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WO1999055911A1 - Identification de ligands peptidiques de proteines cibles par la technique des banques complementaires de cibles tclt - Google Patents

Identification de ligands peptidiques de proteines cibles par la technique des banques complementaires de cibles tclt Download PDF

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Publication number
WO1999055911A1
WO1999055911A1 PCT/US1999/006537 US9906537W WO9955911A1 WO 1999055911 A1 WO1999055911 A1 WO 1999055911A1 US 9906537 W US9906537 W US 9906537W WO 9955911 A1 WO9955911 A1 WO 9955911A1
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peptides
peptide
complementary
target protein
target
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PCT/US1999/006537
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English (en)
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Fang Fang
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Fang Fang
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Priority to AU35465/99A priority Critical patent/AU762711C/en
Priority to EP99917315A priority patent/EP1078099A4/fr
Priority to CA002350163A priority patent/CA2350163A1/fr
Publication of WO1999055911A1 publication Critical patent/WO1999055911A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to methods of screening for peptide ligands of target proteins.
  • This invention features an improved method of screening for peptide ligands of target proteins.
  • This method is more powerful than the random peptide library technology in that it utilizes a predefined pool of peptides containing ligands for a selected target protein.
  • the peptides in the pool exhibit complementary hydropathy to the target protein.
  • Peptide ligands are selected based on their ability to bind to the target protein. This method is hereinafter referred to as Target Complementary Library Technology (TCLT).
  • TCLT Target Complementary Library Technology
  • TCLT can be used in all occasions when random peptide libraries are used to identify ligands for target proteins. By identifying peptide ligands which can influence the biological activities of their target proteins, TCLT can be used to discover peptides of therapeutic utility. In addition, peptide ligands are useful for studying the dynamic structure of their target proteins, and such information can be used for designing functional pharmacological small molecules.
  • TCLT Applications of TCLT include, but are not limited to, the following: 1) Identifying peptides which functionally mimic idratypic antibodies. By mimicking the anti-idiotypic antibodies of the beta type (Ab2beta) (62), a peptide ligand can block the antigen-binding site of an antigen-specific antibody. By mimicking the anti- idiotypic antibodies of the gamma type (Ab2gan ⁇ ma) (62), a peptide ligand can prevent antigen-binding of an entire class of antibodies, such as IgG or IgE.
  • the framework 2 and framework 3 (FR2 and FR3) segments of both the heavy chain and light chain of a immunoglobulin molecular can serve as the target sequence for binding peptides which mimic the function of an Ab2gamma antibody.
  • Each of the complementarity - determining - regions (CDR1, CDR2 and CDR3) can serve as the target sequence for AB2 beta-mimicking peptides.
  • the target proteins include IgE, IgG, IgA, and specific antibodies such as anti-acetylcholine receptor antibodies.
  • Peptide ligands of these target proteins can be used for the prevention and treatment of allergy, and autoimmune diseases including rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosis, and autoimmune nephritis. 2) Identifying peptides which mimic the function of a biological active molecule, such as a hormone, a cytokine, a growth factor, or a chemical compound.
  • a biological active molecule such as a hormone, a cytokine, a growth factor, or a chemical compound.
  • the target proteins include the cell surface receptors and intracellular receptors for these molecules, including, but not limited to, the receptors for insulin, human growth hormone, erythropoietin, thrombopoietin, estrogen, androgen, epidermal growth factor, interferons, interleukins, fibrinogen, endorphin, enkephalin, dopamine, nicotine, acetylcholine, angiotensin, and somatostatin.
  • peptides which mimic the binding domain of a receptor.
  • the peptide ligands act as decoys to compete with the receptor, thereby preventing the biological effects induced by the interaction between the receptor and its native ligand.
  • the target proteins in this case include the biological active peptides and proteins, which include, but are not limited to, interleukin-1, interleukin-2, bradykinin, platelet activating factors, and tumor necrosis factor (TNF).
  • peptides which mimic the receptor-binding domains of viral or bacterial proteins.
  • Such peptides can block viral or bacterial infections by preventing the virus or bacteria from binding to its cellular receptors.
  • the target proteins can either be the known cellular receptors for viruses/bacteria, such as ICAM-1 and LDL receptor which are the major and minor receptors for human rhinoviruses (63, 64), or a viral or bacterial surface protein known to bind to corresponding cellular receptors.
  • peptide ligands for any protein.
  • the peptide ligand can either be used for affinity purification of the target protein, or as a probe for the detection of the target protein in vitro and in vivo.
  • the known ligand/receptor would serve as the target and the corresponding receptor/ligand can be identified by one of the following approaches.
