparations for topical application to the skin comprising a compound of the present invention, typically in concentrations in the range of from about 0.001% to 10%, together with a non-toxic, pharmaceutically acceptable topical carrier.
• topical preparations can be prepared by combining an active ingredient according to this invention with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, cream and aerosol formulations.
• Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and /or gelling agents.
• bases may include water and /or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil.
• Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like.
• Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. Powders may be formed with the aid of any suitable powder base, e.g., talc, lactose, starch, and the like. Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like.
• the topical pharmaceutical compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like.
• the topical pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
• Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
• additional substances e.g., lubricating agents such as magnesium stearate.
• the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
• Preparations according to this invention for parental administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
• compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.
• Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art. It should, of course, be understood that the compositions and methods of this invention can be used in combination with other agents exhibiting the ability to modulate IL-5 synthesis, release, and /or binding and with other agents for the treatment of immunomediated inflammatory disorders, and particularly asthma. ⁇ -Adrenergic agonists are especially useful in these combinations, because they provide symptomatic relief of the initial asthmatic response, whereas the compounds of the present invention provide relief for the late asthmatic response.
• Preferred ⁇ - adrenergic agonists in these solutions include any of the usual ⁇ -agonists employed for the relief of asthma, such as albuterol, terbutaline, formoterol, fanoterol, or prenaline.
• agents useful in combination with the compounds of the instant invention include anticholinergics, such as ipratropium bromide, and antiinflammatory corticosteroids (adrenocortical steroids) such as beclomethasone, triamcinolone, flurisolide, or dexamethasone.
• anticholinergics such as ipratropium bromide
• antiinflammatory corticosteroids such as beclomethasone, triamcinolone, flurisolide, or dexamethasone.
• compositions containing the compounds can be administered for prophylactic and /or therapeutic treatments.
• compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
• An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.
• compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically effective dose.” In this use, the precise amounts again depend on the patient's state of health and weight.
• the quantities of the IL-5 blocking compound necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds), (1990) Goodman and Gilman's: The Pharmacological Basis of Therapeutics. 8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences. (1985) 7th ed., Mack Publishing Co., Easton, Penn.; each of which is hereby incorporated by reference.
• the peptides and peptide mimetics of this invention are effective in treating IL-5 mediated conditions when administered at a dosage range of from about 0.001 mg to about 10 mg/kg of body weight per day.
• the specific dose employed is regulated by the particular condition being treated, the route of administration as well as by the judgement of the attending clinician depending upon factors such as the severity of the condition, the age and general condition of the patient, and the like.
• the resin used was HMP resin or PAL (Milligen/Biosearch), which is a cross-linked polystyrene resin with 5-(4'-Fmoc-aminomethyl-3,5'- dimethyoxyphenoxy)valeric acid as a linker.
• PAL resin results in a carboxyl terminal amide functionality upon cleavage of the peptide from the resin.
• the HMP resin produces a carboxylic acid moiety at the C-terminus of the final product.
• Most reagents, resins, and protected amino acids were purchased from Millipore or Applied Biosystems Inc.
• the Fmoc group was used for amino protection during the coupling procedure.
• Primary amine protection on amino acids was achieved with Fmoc and side chain protection groups were t-butyl for serine, tyrosine, asparagine, glutamic acid, and threonine; trityl for glutamine; Pmc (2,2,5,7,8- pentamethylchroma sulfonate) for arginine; N-t-butyloxycarbonyl for tryptophan; N-trityl for histidine and glutamine; and S-trityl for cysteine.
• Bioactivity of synthetic peptides and MBP-peptide fusions is measured using a Cytosensor microphysiometer (Molecular Devices) to record the metabolic response of TF-1 cells (a human leukemia cell line) to IL-
• IL-5 these cells exhibited a robust increase in metabolic activity when IL-5 is added to the medium. This increase was measured by the microphysiometer as an increase in the rate of acidification of weakly buffered tissue culture medium.
• TF-1 cells were seeded into microphysiometer chambers at a density of 1.5 x 10 ⁇ cells/chamber and grown overnight in DMEM tissue culture medium containing 10% fetal bovine serum , but lacking the 1 ng/ml
• IL-5 (R&D Systems) that is required for long-term maintenance of these cells in culture.
