EP0954531A1 - PEPTIDES IMMUNOREACTIFS PAR RAPPORT AUX aPL, LEURS CONJUGUES ET PROCEDES DE TRAITEMENT DES PATHOLOGIES DEPENDANTES DE L'ANTICORPS aPL - Google Patents

PEPTIDES IMMUNOREACTIFS PAR RAPPORT AUX aPL, LEURS CONJUGUES ET PROCEDES DE TRAITEMENT DES PATHOLOGIES DEPENDANTES DE L'ANTICORPS aPL

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Publication number
EP0954531A1
EP0954531A1 EP97933138A EP97933138A EP0954531A1 EP 0954531 A1 EP0954531 A1 EP 0954531A1 EP 97933138 A EP97933138 A EP 97933138A EP 97933138 A EP97933138 A EP 97933138A EP 0954531 A1 EP0954531 A1 EP 0954531A1
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EP
European Patent Office
Prior art keywords
apl
phage
antibody
peptide
analog
Prior art date
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Withdrawn
Application number
EP97933138A
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German (de)
English (en)
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EP0954531A4 (fr
Inventor
Edward Jess Victoria
David Matthew Marquis
David S. Jones
Lin Yu
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La Jolla Pharmaceutical Co
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La Jolla Pharmaceutical Co
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Priority claimed from US08/660,092 external-priority patent/US6207160B1/en
Application filed by La Jolla Pharmaceutical Co filed Critical La Jolla Pharmaceutical Co
Publication of EP0954531A4 publication Critical patent/EP0954531A4/fr
Publication of EP0954531A1 publication Critical patent/EP0954531A1/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/03—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in epitope analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the field of immunology and relates to compositions and methods for treating and diagnosing antiphospholipid (aPL) antibody-mediated pathologies. More specifically, the invention relates to conjugates of chemically- defined nonimmunogenic valency platform molecules and immunospecific analogs of aPL-binding epitopes as well as methods and compositions for producing these conjugates. Optimized analogs lack T cell epitopes. In addition, the invention relates to diagnostic assays for detecting the presence of and quantitating the amount of antiphospholipid antibodies in a biological sample. The invention also relates to a method of utilizing random peptide libraries to identify immunospecific analogs of aPL-binding epitopes.
  • Antiphospholipid antibodies occur in autoimmune diseases such as systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome (APS) as well as in association with infections and drug therapy.
  • SLE systemic lupus erythematosus
  • APS antiphospholipid antibody syndrome
  • APS is characterized by one or more clinical features such as arterial or venous thrombosis, thrombocytopenia and fetal loss.
  • APS may be primary or it may be associated with other conditions, primarily SLE (PHOSPHOLIPID-BINDING ANTIBODIES (Harris et al , eds., CRC Press, Boca Raton, FL, 1991); McNeil et al. ADVANCES IN IMMUNOLOGY, Vol. 49, pp.
  • Transient aPL antibodies such as those detected in a VDRL test, occur during many infections. Approximately 30% of patients possessing persistent aPL antibodies have suffered a thrombic event. The presence of aPL antibodies defines a group of patients within SLE who display a syndrome of clinical features consisting of one or more of thrombosis, TCP, and fetal loss. The risk of this syndrome in SLE overall is around 25%; this risk increases to 40% in the presence of aPL antibodies and decreases to 15% in their absence.
  • aPL antibodies were thought to be directed at phospholipids in plasma membranes, it has been postulated that they may exert direct pathogenic effects in vivo by interfering with hemostatic processes that take place on the phospholipid membranes of cells such as platelets or endothelium.
  • the fact that aPL antibodies appear to be the only risk factor present is further evidence that these antibodies have a direct pathogenic role.
  • Induction of PAPS by immunizing mice with human anticardiolipin antibodies is the best evidence yet that aPL antibodies are directly pathogenic (Bakimer et al 1992 J Clm Invest 89:1558-1563; Blank et al 1991 Proc Nat! Acad Sci 88:3069-3073)
  • aPL antibodies recognize an antigenic complex comprised of ⁇ 2 -glycoprotein I ( ⁇ 2 -GPI) and negatively-charged phospholipid, e.g., cardiolipin (McNeil et al. (1990) Proc. Natl. Acad. Sci. 87:4120- 4124; Galli et al. (1990) Lancet 1:1544-1547) (hereinafter "aPL immunogen").
  • ⁇ 2 -GPI is a minor plasma glycoprotein found free and in association with lipoprotein lipids where it is also known as apolipoprotein H (apo H). It consists of five independently folding domains referred to as Sushi or short consensus repeat domains that resemble similar domains in other proteins.
  • ⁇ 2 -GPI has been reported to undergo antigenic and conformational changes upon binding phospholipid (Wagenkneckt et al. (1993) Thromb. Haemostas. 69:361-365; Jones et al. (1992) Proc. 5th Intl. Symp. Antiphospholipid Antibodies (Abstract S5)).
  • the fifth domain contains the putative sites of lipid binding and aPL antibody binding (Hunt J. and S. Krilis, (1994) J. Immunol. 152:653-659; Lauer et ⁇ /. (1993) Immunol. 80:22-28).
  • the pathological mechanism for aPL is unknown (McNeil et al, supra).
  • phage libraries are prepared by incorporating randomized oligonucleotide sequences into the phage genome, usually the pill gene, which encode unique peptide sequences on the surface of each phage. Following sequential rounds of affinity purification and amplification, those phage that bind antibody are propagated in E. coli and the binding peptides identified by sequencing the corresponding coding region of viral DNA. In most cases, subsequent study will involve corresponding synthetic peptides after establishing their ability to bind antibody. Phage-based libraries have been used to mimic discontinuous epitopes (Luzzago et al.
  • B cell tolerance entails administering such peptides conjugated to multivalent, stable, non-immunogenic valency platforms in order to abrogate antibody production via B cell anergy or clonal deletion after cross-linking surface immunoglobulin.