  • the complementary DNA fragment selected by TCLT can be used as a probe to screen another library which contains the potential receptor (23).
  • the complementary anti- sense peptide encoded by the complementary DNA fragment can serve as an immunogen to generate antibodies which interact with the potential receptor with specificity (24-37).
  • TCLT involves constructing (i) a peptide library containing a collection of complementary peptides of a target protein peptide, wherein the complementary peptides in aggregation complement all, substantially all (preferably no less than 70%, more preferable no less than 80%, even more preferably no less than 90%), or a significant portion of the target protein/peptide; or (ii) a nucleic acid library containing a collection of nucleic acids encoding the aforesaid complementary peptides.
  • the nucleic acid library is inserted into expression vectors and introduced into genetic packages such as phage particles and the complementary peptides are expressed on the surface of the genetic packages.
  • Peptide ligands are selected from the library by panning against the target protein/peptide or the binding domain of the target protein/peptide.
  • the target protein/peptide or its binding domain is preferably immobilized on a solid support.
  • Genetic packages which bind to the immobilized target protein/peptide can be amplified and subject to several more rounds of panning.
  • Complementary peptides with sufficient specific affinity to the target protein/peptide are selected at the end of this process, and their sequences can be uncovered from the genome of the selected genetic packages.
  • hydropathy character of an amino acid is meant the hydrophobic or hydrophilic nature of the amino acid. Specifically, it is reflected by the hydropathy score of the amino acid in Tables 1-3 of the specification. The hydropathy scores of the twenty amino acids in Table 1-3 range from +4.5 to -4.5, and allow for the grouping of the twenty amino acids as hydrophobic (+4.5 to +1.8, Table 1), hydrophilic (-4.5 to -3.2, Table 2), and slightly hydrophilic (-0.4 to -1.6, Table 3) (Ref. 11).
  • Hydrophobic amino acids in Table 1 complement hydrophilic amino acids in Table 2 (i.e., Arg, Lys, Asn, Asp, Gin, Glu, and His), and vice versa.
  • Slightly hydrophilic amino acids in Table 3 i.e., Gly, Thr, Tip, Ser, Tyr, and Pro
  • each of Arg, Lys, Asn, Asp, Gin, Glu, and His is complementary to He
  • each of Gly, Thr, Tip, Ser, Tyr, and Pro is complementary to Gly.
  • each of Arg, Lys, Asn, Asp, Gin, Glu, and His is a complementary amino acid to He
  • each of Gly, Thr, Tip, Ser, Tyr, and Pro is a complementary amino acid to Gly.
  • hydropathy profile of a peptide or protein is meant its profile as defined by the hydropathic characters of amino acids in the peptide or protein.
  • complementary peptide is meant a peptide containing a contiguous amino acid sequence (in the direction from the amino terminus to the carboxy terminus or in the direction from the carboxy terminus to the amino terminus) which complements the amino acid sequence of the target protein/peptide according to the rule set forth above.
  • Any target protein/peptide has a plethora of complementary peptides because each amino acid in the target protein/peptide has a number of complementary amino acids.
  • anti-sense peptide is meant a peptide encoded by the anti-sense strand of the target gene translated in either 5' to 3' or 3' to 5' directions.
  • the anti-sense peptide would either be a peptide having the sequence Lys-Lys-Asn-Lys as encoded by the anti-sense strand 5' AAA AAG AAC AAG 3' or a peptide having the sequence Glu-Gln-Glu-Lys as encoded by 5'GAA CAA GAA AAA 3'.
  • the matching pattern in Tables 1-3 shows that the anti-sense strand of a target gene usually encodes complementary peptide to the target protein/peptide.
  • Anti-sense peptides which are complementary to the target protein/peptide are called complementary anti-sense peptides.
  • target complementary library (i) a peptide library containing a collection of complementary peptides of a target protein/peptide, wherein the complementary peptides in aggregation complement all, substantially all (preferably no less than 70%, more preferable no less than 80%, even more preferably no less than 90%), or a significant portion of the target protein/peptide; or (ii) a nucleic acid library containing a collection of nucleic acids encoding the aforesaid complementary peptides.
  • the complementary peptides are linked to flanking sequences.
  • anti-sense target complementary library (anti-sense TCL) is meant a target complementary library wherein the complementary peptides are also anti-sense peptides of the target protein/peptide. These anti-sense peptides in combination complement the amino acid sequence of the target protein/peptide in its entirety or
  • the anti-sense TCL is a gene fragment library wherein fragments of the anti-sense strand of the target protein are operatively linked to a promoter for expressing the anti-sense peptides.