• the chambers were then placed in the microphysiometer and incubated with weakly buffered DMEM/F12 medium containing 1% human serum albumin until a baseline rate of medium acidification was established.
• Varying dilutions of test peptide were then introduced for 15 min. None of the peptides tested had any effect on the baseline acidification rate. IL-5 at 10 ng/ml was then introduced for 25 minutes in the continued presence of test peptide. The chambers were then flushed with fresh medium.
• maximal response to IL-5 occurred within 20 min. of the onset of IL-5 addition to the medium. In the absence of test peptide this response was typically a 1.5 to 2-fold increase in the rate of medium acidification. All peptides tested were able to reduce or completely block the response of the TF-1 cells to IL-5. Other, randomly chosen control peptides, at the same or higher concentrations, had no effect. The test peptides also had no effect on the robust microphysiometer response of TF-1 cells to TNF ⁇ , indicating that the test peptides were exhibiting their effect by specifically antagonizing IL-5 action. The IC50 for test peptides was defined as that peptide concentration which gave a 50% reduction in the maximal IL-5 response when compared to the response to IL-5 alone.
• Binding Affinity Binding affinities of synthetic peptides for IL-5R ⁇ were measured in a competition binding assay using radio-iodinated IL-5.
• Immulon 4 (Dynatech) microtiter wells were coated with streptavidin (Sigma) by incubating 100 ⁇ l of a 50 ⁇ g/ml solution in PBS for 30 min. at 37°. The wells were blocked with 200 ⁇ l of 1 % BSA in PBS for 15 min. at 37°, followed by 100 ⁇ l of biotinylated mAb 179 at 5 ⁇ g/ml in PBS.
• Soluble IL-5R ⁇ was then immobilized in the wells by incubating 100 ⁇ l of a solution of soluble receptor harvest diluted 1:5000 in PBS/0.1% BSA for 1 hr. at 4°. After washing away unbound receptor, 50 ⁇ l of various concentrations of test peptide diluted in PBS/ 0.1% BSA were added to the wells, followed by 50 ⁇ l of a fixed concentration of [ ⁇ 5i] IL-5 (Amersham). The binding reactions were incubated at 4°C for 2 hr., then washed with PBS to remove unbound [125 ⁇ IL_ 5. Bound [125i] j -5 was determined by counting.
• oligonucleotide sequences are needed for library construction: ON-829 (5' ACC ACC TCC GG) (SEQ ID NO:49); ON-830 (5' TTA CTT AGT TA) (SEQ ID NO:50) and a library specific oligonucleotide of interest (5' GA GGT GGT ⁇ NNK ⁇ n TAA CTA AGT AAA GC), where (NNK ⁇ n denotes a random region of the desired length and sequence.
• the oligonucleotides can be 5' phosphorylated chemically during synthesis or after purification with polynucleotide kinase. They are then annealed at a 1:1:1 molar ratio and ligated to the vector.
• the strain of E. coli which is preferably used for panning has the genotype: ⁇ (srl-recA) endAl nupG lon-11 sulAl hsdR17 ⁇ (ompT-fepC)266 ⁇ clpA319::kan ⁇ lacI lac ZU118 which can be prepared from an E. coli strain from the E. coli Genetic Stock Center at Yale University (E. coli b/r, stock center designation CGSC:6573) with genotype lon-11 sulAl.
• the above E. coli strain is prepared for use in electroporation as described by Dower et al. (1988) Nucleic Acids Res.
• Peptides on plasmids are released from cells for panning by gentle enzymatic digestion of the cell wall using lysozyme. After pelleting of the cell debris, the crude lysate can be used directly on most receptors. If some additional purification of the plasmid complexes is needed, a gel filtration column can be used to remove many of the low molecular weight contaminants in the crude lysate.
• Panning is carried out in a buffer (HEKL) of a lower salt concentration than most physiological buffers.
• the panning can be conducted in microtiter wells with a receptor immobilized on a nonblocking monoclonal antibody (MAb) or by panning on beads or on columns. More specifically, in the first round of panning, 24 wells, each coated with receptor, can be used. For the second round, six wells coated with receptor (PAN sample) and 6 wells without receptor (NC sample) are typically used.
• Enrichment is defined as the ratio of PAN transformants to those recovered from the NC sample. Enrichment of 10 fold is usually an indication that receptor specific clones are present.