  • This invention resides in the discovery of a method for identifying analogs of key epitopes recognized by aPL antibodies in patients suffering from PAPS, APS and other aPL antibody-mediated diseases, such as recurrent stroke and recurrent fetal loss, using random peptide phage libraries.
  • one aspect of the invention is an improved method for screening random peptide phage libraries in order to identify the peptide sequences which best mimic the epitopes recognized by aPL antibodies.
  • This method comprises the steps of: (a) biopanning the library using methods modified from those known in the art; (b) eliminating very weakly-binding phage by micropanning the phage screened from step (a) by (i) incubating the phage in microplate wells coated with aPL antibody bound to Protein G, (ii) washing the microplate wells to remove unbound phage, (i ⁇ ) eluting the bound phage, and (iv) infecting a microorganism such as E.
  • the invention encompasses a method for identifying analogs of epitopes which specifically bind aPL antibodies isolated from humans suffering from an aPL antibody-mediated disease comprising: (a) preparing phage random peptide libraries; (b) screening said libraries with aPL antibodies to identify aPL mimetic epitopes, wherein said screening comprises (i) screening said libraries by biopanning; (ii) further screening phage isolated by biopanning in (i) by micropanning; and (iii) identifying phage containing aPL antibody high-affinity binding peptides recovered in (ii) by immunoassay.
  • the invention also encompasses a method of biopanning phage random peptide libraries to identify and isolate peptides which bind to aPL antibody comprising: (a) reacting affinity-purified aPL antibody with phage bearing random peptide inserts; (b) recovering phage bearing random peptide inserts which bind to the aPL antibody; (c) infecting a microorganism with phage recovered in (b); and (d) culturing the infected microorganism in an antibiotic-containing medium in order to isolate the phage.
  • the invention further encompasses a method of micropanning phage random peptide libraries to identify and isolate peptides having a high binding affinity to aPL antibodies comprising: (a) isolating phage bearing random peptide inserts by biopanning; (b) incubating the phage recovered in step (a) in microplate wells coated with aPL antibody bound to Protein G; (c) washing the microplate wells to remove unbound phage;
  • the invention also encompasses the above method described wherein the immunoassay is a phage-capture ELISA comprising: (a) incubating phage bearing random peptide inserts isolated by micropanning in the microplate wells coated with aPL antibody; (b) washing away unbound phage;(c) incubating an enzyme-labeled anti-phage antibody to the wells; (d) washing away unbound enzyme-labeled anti-phage antibody;
  • the invention also encompasses the method described above wherein the immunoassay is a colony-blot immunoassay comprising: (a) culturing a microorganism infected with phage bearing random peptide inserts on a nitrocellulose membrane atop an agar-containing culture medium; (b) replicate transferring the microorganism cultured in (a) by blotting the microorganism on a second nitrocellulose membrane atop an agar- containing culture medium; (c) incubating the transferred microorganism;(d) lysing the microorganism; (e) digesting the microorganism with lysozyme; (f) blocking the membrane with a gelatin solution; (g) incubating the membrane with aPL antibody; (h) washing away unbound aPL antibody; (i) incubating a enzyme-labeled anti-aPL antibody with the nitrocellulose membrane; (j) washing away unbound enzyme-labeled anti-aPL antibody; (k)
  • a method for assaying and ranking, for affinity-binding characteristics, epitopes which specifically bind aPL antibodies isolated from humans suffering from an aPL antibody-mediated disease comprising: (a) coating wells of a microtitration plate with cardiolipin; (b) adding adult bovine or human serum as a source of ⁇ 2 -GPI to bind to the cardiolipin and to prevent non-specific binding to the wells of the plate; (c) incubating a solution of monomeric analog and a high-titere aPL antibody for a pre-determined time; (d) adding the aPL antibody/analog mixture to wells of the microtitration plate and incubating for a pre-determined time; (e) washing the wells to wash away unbound aPL antibody; (f) adding anti-human IgG conjugated with a label (e.g., an enzyme) to the wells of the plate and incubating for a pre-determined time; (g) washing the wells to
  • Another aspect of the invention encompasses a fluorescence polarization peptide binding assay for determining the dissociation constants for peptides that bind to aPL antibodies. This assay detects direct binding of peptides to aPL antibodies.
  • the invention also encompasses a diagnostic immunoassay for determining the presence of aPL antibody in body fluids taken from subjects suspected of suffering from an aPL antibody-mediated disease comprising contacting a sample of a body fluid with an analog of an epitope which specifically binds aPL antibodies and determining by methods well known in the art whether aPL antibodies are present in the body fluid and, if present, quantitating the amount of aPL antibodies present in the fluid.
  • One such immunoassay comprises: (a) coating wells of a microtitration plate with an analog of an epitope which specifically binds aPL antibodies; (b) washing the wells to wash away unbound analog; (c) adding a test sample of a body fluid to the wells and incubating for a pre-determined time; (d) washing the wells to remove unbound test sample; (e) adding anti-human IgG conjugated with a label to the wells of the plate and incubating for a pre-determined time;(f) washing the wells to wash away unbound anti-human IgG conjugate; (g) adding a substrate for the labeled conjugate and developing the substrate/label reaction for a pre-determined time; (h) measuring the end-product of the substrate/label reaction to determine the presence of anti-aPL antibody in the test sample.
  • a diagnostic immunoassay as described above wherein the immunoassay is quantitative is also encompassed.
  • the phage-ELISA assay consists of (i) coating a uniform amount of different clones on wells of a microtitration plate followed by (ii) identifying the peptide inserts which most strongly bind aPL antibody by adding antibody to the wells and developing the reaction with an enzyme-labeled anti-human IgG conjugate.
  • the random peptides displayed by the phage which have a high binding affinity to aPL antibody as measured by phage-ELISA, colony blot or phage-capture-ELISA represent the analogs of the aPL-specific epitope. These peptides are then synthesized and ranked for strength of binding using competition assays.
  • Another aspect of the invention is aPL antibody-binding analogs that bind specifically to B cells to which an aPL epitope binds. Optimized analogs lack T cell epitope(s).