  • the sizes of the nucleic acid fragments encoding anti-sense peptides may be defined or random.
  • the anti- sense peptides encoded by the gene fragments may overlap each other randomly or in a predefined manner.
  • fragments of the anti-sense strand are linked to flanking sequences.
  • cTCL comprehensive target complementary library
  • cTCL a target complementary library containing or encoding a collection of complementary peptides.
  • Each complementary peptide of the collection complements the entire length of the target protein/peptide.
  • this collection includes all possible complementary peptides to the target.
  • each amino acid in the target is complemented by all its possible complementary amino acids at the corresponding position respectively in different complementary peptides of the collection.
  • the cTCL of the target peptide Ile- Gly-Arg would contain a collection of complementary peptides represented by the formula X-(Arg, Lys, Asn, Asp, Gin, Glu, or His)-(Gly, Thr, Tip, Ser, Tyr, or Pro)-(Ile, Val, Leu, Phe, Cys, Met, or Ala)-Y, wherein each position of the complementary peptides is fully randomized according to the formula and X and/or Y represent either nothing or one or more amino acid residues.
  • target protein/peptide is meant any protein/peptide or a domain within any protein/peptide.
  • binding domain or “interaction domain” is meant a domain or region of a protein which is primarily responsible for the protein's ability to bind to another protein.
  • genetic packages replicable entities capable of carrying the nucleic acids of a target complementary library (TCL), including, but not limited to, vegetative bacterial cells, bacterial spores, bacterial viruses and eukaryotic cells. Genetic packages allow the amplification and recovery of the encapsulated genetic message following selection.
  • a preferred genetic package is a phage.
  • a phage display library expresses a collection of cloned proteins on the surface of phage particles as fusion with a phage coat protein.
  • selection means
  • Two peptides encoded by complementary sequences in the same reading frame show a total interchange of their hydrophobic and hydrophilic amino acids when the amino terminus of one is aligned with the carboxy terminus of the other.
  • the molecular recognition theory predicts that such inverted hydropathic pattern may allow pairs of complementary peptides to assume conformations that enable the pair to interact with each other specifically (11).
  • a peptide or protein that does not interact with another peptide or protein is of little value to cells and organisms.
  • the molecular recognition theory suggests that nature may have evolved a fail-safe system to ensure that this does not occur. Because of the complementarity of nucleic acids and the interactive qualities of the peptides encoded thereby, a strand of nucleic acid contains not only the information for one peptide but also that for a second peptide which is meant to interact with the first (38). It has been postulated that the interacting domains (encoded by the functional exons) of two proteins, such as a receptor and its ligand, co-evolved in this manner (38-40).
  • complementary anti-sense peptides which bind to a target protein would affect the biological activities of the target protein.
  • biologically active proteins such as antibodies, cytokines. and hormones can be functionally affected by the binding of complementary anti-sense peptides (42-56).
  • peptide ligands for any target protein could be identified among the complementary peptides of the target protein, numerous experimental observation also reveal the specific binding between peptides with opposite hydropathy profiles. Therefore, an efficient way of identifying ligands for a target is to screen a pool of complementary peptides.
  • Sequences of complementary peptides to a target protein/peptide can be constructed by using computer programs (e.g., see 15, 70). However, because the "native" hydropathy of a peptide is not only determined by its sequence, but also strongly influenced by the flanking regions, a better TCL is prepared by linking appropriate flanking amino acids to complementary peptides (15).
  • a relatively simple way of constructing a TCL is to prepare a gene fragment anti-sense TCL.
  • the TCL is prepared by amplifying fragments of the target gene by polymerase chain reaction (PCR) using random hexamer oligonucleotide primers or other universal primers (57). Because sequences encoded by the anti-sense DNA strand of the target gene is just as well represented as that of the sense strand, a complete collection of the anti-sense peptides with their various flanking sequences are encoded by approximately one half of the genetic packages so prepared.
  • the fraction encoding the sense strand peptides could be excluded by subtractive hybridization following the generation of a single stranded nucleic acid (e.g., by transcription).
  • a cTCL can be synthesized as follows: residues with complementary hydropathy scores (as defined in Tables 1-3 and refs. 11-13) to each residue in the target sequence are randomly incorporated into the corresponding positions in the library. As a result, the TCL contains all the peptides which complement the target sequence hydropathically. This method is particularly suitable for screening for peptide ligands when the target is a short region in a protein of known sequence.
  • TCL can be displayed on phage as either fusion proteins with the minor phage coat protein pill or the fusion proteins with the major phage coat protein pVIII.
  • fusion proteins with the minor phage coat protein pill or the fusion proteins with the major phage coat protein pVIII.