• the binding properties of the peptides encoded by individual clones are typically examined after 3, 4, or 5 rounds of panning, depending on the enrichment numbers observed.
• an ELISA that detects receptor specific binding by Lacl-peptide fusion proteins is used.
• Lad is normally a tetramer and the minimum functional DNA binding species is a dimer.
• the peptides are thus displayed multivalently on the fusion protein. Assuming that a sufficient density of receptor can be immobilized in wells, the peptides fused to La will bind to the surface in a cooperative, multivalent fashion. This cooperative binding permits the detection of binding events of low intrinsic affinity.
• the sensitivity of this assay is an advantage in that initial hits of low affinity can be easily identified, but is a disadvantage in that the signal in the ELISA is not correlated with the intrinsic affinity of the peptides.
• MBP maltose binding protein
• DNA from clones of interest can be prepared in double stranded form using any standard miniprep procedure.
• the coding sequences of interesting single clones or populations of clones can be transferred to vectors that fuse those sequences in frame with the gene encoding MBP, a protein that generally occurs as a monomer in solution.
• the cloning of a library into pJS142 creates a BspEI restriction site near the beginning of the random coding region of the library.
• Digestion with BspEI and Seal allows the purification of a " 900 bp DNA fragment that can be subcloned into one of two vectors, pELM3 (cytoplasmic) or pELM15 (periplasmic), which are simple modifications of the pMALc2 and pMALp2 vectors, respectively, available commercially from New England Biolabs. See Figure 2A-B.
• Digestion of pELM3 and pELM15 with Agel and Seal allows efficient cloning of the BspEI- Seal fragment from the pJSl42 library. The BspEI and Agel ends are compatible for ligation.
• lysates for the La or MBP ELISAs are prepared from individual clones by lysing cells using lysozyme and removing insoluble cell debris by centrifugation. The lysates are then added to wells containing immobilized receptor and to control wells without receptor.
• Binding by the Lad or MBP peptide fusions is detected by incubation with a rabbit polyclonal antiserum directed against either Lad or MBP followed by incubation with alkaline phosphatase labeled goat anti rabbit second antibody. The bound alkaline phosphatase is detected with p-nitrophenyl phosphate chromagenic substrate.
• EXAMPLE 5 "Headpiece Dimer" System A variant of the Lad peptides-on-plasmids technique utilizes a DNA binding protein called "headpiece dimer". DNA binding by the E. coli lac repressor is mediated by the approximately 60 amino acid "headpiece" domain.
• the dimer of the headpiece domains that binds to the lac operator is normally formed by association of the much larger approximately 300 amino acid C-terminal domain.
• the "headpiece dimer” system utilizes headpiece dimer molecules containing two headpieces connected via short peptide linker. These proteins bind DNA with sufficient stability to allow association of a peptide epitope displayed at the C-terminus of the headpiece dimer with the plasmid encoding that peptide.
• the random peptides are fused to the C-terminus of the headpiece dimer, which binds to the plasmid that encoded it to make a peptide-headpiece dimer-plasmid complex that can be screened by panning.
• the headpiece dimer peptides-on-plasmids system allows greater selectivity for high affinity ligands than the Lad system.
• the headpiece dimer system is useful for making mutagenesis libraries based on initial low-affinity hits, and selecting higher affinity variants of those initial sequences.
• the libraries are constructed as with peptides on plasmids using headpiece dimer vector pCMG14 (see Figure 3). The presence of the lac operator is not required for plasmid binding by the headpiece dimer protein.
• the libraries were introduced into bacterial strain comprising E. coli (lon-11 sulAl hsdR17 (ompT-fepC) ⁇ clpA319::kan ⁇ lacI lac ZU118 ⁇ (srl-recA) 306::Tnl0 .and amplified under conditions of basal (A) promoter induction.
• Panning of headpiece dimer libraries is carried out by similar procedures to those used for Lad libraries, except that HEK buffer is used instead of HEKL buffer and elution of plasmids from the wells is performed with aqueous phenol instead of with IPTG. Sequences from headpiece dimer panning are often characterized after transfer to the MBP vector so that they can be tested in the affinity sensitive MBP ELISA and also so that populations of clones can be screened by colony lifts with labeled receptor.
• ADDRESSEE Affymax Technologies, N.V.

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