  • compositions for inducing specific B cell tolerance to an aPL immunogen comprising a conjugate of a nonimmunogenic valency platform molecule and an aPL antibody-binding analog that (a) binds specifically to B cells to which an aPL immunogen binds and (b) lacks T cell epitope(s).
  • Figure 1 shows that the substitution offish gelatin for adult bovine serum abolished all anticardiolipin (ACA) activity in an ELISA assay of a commercial aPL antibody standard. This result supported the findings of McNeil et al, supra, and Galli et al, supra, concerning the importance of ⁇ 2 -glycoprotein I ( ⁇ 2 -GPI) in defining the target epitope(s) of ACA.
  • Figure 2 shows that resin-bound analog 5A12 immunospecifically binds to affinity-purified IgG designated ACA-6501.
  • Figure 3 illustrates that the aPL antibody-binding analogs derived from screening with ACA-6626 bound aPL antisera but did not bind normal sera.
  • Figure 4 also illustrates that the ACA-6501/5A12 analog immunospecifically binds ACA-6501 antiserum and is crossreactive with ACA-6626.
  • Figures 4 and 5 illustrate that while the ACA-6501/5A12 and ACA-6626/4D3 aPL antibody-binding analogs derived from screening with methods within the instant invention bind preferentially with the screening antibody, a significant degree of crossreactivity was detected.
  • Figure 6 illustrates method for calculating the GPL value for ACA-6501 aPL antibody.
  • Figure 7 shows the activity of affinity-isolated ACA-6501 compared to GPL standard sera.
  • Figure 8 illustrates the dramatic drop in sequence diversity of the isolated clones by the fourth round of biopanning. ⁇ 0
  • Figure 9 illustrates that three clones (3A12, 3B3 and 3A5) exhibited a very strong immunospecific signal in the phage-capture ELISA using ACA-6635 whereas all clones tested were unreactive with normal IgG.
  • Figure 10 shows the strong signal exhibited by seven clones in a phage-ELISA using ACA-6501.
  • Figure 11 shows the results of a competitive-binding ELISA obtained with peptides 5A12, CB2 and 3B10 using ACA-6501.
  • 0.16 ⁇ g of Peptide 5A12 produced 50% inhibition of binding of ACA-6501 aPL antibody to tetravalent peptide ACA- 6501/3B10 bound to polystyrene microplate wells, whereas 0.08 ⁇ g of CB2 and 0.5 ⁇ g of 3B10 were required to produce 50% inhibition.
  • Figure 12 illustrates the comparative activity of modified ACA-6641/3G3 analogs.
  • Figure 13 illustrates that the 50% inhibition values for peptides 139, 142 and 143 in a competitive-binding ELISA using ACA-6501 aPL antibody were 6.9, 0.7 and 0.9 ⁇ g, respectively.
  • Figure 14 illustrates the effects of substituting ⁇ -Me-Pro at positions 3, 9 and both 3 and 9 in peptide 3B10. Substitution of ⁇ -Me-Pro at position 9 increased activity of the peptide four-fold compared to the "native" peptide.
  • Figure 15 shows that peptide 6641/3G3 (LJP 688) is highly cross-reactive with nine affinity-purified ACA antibodies.
  • Figures 16 and 17 show the dose-dependent reduction in anti-685 antibody ABC at 10 8 M using spleen cells from mice immunized with LJP 685-KLH after the spleen cells were incubated with 100, 20 and 4 ⁇ M of LJP 685-MTU-D ABA-PEG conjugate (compound 3 > and LJP 685-ATU-MTU-AHAB-TEG conjugate (compound 3_5_), respectively, for 2 hours.
  • Figure 18 displays the NMR structure closest to the centroid of the nine structures elucidated for peptide 925 and is a reasonable representation of the shape of the peptide 925 molecule.
  • Figure 19 compares the structure of peptide 925 (labeled at the bottom of the figure as 3G3) with the structure of peptide 5A12. Both peptides have turns at approximately the same positions in the peptide sequence.
  • Figures 20A and 20 B illustrate that the pharmacophore of aPL analogs has been tentatively identified as a small hydrophobic group and a positively charged group.
  • the gem-dimethyl and amino groups of peptide 925 are tentatively identified as the pharmacophore of this peptide as shown in Figure 20 A.
  • the lengths of the hydrocarbon linkers that tether the pharmacophore groups to some scaffold are specified in Figure 20A as well as the distances separating the points at which these linkers are attached to the scaffold.
  • Figure 21 illustrates inhibition of ⁇ 2 GPI by 6501 -derived peptides with diluted 6501 serum.
  • Figure 22 shows the ACA-6701 titration of CB2*-F; FITC- GPCILLARDRCG.
  • Figure 23 shows the ACA-6501 titration of CB2*-F: FITC- GPCILLARDRCG.
  • Figure 24 shows complete ACA-6501 titration of CB2*-F: FITC- GPCILLARDRCG.
  • Figure 25 shows the displacement of CB2*-F from ACA-6701 using 1.04 equivalents of CB2*.
  • Figure 26 shows cFP titration using CB2* to displace CB2*-F from ACA- 6701.
  • Figure 27 shows cFP titration using 3B10 to displace CB2*-F from ACA- 6701.
  • Figure 28 shows the dose response of the (LJP685) 4 /MTU-AHAB-TEG conjugate for tolerance activity.
  • Figure 29 shows the dose response of the (LJP685) 4 /MTU-DABA-TEG conjugate for tolerance activity.
  • Figure 30 shows the dose response of tolerance activity of the (LJP685) 4 /MTU-AHAB-TEG conjugate tested in the in vitro model.
  • Figure 31 shows the dose response of tolerance activity of the (LJP685) 4 /MTU-DABA-TEG conjugate tested in the in vitro model.
  • Figure 32 shows the tolerizing effect of the (LJP685) 4 /MTU-AHAB-TEG conjugate comparing various administrative routes and dosage ranges.
  • aPL antibody means any antibody which specifically binds ⁇ 2-GPI that mediates disease.