  • Three to five copies of pill fusion proteins are expressed per phage particle, therefore they are used to display peptides with good binding affinities.
  • pVIII fusion proteins are expressed at a much higher level at about 2,700 copies per particle, they can be used in a first screen when the binding affinities are suspected to be weak (1).
  • the complementary anti-sense peptide is synthesized and included in the elution buffer used to elute phage particles from the immobilized target protein/peptide.
  • the concentration of the complementary anti-sense peptide determines the stringency of the elution buffer, the higher the concentration, the higher the stringency. Phage particles which withstand the high stringency elution carry peptides with high affinity to the target protein/peptide.
  • Peptides with higher affinity can also be generated by linking a peptide identified by TCLT to the polymerization domain of a coiled-coil protein (58), such as GCN4(59) or COMP(60), to create a tetravalent or pentavalent binding molecule.
  • a coiled-coil protein such as GCN4(59) or COMP(60)
  • a constrained TCL can be prepared by carrying PCR reaction with cytosine-containing random hexamer oligonucleotide primers in which either cytosine is present at various positions in every hexamer oligonucleotide primers, or one additional cytosine is added at the 5'- or 3'- end of the random hexamer oligonucleotide sequences. Cystein can be synthesized at both ends of the synthetic library.
  • Phage fUSE5 is used as the vector for the cTCL.
  • the library is made by ligation of synthetic degenerate Bgll deoxyoligonucleotides fragments into the Sfi I site of the fUSE5, and transfection of E. coli with the ligation products by electroporation.
  • the fUSE5 vector and the cloning procedure is described in detail in ref. 71, 72 and 89.
  • Bgll deoxyoligonucleotides fragments are synthesized by solid-phase phosphoramidite chemistry (90), and carry the following sequence:
  • n is the number of amino acid residues in the target peptide.
  • the degenerate sequence of (XXX)n is designed by the following principle: hydrophobic amino acids which are complementary to the hydrophilic amino acids in the target peptide are randomly incorporated in the same positions in cTCL; hydrophilic amino acids which are complementary to the hydrophobic amino acids in the target peptide are randomly incorporated in the same positions in sTCL. Slightly hydrophilic amino acids are used in sTCL corresponding to similar residues in the target peptide.
  • the hydrophilic and hydrophobic amino acid residues are defined by their hydropathic scores as given in Tables 1-3.
  • the second base comprises a mixture containing A and G at molar ratio 6: 1 ; the first base consists of equimolar mixture of A, G, and C. Thymine is not included in the mixture because its presence in the first position would give rise to the stop codon TAA or TAG (91).
  • the third base includes only an equimolar mixture of G and C which is designed to favor codons used by E. coli to express its most abundant proteins (91).
  • the second base comprises a mixture containing T and C at molar ratio 5:2; the first base consists of mixture containing an A:T:C:G molar ratios of 3:3:3:1.
  • the third base again includes only an equimolar mixture of G and C.
  • the first base consists of an equimolar mixture of A, T, C and G; the second base contains a mixture of G, C, and A molar ration 2:2: 1, and third base includes an equimolar mixture of G and C.
  • the oligonucleotides are purified by denaturing PAGE, their complementary strands are synthesized by Klenow DNA polymerase (91). They are subsequently digested with Bgll and ligated with fUSE5 (71, 72, 89).
  • a cTCL carrying peptides with ten amino acid residues will contain approximately 2.8x10 independent clones, which can be completely amplified as a phage display library.
  • a cTCL carrying peptides with eleven amino acids will have approximately 2x10 clones.
  • Variable regions of both the heavy chain and light chain of an immunoglobulin molecule are composed of four framework segments (FRl, FR2, FR3 and FR4) and three complementarity-determining segments (CDR1, CDR2 and CDR3) with the three CDRs separating the four FRs.
  • the residue numbers for these segments were defined by Kabat et al. (80).
  • the CDRs are the segments with highly variable amino acid sequences and are primarily responsible for antigenic specificity of each antibody.
  • the FRs are the relatively conserved segments which provides structural framework for the antigen-binding domain of an antibody.
  • the FR2 of light chain constitutes amino acid 35 to 49
  • FR2 of heavy chain constitutes amino acid 36 to 49 (80).
  • the antigen-binding site is formed by the convergence of six hyper- variable peptide loops which represent six CDRs of both heavy and light chains (81).
  • the FR2 segments of each chain also directly contribute to an antigen-binding site.
  • the FR2 segments of heavy and light chains constitutes the side and base of the antigen-binding surface formed by six CDRs, and appear to be an integral part of the site. Parts of the FR2 seem to be as exposed as the CDR3 loops.
  • the closest contact points between VH and VL domains of Fab are between the FR2 of the heavy chain and the CDR3 of the light chain, and vice versa.
  • the FR2 plays an important role in keeping the structural integrity of Fab since VH and VL need to be closely apposed to each other to form a complete antigen-binding site. Consequently, a polypeptide (or a chemical compound) which specifically binds to the FR2 segment can disrupt the antigen-binding ability of an antibody molecule by (1) altering the topography of an antigen-binding site and thereby preventing antigen binding; and/or (2) acting as a structural hindrance to prevent close contact between the FR2 and the CDR3s in both chains and thereby disrupting the structural integrity of an antigen-binding site.
  • FR2 is the most conserved segment in the variable region, a peptide targeting FR2 may mask antigen-binding activity of antibodies regardless of their antigenic specificities. Furthermore, peptides targeting the FR2 segments in antibodies of different isotypes can be used to mask antigen-binding activities of all the antibodies of the same isotype, i.e., IgE, IgG, IgA, IgM and IgD.
  • An FR2 targeting peptide can be identified by the following approaches:
  • ICAM-1 serves as the cellular receptor for majority of HRVs (82).
  • the extracellular part of the ICAM-1 molecule is composed by five immunoglobulin-like domains (D1-D5).
  • D1-D5 immunoglobulin-like domains
  • Mutational analysis of ICAM-1 has shown that domain Dl contains the primary binding site for rhinoviruses as well as the binding site for its natural ligand lymphocyte function-associated antigen 1 (LFA-1) (83-86).
  • LFA-1 lymphocyte function-associated antigen 1
  • the regions in Dl which have been implicated as the contact sites with HRVs include residues 1, 2, 24-29, 40-49, and 70-77 (83-87). Accordingly, peptide ligands of ICAM- 1 targeting these regions may prevent the binding of HRVs to ICAM-1.
  • Suitable peptide targets include, but are not limited to the following: Residues 1-5: QTSVS (SEQ ID NO. 2) Residues 24-29: SCDQPK (SEQ ID NO. 3) Residues 40-49: KELLLPGNNR (SEQ ID NO. 4) Residues 70-77: PDGQSTAK (SEQ ID NO. 5) Peptide ligands capable of binding to each of the above target peptides are identified by the following procedure:
  • Example 4 Identifying anti-IgE peptides with TCLT
  • allergic rhinitis nonasal allergy
  • Allergic rhinitis can be either seasonal which indicates pollen-allergen sensitivity (also referred to as hay fever), or throughout the year (perennial rhinitis) which tend to be caused by house dust, dust mites, and animal danders (66).
  • Allergic rhinitis is associated with irritating, sometimes disabling symptoms involving upper respiratory tract and eyes, it also imposes morbidity by leading to frequent secondary infections and even asthma attacks.
  • nasal allergy is caused by inflammatory mediators released by the mast cells, such as histamine, serotonin, prostaglandins, and various cytokines.
  • Nasal allergy is also associated with basophils which have been presensitized by IgE.
  • IgE is the least component of the immunoglobulin family in human and animals. Its concentration in normal human serum is very low.
  • Most IgE antibodies bind to the surface of mast cells and basophils through the Fc type 1 receptors (FcRI). Binding of IgE to the corresponding allergen causes clustering of IgE molecules which in turn activates mast cells and basophils and results in the release of inflammatory mediators.
  • FcRI Fc type 1 receptors
  • One approach of preventing allergy is to mask the antigen-binding site of IgE with a peptide which only binds to a single IgE molecule and does not cause cross-
  • Such peptides target IgE at or near the antigen binding site and prevent antigen binding by causing steric hindrance to the binding site.
  • the peptides target the conserved framework regions rather than the hypervariable regions in order to affect IgE molecules of a wide range of antigenic specificities.
  • TCLT is suitable for finding such peptides.
  • Abl antigen binding site in an antibody molecule
  • Ab2beta anti-idiotype antibodies
  • Ab2gamma Another type of anti- idiotype antibody, Ab2gamma, is directed against an idiotype near the binding site and causes steric hindrance to the antigen binding site (62).
  • idiotypes of an antibody are composed by peptide sequences found in both the hypervariable regions (CDRs) and the conserved framework regions of a antibody molecule (62, 69, 70).
  • a peptide targeting the conserved region of an idiotype can alter the idiotype and affect the antigen- binding ability of an entire class of antibody, such as IgE, IgG and IgA.
  • Kang et al. have demonstrated the presence of complementary sequences in human immunoglobulin (52).