  • B cell anergy intends unresponsiveness of those B cells requiring T cell help to produce and secrete antibody and includes, without limitation, clonal deletion of immature and/or mature B cells and/or the inability of B cells to produce antibody.
  • Unresponsiveness means a therapeutically effective reduction in the humoral response to an immunogen. Quantitatively the reduction (as measured by reduction in antibody production) is at least 50%, preferably at least 75%, and most preferably 100%.
  • Antibody means those antibodies which are T cell dependent.
  • immunogen means an entity that elicits a humoral immune response comprising aPL antibodies. Immunogens have both B cell epitopes and T cell epitopes. aPL immunogens that are involved in aPL antibody-mediated pathologies may be external (foreign to the individual) immunogens such as drugs, including native biological substances foreign to the individual such as therapeutic proteins, peptides and antibodies, and the like or self-immunogens (autoimmunogens) such as those associated with antibody-mediated hypercoagulability (stroke).
  • drugs including native biological substances foreign to the individual such as therapeutic proteins, peptides and antibodies, and the like or self-immunogens (autoimmunogens) such as those associated with antibody-mediated hypercoagulability (stroke).
  • analog of an immunogen intends a molecule that (a) binds specifically to an antibody to which the immunogen binds specifically and (b) lacks T cell epitopes.
  • the analog will normally be a fragment or derivative of the immunogen and thus be of the same chemical class as the immunogen (e.g., the immunogen is a polypeptide and the analog is a polypeptide), chemical similarity is not essential. Accordingly, the analog may be of a different chemical class than the immunogen (e.g., the immunogen is a carbohydrate and the analog is a polypeptide) as long as it has the functional characteristics (a) and (b) above.
  • the analog may be a peptide, carbohydrate, lipid, lipopolysaccharide, nucleic acid or other biochemical entity. Further, the chemical structure of neither the immunogen nor the analog need be defined for the purposes of this invention.
  • the term “analog” of an immunogen also encompasses the term “mimotope.”
  • the term “mimotope” intends a molecule which competitively inhibits the antibody from binding the immunogen. Because it specifically binds the antibody, the mimotope is considered to mimic the antigenic determinants of the immunogen.
  • valency platform molecule means a nonimmunogenic molecule containing sites which facilitate the attachment of a discreet number of analogs of immunogens.
  • Nonimmunogenic is used to describe the valency platform molecule and means that the valency platform molecule elicits substantially no immune response when it is administered by itself to an individual.
  • mammals denotes a member of the mammalian species and includes humans, primates, mice and domestic animals such as cattle and sheep, sports animals such as horses, and pets such as dogs and cats.
  • pharmacophore means the three dimensional orientation and chemical properties of key groups involved in binding of an aPL analog to the antibody target.
  • aPL antibody-binding analogs may be identified by screening candidate molecules to determine whether or not they (a) bind specifically to aPL antibodies and (b) lack T cell epitopes. Specific binding to aPL antibodies may be determined using conventional immunoassays such as the ELISA assays described in the examples below and the presence or absence of T cell epitopes may be determined by conventional T cell activation assays also described in the examples. In this regard, an analog which "binds specifically" to serum antibodies to the immunogen exhibits a reasonable affinity thereto, e.g., 10 M .
  • T cell epitopes may be determined using a tritiated thymidine incorporation assay disclosed in Serial No. 08/118,055.
  • the presence of T cell epitopes can also be determined by measuring secretion of T cell-derived lymphokines by methods well known in the art. Analogs that fail to induce statistically significant incorporation of thymidine above background are deemed to lack T cell epitopes. It will be appreciated that the quantitative amount of thymidine incorporation may vary with the immunogen.
  • a stimulation index below about 2-3, more usually about 1-2, is indicative of a lack of T cell epitopes.
  • aPL antibody-binding analogs are coupled to a nonimmunogenic valency platform molecule to prepare the conjugates of the invention.
  • Preferred valency platform molecules are biologically stabilized, i.e., they exhibit an in vivo excretion half-life often of hours to days to months to confer therapeutic efficacy, and are preferably composed of a synthetic single chain of defined composition. They will normally have a molecular weight in the range of about 200 to about 200,000, usually about 200 to about 20,000.
  • Examples of valency platform molecules within the present invention are polymers such as polyethylene glycol (PEG), poly-D_-lysine, polyvinyl alcohol and polyvinylpyrrollidone. Preferred polymers are based on polyethylene glycols (PEGs) having a molecular weight of about 200 to about 8,000.
  • valency platform molecules suitable for use within the present invention are the chemically-defined, non-polymeric valency platform molecules disclosed in co-owned, co-pending U.S. patent application Serial No. 08/152,506, filed November 15, 1993, which is incorporated by reference herein in its entirety.
  • Particularly preferred homogeneous chemically-defined valency platform molecules suitable for use within the present invention are derivatized 2,2'-ethylenedioxydiethylamine (EDDA) and triethylene glycol (TEG).
  • Suitable valency platform molecules include tetraaminobenzene, heptaaminobetacyclodextrin, tetraaminopentaerythritol, 1,4,8,1 1- tetraazacyclotetradecane (Cyclam) and 1,4,7,10-tetraazacyclododecane (Cyclen).
  • Conjugation of the aPL antibody-binding analog to the valency platform molecule may be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the analog and valency platform molecule.
  • Polypeptide analogs will contain amino acid sidechain moieties containing functional groups such as amino, carboxyl, or sulfhydryl groups that will serve as sites for coupling the analog to the carrier. Residues that have such functional groups may be added to the analog if the analog does not already contain these groups. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. In the case of carbohydrate or lipid analogs, functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry.
  • primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent.
  • the valency platform molecule may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.
  • Linkers of variable lengths are useful for connecting peptides or other bioactive molecules to valency platform molecules.
  • Suitable linkers include linear oligomers or polymers of ethylene glycol.
  • linkers are useful in connecting a molecule containing a thiol reactive group such as haloaceyl, maleiamide, etc., via a thioether to a second molecule which contains an amino group via an amide bond.
  • linkers are flexible with regard to the order of attachment, i.e., the thioether can be formed first or last.
  • the conjugates will normally be formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, they will typically be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • the conjugate will normally constitute about 0.01% to 10% by weight of the formulation.
  • the conjugate is administered to an individual in a "therapeutically effective amount", i.e., an amount sufficient to produce B cell anergy to the involved immunogen and effect prophylaxis, improvement or elimination of the antibody-mediated condition being addressed.
  • the particular dosage regimen i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history.
  • a dose of about 1 ⁇ g to about 100 mg conjugate/kg body weight, preferably about 100 ⁇ g to about 10 mg/kg body weight, will be given weekly.
  • Other appropriate dosing schedules would be as frequent as daily or 3 doses per week, or one dose per week, or one dose every two to four weeks, or one dose on a monthly or less frequent schedule depending on the individual or the disease state.
  • Repetitive administrations normally timed according to B cell turnover rates, may be required to achieve and/or maintain a state of humoral anergy. Such repetitive administrations will typically involve treatments of about 1 ⁇ g to about 10 mg/kg body weight or higher every 30 to 60 days, or sooner, if an increase in antibody titer is detected.
  • sustained continuous release formulations of the conjugates may be indicated for some pathologies.
  • Various formulations and devices for achieving sustained release are known in the art.
  • Anti-helper T cell treatments may be administered together with the conjugates.
  • Such treatments usually employ agents that suppress T cells such as steroids or cyclosporin.
  • NMR nuclear magnetic resonance
  • multilamellar, cardiolipin- - containing dispersions liposomes; also containing cholesterol and dicetylphosphate
  • liposomes are pelleted from the serum by centrifugation. After washing, the liposome mixture is disrupted by 2% octylglucoside detergent and applied to a protein A-agarose column. Following extensive washings to first remove lipids and then to remove non-IgG components, IgG aPL antibody is eluted from protein A with mild acid, neutralized, buffer- exchanged, and tested in the ACA ELISA.
  • This procedure yields aPL antibody enriched up to 10,000-fold that is devoid of any contaminating ⁇ 2 -GPI as shown by western blotting with rabbit IgG anti-human ⁇ 2 -GPI antisera.
  • An additional affinity- purification step is performed by chromatography of the affinity-purified antibody on solid phase ⁇ 7 -GPI
  • This second affinity-purification step is recommended as a result of the new awareness regarding the greater clinical relevance of aPL antibodies that directly bind to ⁇ 2 -GPI. It also serves to further ensure a final preparation devoid of contaminants, in particular ⁇ 2 -GPI.
  • the "y” library is the same as the “y”' library except that it lacks the 6 and 8 amino acid inserts. These peptide inserts for both “y” and “y' “ libraries are flanked by cysteine residues at both the amino and carboxyl ends to form cyclic, more rigid structures. Proline residues are incorporated outside these cysteine residues for reasons similar to those for the "x” libraries above.
  • the "x,” “y' “, and “y” libraries are located five residues from the amino terminus of the native p-III protein.
  • the "z” library consists of random eight amino acid inserts located at the amino terminus of the p-III protein and do not contain any flanking proline or cysteine residues. A combination of the "x,” “y 1 " and “z” libraries represents eleven different libraries each with approximately one hundred million different peptide inserts.
  • libraries are constructed by incorporation of random oligonucleotide sequences of the length appropriate to give the desired length insert into the p-III gene of fUSE 5 using standard molecular biology techniques. Following restriction endonuclease digestion of the fUSE 5 DNA, an excess of kinased oligonucleotides provided as gapped duplexes is added and ligated. The DNA is then electroporated into E. c ⁇ li and inserts are selected by culturing in tetracycline-containing media. The phage from this culture (which contain the peptide insert) are isolated from the supernatant, washed and resuspended in buffer. Typically libraries are shown to have 7 X 10 8 independent clones at 8 X 10 transducing units per mL.
  • Biopanning describes the technique wherein affinity-purified aPL antibody and phage bearing random peptide inserts are allowed to mix, following which antibody-specific recovery captures the bound phage.
  • the phage confer tetracycline resistance to E. coli that are propagated in a tetracycline-containing medium and then isolated. Multiple rounds of biopanning enrich the number of immunospecific phage in a sample. Phage are always recovered at the end of three to five rounds of selection but may represent only sequences that are nonspecifically bound at low affinities for the selecting antibodies. A method for further evaluating these phage (micropanning) is required.
  • AO anti-purified aPL antibody and phage bearing random peptide inserts
  • Micropanning is carried out following three or more rounds of biopanning and uses the same antibody as employed in the biopanning method. The method consists of dilution of the phage from the last round of biopanning and analyzing fifty or more of these clones by micropanning. Micropanning is accomplished by growing each clone to a similar density and then incubating dilute phage at an optimal single concentration in microtitration wells previously coated with a constant amount of antibody.
  • the optimal single concentration of phage is that concentration most likely to reveal the widest range of micropanning scores (from 0 to 4+) and, thus, permit the greatest discrimination among the clones being tested. It is based on the micropanning behavior of six randomly selected clones where the score is determined at each of several concentrations of phage obtained by serial dilution. Following the incubation with antibody, the unbound phage are washed away and the amount of bound phage is used as an indication of the affinity of the phage insert for the antibody. The amount of bound phage is determined by elution with mild acid followed by neutralization and infection of E. coli. The number of infected E. coli are then quantitated by plating the microorganisms on agar plates containing tetracycline and then determining colony densities achieved by each clone, (iii) Phage-Capture ELISA
  • the phage-capture ELISA test was developed to provide an intermediate level assay to bridge the gap between the relatively low stringency of the micropanning assay and the high stringency of the phage- or peptide-ELISA assays.
  • Preliminary studies show that some antibody preparations give too many positive clones by micropanning but none by phage-ELISA or peptide-ELISA.
  • the limitation of the phage-ELISA described below is that only five copies of p-III are located on each phage and even with a large number of phage coated on a well, few copies of the insert are represented and detection requires that the antibody have a very high affinity for the insert.