  • Peptides affecting antigen-binding ability of human IgE molecules can be identified with TCLT as described in the following examples.
  • Example 5 Blocking antigen-binding ability of an antibody molecule by targeting the framework 2 (FR2) region of the antibody
  • the heavy chain (H) of an antibody can be classified into six families (V H I to V H 6).
  • the general sequences of FR2 region in each family are in the following:
  • V H 1 WVR/QQA P/H/T G/A K/Q G/ER/A LE/GWM/IG (SEQ ID NO.6)
  • V H 2 WIRQPPGKALEWLA (SEQ ID NO.7)
  • V H 3 W V/I R/H Q A P/Q G K G L/P E/V W/YL V S/A/G (SEQ ID NO.8)
  • V H 4 WIVRQPPGKGLEWIG (SEQ ID NO.9)
  • V H 5 WVRQMPGKG/ELEWMG (SEQ ID NO.10)
  • V H 6 WIRQSPSRGLEWLG(SEQIDNO.11)
  • the sequence of the light chain (L) of an antibody is:
  • V L kappa W Y Q Q K P G Q/K P/S/A P K L L I Y (SEQ ID NO.12)
  • Human IgE molecules are mostly composed of heavy chain genes from the V H 5, V H 3, V H 4 and V H 6 families (92,93), therefore the FR2 sequences from these families plus
  • the IgE-blocking peptide can be designed directly based on the molecular recognition theory as in the following: Peptide 1 (binds to FR2 in V H 5): P D A L H G P F A Q(or D) L P H P (SEQ ID NO:
  • Peptide 2 (binds to FR2 in V H 3, V H 4 and V H 6): P D A L G/R G P F A Q/D L P N P (SEQ ID NO. 14)
  • Peptide 3 (binds to FR2 in V L kappa chain): P V L L F R P L R G F E E D I (SEQ ID NO. 15)
  • a human IgE anti- sense TCL or comprehensive TCL library can be screen using the above FR2 target sequences.
  • the human IgE anti-sense TCL is prepared by using coding sequences of the cDNA clones of human IgE light chain and heavy chain.
  • the cDNAs of human IgE are separated from the vector sequences by agarose gel electrophoresis. Equal amounts of each purified inserts are mixed and used as templates for a random priming reaction.
  • the mixed template is boiled with 1 pmol of the primer No. 1 (5'-GACGTGGCCN3-3 ⁇ N can be A, T, C, or G) for 3 min, cooled on ice, mixed with a reaction mixture containing 10 mM Tris.HCl (pH 7.5), 10 mM MgC12, 1 mM dithiothreitol, 0.125 mM each dNTP, and 2.5 units of Klenow fragment of DNA polymerase and incubated at 37 °C for 15 min. Then the reaction mixture is boiled and cooled again, another 2.5 units of Klenow fragment is added, and the reaction mixture is incubated at 37 °C for 15 min.
  • the primer No. 1 5'-GACGTGGCCN3-3 ⁇ N can be A, T, C, or G
  • reaction is stopped by boiling and diluted 1: 10 with TE buffer and products are separated from primers using Microcon-100 microconcentrators (Amicon) with two washes of the retained solution with 200 ml of the TE buffer. After two cycles of priming, some reaction products will incorporate primer sequences at both ends. They are
  • Primer No.l 5'-GACGTGGCCTGTN6-3' (SEQ ID NO. 17) and primer No. 2: 5'-GGCCGACGTGGCCTGT-3' (SEQ ID NO. 18) are used to generate a constrained IgE-TCL.
  • Example 7 Selection of the IgE binding complementary anti-sense peptide by panning the human IgE TCL
  • Human IgE is diluted in PBS to a concentration of 20 mg/ml and is used to coat 3.5 cm wells by incubating for 1 h at 4 °C. The remaining binding sites are saturated by bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • a portion of the amplified IgE-TCL is first incubated for 2 h at 4 °C in a 3.5 cm well precoated with 1 mg/ml BSA in PBS and 1 mM MnC12.
  • the phage unbound to BSA are transferred to a similar well precoated with human IgE.
  • the unbound phage are removed by washing 10 times with PBS buffer containing 0.5% Tween 20.
  • the bound phage are eluted with 0.1 M glycine buffer, pH 2.2, containing 1 mg/ml BSA and 0.1 mg/ml phenol red.
  • the phages are amplified using the K91kan bacteria and partially purified by precipitation with polyethylene glycol (72). The panning is repeated for two more rounds. Sequences carried by the selected phage are then determined using the Sequenase kit (United States Biochemical) with the primer 5'-CCCTCATAGTTAAGCGTAACG-3' (73) (SEQ ID NO. 19).