  • the phage-capture ELISA With the phage-capture ELISA, the signal is amplified many times which facilitates the detection of lower affinity, stable interactions between the antibody and the insert.
  • the phage-capture ELISA consists of the following steps. Microtitration wells are coated with aPL antibody and phage clones are added as in the micropanning assay. Unbound phage are washed away and the amount of bound phage is quantitated using an enzyme-conjugated goat antiserum which binds the major coat protein of the phage. Phage screened using phage-capture ELISA react with many aPL antibodies and provide a strong signal in subsequent ELISA assays.
  • Phage are directly coated onto wells of a microtitration plate and incubated with the screening antibody. Following washes to remove unbound antibody, an anti-human IgG alkaline phosphatase conjugate is added to bind any aPL antibodies bound to the phage. APL antibodies are then detected by adding a colorimetric substrate to the well which will react with alkaline phosphatase according to methods well known in the art.
  • This assay allows large-scale colony screening of E. coli infected by biopanned phage.
  • This procedure is an alternative to phage-ELISA for identifying immunoreactive clones and exhibits a comparable level of sensitivity without requiring culturing of individual phage clones prior to testing.
  • E. coli infected with phage from a round of biopanning are spread on a large diameter nitrocellulose (NC) membrane and cultured overnight on the surface of an agar plate containing tetracycline (Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978- 7982). Each colony results from infection by phage containing identical sequences.
  • the peptides are made using standard Fmoc peptide chemistry as is well known in the art.
  • the peptides can be made, for example, as branched tetravalent molecules, i.e., each molecule has four copies of the insert. Such a molecule can coat the well of a microtitration plate and still have epitopes exposed to the solution to allow binding by an antibody.
  • the tetravalent peptides are synthesized by incorporating lysines as branch points at the first two couplings analogous to the methods used for Multiple Antigenic Peptides (MAPS) (Posnett et al. (1988) J. Biol Chem. 263:1719-1725).
  • a spacer consisting of glycine-serine-glycine-serine is added on each arm after the lysines and then the insert, including the framework amino acids found in the phage, proline-glycine at the carboxyl terminus and alanine-glycine- proline at the amino terminus. All amino acids in this synthesis are added one at a time using standard Fmoc methods.
  • peptides are then assayed by ELISA which is carried out by coating the peptides on microtitration wells and then assaying their reactivity with aPL antibody in a standard ELISA format.
  • ELISA electrospray sorbent assay
  • the peptides usually bind very strongly to the original screening antibody and show some cross-reactivity with other aPL antibodies. Controls of non-aPL antibodies are included to eliminate nonspecific binding peptides.
  • ELISA-positive peptides are identified, it is necessary to quantitate their relative binding affinity to the aPL antibodies and to determine whether or not two peptides bind the same population of antibodies in a given patient serum via a peptide-competition ELISA assay.
  • various monomeric peptides compete with tetravalent peptides coated on a microtitration plate well.
  • the peptides to be evaluated are synthesized as monomers, i.e., without the lysine branches employed in the synthesis of the tetravalent peptides, using standard Fmoc chemistry. The monomeric peptides are then purified and dissolved at known concentrations.
  • Wells of a microtitration plate are coated with a tetravalent peptide known to bind to the aPL antibody.
  • Serial dilutions of the monomeric peptides are incubated with a constant dilution of the aPL antibody.
  • the dilution of the aPL antibody was previously determined by titering the antibody against the tetravalent peptide and selecting a dilution on the downslope of the titration curve.
  • the antibody/peptide solutions are added to the microtitration wells and a standard colorimetric ELISA is performed.
  • the concentration of each monomeric peptide that decreases binding of the aPL antibody with the tetravalent peptide is determined by plotting the colorimetric readings obtained for each well. The 50% inhibition point is used as the measure of the relative strength of binding for the monomeric peptides.
  • a variation of this assay uses microtitration plates coated with human ⁇ 2 - glycoprotein I/cardiolipin ( ⁇ 2 -GPI/CL) instead of tetravalent peptide and tests the ability of monomeric peptides to block the binding of aPL antibody to the epitope(s) on ⁇ 2 -GPI/CL.
  • IgG-depleted human serum at an optimized concentration is used as a source of ⁇ 2 -GPI.
  • the monomeric peptides at several concentrations are incubated with an optimized concentration of aPL antibody in a manner analogous to the assay which employs tetravalent peptide as a plate substrate. Following the incubation of aPL/peptide in ( ⁇ 2 -GPI/CL) plates, antibody binding and the peptide concentration required for 50% inhibition is determined at half-maximal absorbance as in the tetravalent assay.
  • An additional variation of this assay tests the ability of monomeric peptides to block the binding of aPL antibody to ⁇ 2 -GPI coated directly on the wells of Nunc Maxisorp microtitration plates.
  • the use of cardiolipin is omitted and instead offish gelatin, the reagent diluent and blocker used is nonfat milk/Tween.
  • This assay detects direct binding of the peptide to aPL antibody. Since aPL antibodies bind to ⁇ 2 -GPl (the antigen), the ELISA competitive inhibition assay can show inhibition due to binding to ⁇ r GPI as well inhibition due to binding of the peptide to the aPL antibody. Because binding to antibodies is required in order for the peptide to function as a Toleragen, it is essential to establish that a peptide can directly bind to an aPL antibody. This assay is used to determine the dissociation constants for peptides that bind to two aPL antibodies, ACA-6501 and ACA-6701.
  • ELISA-positive peptides should be further evaluated by the Fluorescence Polarization assay to determine whether they are capable of directly binding with aPL antibody.
  • the desired epitope for tolerance induction should have as strong an interaction with as many of the aPL antibodies as possible but not contain any unnecessary residues.
  • analogs of each peptide are made (i) that lack given residues, for example, the framework residues at the carboxyl and/or amino termini are deleted, or (ii) in which amino acid substitutions have been made which differ from sequences found in the epitope library screen.
  • amino acid substitutions may be either natural, e.g., isoleucine for leucine, or unnatural, e.g., alpha methyl proline for proline.
  • the effect of these deletions and/or substitutions are then measured via peptide-competition ELISA.
  • a Tolerogen For a Tolerogen to be generally effective, it must bind a major portion of the aPL antibodies in the majority of patients. It is important to determine if several antibodies from different patients bind identical residues within the eighty-four amino acid 5th domain of ⁇ 2 -GPI which has been suggested by others to contain the target epitope. If several antibodies bind identical residues, a single mimotope derived from the structural data of the peptides can be constructed which will react with all the antibodies. On the other hand, if the antibodies bind to different residues, a unique tolerogen would be required for each antibody. Site-directed mutagenesis was performed to identify if key residues involved in aPL antibody binding reside in the 5th domain of ⁇ 2 -GPI.
  • a fusion protein comprising the 5th domain of ⁇ 2 -GPI and glutathionine S transferase (GST) was obtained from A. Steinkasserer and expressed in E. coli (Steinkasserer et al (1992) FEBS Lett. 313: 193-197). This fusion protein was successfully substituted for native ⁇ 2 -GPI in the ACA ELISA. Amino acid substitutions are engineered using standard site- directed mutagenesis.
  • Antibody ACA-6501 from a patient with a GPL score of 151 (high titer) and a history of recurrent stroke, fetal loss, lupus and three aortic valve replacements was immunoaffinity purified on cardiolipin liposomes.
  • the antibody was used in four separate phage library screens, using the xy, the xyz, the xy'z, and a special pro/cys- bounded 7-mer library where, based on the previous screens, Arg was fixed at the seventh position. As shown in Table 1, 36 sequences were obtained in phage that micropanned (out of 140 tested). These appear quite homologous and the conserved DR residues at positions 6 and 7 are notably striking.
  • the consensus sequence is CLLLAPDRC. Despite this homology, only seven phage (see Table 2) were positive after phage-ELISA colorimetric testing. Screening the xy'z phage with affinity purified ACA-6626 (from a patient with a high, but lower than ACA-6501, titer) yielded five unique sequences that were phage-ELISA tested. None were color positive but two were positive when the ELISA irnmunoconjugate was developed with a chemiluminescent substrate. The sequence motif associated with ACA-6626 appears related but is different from that seen with ACA-6501 (see Table 3). Both antibodies preferred the cys-bounded (probably cyclized and constrained) epitopes over the open pro-bounded sequences.
  • AC A-6644 is another high-titered aPL antibody that was used to screen the pooled p-III phage libraries according to methods described herein. The following sequences were discovered:
  • sequences all were derived from the component "z” epitope library that lacks phage framework residues at the N-terminus.
  • sequences were immunoreactive with several ACA sera including ACA-6644 and ACA-6501. Analysis revealed unsuspected homologies with the sequences previously obtained with ACA-6501 as illustrated in Table 4.
  • sequence homology from two very dissimilar source libraries screened by these two aPL antibodies suggests that the sequences may mimic a major, perhaps immunodominant, region in the native target antigen.
  • ACA-6644/CBf GILALDYV GG Subsequent testing determined that this peptide, ACA-6641/3G3 having the sequence AGP-CLGVLGKLC-PG (LJP 688) was cross-reactive with a number of ACA antisera. Chemical optimization of this peptide was pursued by truncation, systematic amino acid substitution, and non-disulfide cyclization studies.
  • Phage sequences obtained with affinity-purified ACA-6501 and found to be phage-ELISA-positive were synthesized on a solid support recently developed for combinatorial synthetic peptide libraries.
  • This support a Rapp resin, has a high peptide density and uses a hydrophilic polyethylene glycol spacer before the first amino acid is coupled.
  • the synthesis resulted in resin-bound peptide that was ideally suited for antibody binding studies.
  • peptide 5A12 sequence CLILAPDRC
  • Similar results were obtained with the other phage- ELISA-positive peptides tested.
  • resin peptide-bound affinity-purified ACA-6501 aPL antibody was detected by an immunoconjugate color reaction.
  • 6641/3G3 was tested against several high titer ACA antisera. This peptide appeared to be crossreactive with the vast majority of ACA antisera tested as illustrated in Figure 14. 6641/3G3 demonstrated dose- dependent cross-reactivity with 10 of 10 ACA antibodies with complete inhibition (>80%) at 1.8 mg/mL and appeared to be specific for ACA antibodies as demonstrated in Table 7 below. *0
  • Peptide synthesis allows the molecular dissection of the mimotope by selective synthesis. This includes the modification of each amino acid along the chain with the goal of enhancing antibody binding. Selective synthesis reveals the relative importance of each amino acid in the sequence. If necessary, selective substitution at particular residue locations can be designed to maintain B cell reactivity while abolishing any T cell proliferative reactivity discovered during T cell assays.
  • Peptide 6641/3G3 (LJP 688) was subjected to a number of analyses.
  • the analyses included truncation at both the N-terminus and the C-terminus, disulfide substitution, substitution of alanine and glycine for amino acids in positions 2 through 8, substitution of branched aliphatic amino acids in positions 2, 4, 5 and 8, substitution of amino acids affecting conformation of the peptide, including substitution of ⁇ -methyl amino acids, substitution of basic amino acids in position 7, substitution of D-amino acids in positions 2 through 9, and the substitution of N- ⁇ -methyl amino acids in positions 1 through 9.
  • Table 9 The structure/activity relationship of these substitutions and truncations is shown in Table 8.
  • N-Me amino acids (nAA)
  • Proline residues have a special significance due to their influence on the chain conformation of polypeptides. They often occur in reverse turns on the surface of globular proteins. In the phage epitope libraries of the present invention, all random peptide inserts are flanked by boundary prolines. In addition, most of the mimotopes discovered with ACA-6501 have a third proline which, based on computer-based predictions, likely exists as part of a ⁇ -turn. ⁇ -turn mimetics can be used to enhance the stability of reverse turn conformations in small peptides. Such a mimetic is (S)- ⁇ -methyl proline ( ⁇ --t ⁇ ePro), a proline analog that, in addition to stabilizing turn conformations, confers resistance to protease degradation.