  • Example 8 Characterizing anti-IgE activities of the complementary anti-sense peptides The peptides selected from the previous step are synthesized and individually tested in the following assays.
  • Assay 1 Histamine release by passively sensitized basophils.
  • reaginic sera are obtained from allergic patients who are allergic to ragweed, rye grass, Chinese elm or other preidentified allergens.
  • Peripheral blood mononuclear cells are isolated from normal individuals by differential centrifugation on Ficoll-Pague (Pharmacia). Aliquots of a cell suspension are incubated with 2- to 10- fold dilution of a reaginic serum, Tris A buffer
  • Tris- ACM buffer pH 7.6 which consists of Tris A buffer plus 0.6 mM CaC12 and 0.01 mM MgC12. They are incubated with peptides at various concentrations for 30 min at 37 °C and then are challenged with either purified ragweed antigen E (0.1 mg/ml) or crude ragweed antigen, crude Chinese elm extracts, and rye grass extracts (Hollister-Stier, Spokane, WA).
  • histamine content in supernatants is measured by the method of Siraganian (75).
  • the peptides which can prevent histamine release triggered by all allergens will be selected.
  • the procedure is carried out as described previously (74, 75). The reactions are done in a single healthy individual whose serum IgE concentration is at low level (around 10 ng/ml). To determine the abilities to block passive sensitization, 20 ml of various concentrations of the peptides is injected intracutaneously into skin sites 1 hr before sensitization of the skin sites with 20 ml of a 50-fold dilution of allergic serum (IgE at 912 ng/ml).
  • control site receives only the diluting buffer before the sensitization. All skin sites are challenged 48 hr after the passive sensitization with allergen extract, such as crude ragweed antigen. The size of wheat and erythema reactions are measured 20 min later by using transparent tape to transfer the outline of the reactions to a paper and expressed by the weight of the paper (76). It has been demonstrated that unsensitized skin sites do not show any wheat or flare reaction upon antigen challenge. Peptides which can prevent or lessen the reactions will be selected.
  • Example 9 Measuring affinity of the IgE-binding peptides and improvement of the peptides
  • BIAcore biosensor Pharmacia Biosensor AB, Uppsala, Sweden
  • HBS HEPES buffered saline
  • Surfactant P20 Pharmacia
  • Human IgE is coupled to the sensor chip by activating the carboxymethylated dextran matrix with 0.2 M N-ethyl-N'-(3-diethylaminopropyl)-
  • [SL] KA[S]t[L]/(l+KA[L]) (79) where [SL] is the concentration of the IgE-peptide complex at equilibrium, [L] is the concentration of the free peptide, and [S]t is the total concentration of IgE.
  • peptides selected in the present invention prevent the binding of allergens to IgE by steric hindrance rather than directly competing with the allergens for binding sites on IgE
  • peptides with apparent affinity comparable or even lower than the affinity between IgE and allergens can be effective in preventing the binding of allergens as long as enough concentration of the peptides is maintained locally so that they remain bound to IgE when the presensitized mast cells or basophils encounter the allergens.
  • an in vitro affinity maturation process can be performed using the TCLT-selected peptides as templates. The details of this approach are described in U.S. patent 5,223,409, incorporated by reference herein in its entirety.
  • the human IgE FR2 segment binding peptides can also be selected by constructing a cTCL against the IgE FR2 segment, and screening the cTCL with a synthetic peptide containing the IgE FR2 sequence. The screening procedure and characterization of the selected peptides are carried out as described above.
  • ICAM-1 intercellular adhesion molecule 1
  • Cell 61:243-254 85. McClelland, A. et al (1991) Identification of monoclonal antibody epitopes and critical residues for rhinovirus binding in domain 1 of intercellular adhesion molecule 1. Proc. Natl. Acad. Sci USA 88:7993-7997 86, Lineberger, D. W. et al (1990) Antibodies that block rhinovirus attachment map to domain 1 of the major group receptor. J. Virol 64:2582-2587

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Abstract

L'invention concerne des méthodes et une composition d'identification de ligands peptidiques de protéines cibles avec des banques complémentaires de cibles. Cette technique permet la sélection de peptides complémentaires présentant une grande affinité de liaison pour les protéines cibles. L'invention concerne également des méthodes et des compositions d'identification de peptides prophylactiques et thérapeutiques contre les allergies et le rhume banal.
PCT/US1999/006537 1998-04-24 1999-04-19 Identification de ligands peptidiques de proteines cibles par la technique des banques complementaires de cibles tclt WO1999055911A1 (fr)

Priority Applications (3)

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AU35465/99A AU762711C (en) 1998-04-24 1999-04-19 Identifying peptide ligands of target proteins with target complementary library technology (TCLT)
EP99917315A EP1078099A4 (fr) 1998-04-24 1999-04-19 Identification de ligands peptidiques de proteines cibles par la technique des banques complementaires de cibles tclt
CA002350163A CA2350163A1 (fr) 1998-04-24 1999-04-19 Identification de ligands peptidiques de proteines cibles par la technique des banques complementaires de cibles tclt

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042279A3 (fr) * 1999-12-13 2001-11-08 Proteom Ltd Ligands peptidiques complementaires produits a partir de genomes vegetaux
WO2001042278A3 (fr) * 1999-12-13 2001-11-08 Proteom Ltd Ligands peptidiques complementaires produits a partir de sequences genomiques microbiennes
WO2001042277A3 (fr) * 1999-12-13 2002-02-21 Proteom Ltd Ligands peptidiques complementaires produits a partir du genome humain
WO2001037191A3 (fr) * 1999-11-19 2002-03-21 Proteom Ltd Procede de manipulation de donnees de sequences de proteines ou d'adn servant a generer des ligands peptidiques complementaires
WO2002032934A1 (fr) * 2000-10-14 2002-04-25 Proteom Limited Peptides antisens bloquant l'interaction avec les recepteurs fc humains et leurs utilisations
US7041288B2 (en) 1999-04-14 2006-05-09 Novartis Ag Anti-idiotypic antibodies against antibodies which inhibit the binding of immunoglobulin E to its high affinity receptors
EP2609934A1 (fr) * 2007-02-16 2013-07-03 KTB Tumorforschungsgesellschaft mbH Récepteur et promédicament ciblé antigène

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077195A (en) * 1985-03-01 1991-12-31 Board Of Reagents, The University Of Texas System Polypeptides complementary to peptides or proteins having an amino acid sequence or nucleotide coding sequence at least partially known and methods of design therefor
US5081584A (en) * 1989-03-13 1992-01-14 United States Of America Computer-assisted design of anti-peptides based on the amino acid sequence of a target peptide
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5723286A (en) * 1990-06-20 1998-03-03 Affymax Technologies N.V. Peptide library and screening systems

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077195A (en) * 1985-03-01 1991-12-31 Board Of Reagents, The University Of Texas System Polypeptides complementary to peptides or proteins having an amino acid sequence or nucleotide coding sequence at least partially known and methods of design therefor
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5081584A (en) * 1989-03-13 1992-01-14 United States Of America Computer-assisted design of anti-peptides based on the amino acid sequence of a target peptide
US5723286A (en) * 1990-06-20 1998-03-03 Affymax Technologies N.V. Peptide library and screening systems

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1078099A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7041288B2 (en) 1999-04-14 2006-05-09 Novartis Ag Anti-idiotypic antibodies against antibodies which inhibit the binding of immunoglobulin E to its high affinity receptors
WO2001037191A3 (fr) * 1999-11-19 2002-03-21 Proteom Ltd Procede de manipulation de donnees de sequences de proteines ou d'adn servant a generer des ligands peptidiques complementaires
US6721663B1 (en) 1999-11-19 2004-04-13 Proteom Limited Method for manipulating protein or DNA sequence data in order to generate complementary peptide ligands
WO2001042279A3 (fr) * 1999-12-13 2001-11-08 Proteom Ltd Ligands peptidiques complementaires produits a partir de genomes vegetaux
WO2001042278A3 (fr) * 1999-12-13 2001-11-08 Proteom Ltd Ligands peptidiques complementaires produits a partir de sequences genomiques microbiennes
WO2001042277A3 (fr) * 1999-12-13 2002-02-21 Proteom Ltd Ligands peptidiques complementaires produits a partir du genome humain
WO2002032934A1 (fr) * 2000-10-14 2002-04-25 Proteom Limited Peptides antisens bloquant l'interaction avec les recepteurs fc humains et leurs utilisations
EP2609934A1 (fr) * 2007-02-16 2013-07-03 KTB Tumorforschungsgesellschaft mbH Récepteur et promédicament ciblé antigène
US9216228B2 (en) 2007-02-16 2015-12-22 KTB Tumorforschungsgesellschaft MBM Receptor and antigen targeted prodrug

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AU762711C (en) 2004-05-27
EP1078099A1 (fr) 2001-02-28
AU762711B2 (en) 2003-07-03
AU3546599A (en) 1999-11-16
CA2350163A1 (fr) 1999-11-04

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