  • Protease resistance is a desirable property for a potential drug designed to act in the plasma.
  • Peptide ACA- 6501/3B10 AGPC£ . 4PZ)RCPG (insert highlighted) is a consensus peptide. It has a sequence featuring the most prevalent residue at each position based on a comparison with 35 other homologous sequences. Due to its representative character, the sequence was subjected to a number of systematic modifications and deletions and its activity subsequently evaluated by aPL antibody binding. Among the most important findings was the discovery that the prolines at the 3 and 9 positions are important for activity. Proline-3 is derived from the phage framework and is not part of the random insert. The most dramatic effect was obtained by the substitution of ⁇ -MePro for proline at the 9-position. This substitution led to a six-fold enhancement in immunoreactivity.
  • alpha amino acids can be employed in the present invention.
  • these other classes include d-air ⁇ no acids, .V-alkyl amino acids, alpha-alkyl amino acids, cyclic amino acids, chimeric amino acids, and miscellaneous amino acids.
  • These non-natural amino acids have been widely used to modify bioactive polypeptides to enhance resistance to proteolytic degradation and/or to impart conformational constraints to improve biological activity (Hruby et al (1990) Biochem. J. 268:249-262; Hruby and Bonner (1995) Methods in Molecular Biology 35:201-240).
  • the most common /"-alkyl amino acids are the //"-methyl amino acids, such as .V-methyl cysteine (nC), N"-methyl glycine (nG), / "-methyl leucine (nL), //"- methyl lysine (nK), and "-methyl valine (nV).
  • alpha-alkyl amino acids examples include alpha-methyl alanine (mA), alpha-aminoisobutyric acid (aiB), alpha-methyl proline (mP), alpha-methyl leucine (mL), alpha-methyl valine (mV), alpha-methyl- alpha-aminobutyric acid (tv), diethylglycine (deG), diphenylglycine (dpG), and dicyclohexyl glycine (dcG) (Balaram (1992) Pure & Appl. Chem. 64:1061-1066; Toniolo et al. (1993) Biopolymers 33:1061-1072; Hinds et al. (1991) Med. Chem. 34:1777-1789).
  • mA alpha-methyl alanine
  • aiB alpha-aminoisobutyric acid
  • mP alpha-methyl proline
  • mL alpha-methyl leucine
  • mV
  • cyclic amino acids examples include 1 -amino- 1 -cyclopropane carboxylic acid (cG), 1 -amino- 1-cyclopentane carboxylic acid (Ac5c), 1 -amino- 1-cyclohexane carboxylic acid (Ac6c), aminoindane carboxylic acid (ind), tetrahydroisoquinoline carboxylic acid (Tic), and pipecolinic acid (Pip) (C. Toniolo (1990) Int'l. J. Peptide Protein Res. 35:287-300; Burgess et al (1995) J. Am. Chem. Soc. 117:3808-3819).
  • chimeric amino acids examples include penicillamine (Pe), combinations of cysteine with valine, 4R- and 4S-mercaptoprolines (Mpt), combinations of homocysteine and proline and 4R- and 4S-hydroxyprolines (hyP) and a combination of homoserine and proline.
  • miscellaneous alpha amino acids include basic amino acid analogs such as ornithine (Or), ⁇ -methyl lysine (mK), 4-pyridyl alanine(pyA), 4-piperidino alanine (piA), and 4-aminophenylalanine; acidic amino acid analogs such as citrulline (Cit), and 3-hydroxyvaline; aromatic amino acid analogs such as 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), phenylglycine (pG), 3,3-diphenylalanine (dpA), 3-(2-thienyl)alanine (Thi), and halophenylalanines (e.g., 2-fluorophenylalanine and 4-chlorophenylalanine); hydrophobic amino acid analogs such as t-butylglycine (i.e., tertiary leucine (tL)), 2-aminobutyric acid
  • beta amino acids In addition to alpha-amino acids, others such as beta amino acids can also be used in the present invention.
  • these other amino acids include 2- aminobenzoic acid (Abz), ⁇ -aminopropanoic acid ( ⁇ -Apr), ⁇ -aminobutyric acid ( ⁇ - Abu), and 6-aminohexanoic acid ( ⁇ -Ahx).
  • Carboxylic acids such as 4-chlorobutyric acid (By) and 3-chloropropionic acid (Pp) have also been used as the first residue on the N-terminal in the synthesis of cyclic thioether peptides.
  • the mimetic peptides identified by the methods of the instant invention can be further modified to contain thioether substitutions. Modification of cyclic disulfide analogs to cyclic thioether analogs will extend the plasma half-life of the analog conjugates and, therefore, require a lower dosage. Cyclic thioether analogs also eliminate the problem of disulfide bond exchange which often occurs with cyclic disulfide polypeptides. In addition, the cyclic thioether analogs may also interfere with MHC Class II presentation to T cells and, thus, facilitate induction of anergy. Finally, the cyclic thioether analogs are useful in the thiol-dependent conjugation reactions used in the production of valency platform molecule conjugates.
  • cyclic thioether analogs were prepared according to the methodology described in co-owned, co-pending patent application, attorney docket number 252312006600, which is incorporated herein in its entirety. Using either a RinkTM amide 4-methyl benzhydrylamino resin or MBHA resin, full length peptide analogs of 6641/3G3 were prepared, converted into chloro-peptides, cleaved from a solid support and then cyclized. See Thioether Reaction Scheme below. Suitable cyclic thioether analogs include the analogs shown below.
  • thioether analogs are prepared according to the reaction scheme below.
  • the aPL standards (APL Diagnostics, Inc., Louisville, KY) were reconstituted according to the manufacturer's instructions and diluted 1 :50 with 10% ABS-PBS.
  • the plate was washed five times with TBS and 100 ⁇ L of 1:1,000 goat-anti-human-IgG/alkaline phosphatase conjugate (Zymed, South San Francisco, CA, Cat. No. 62,8422), in 10% ABS-PBS was added and incubated for 1 hour at RT.
  • phenolphthalein monophosphate (PPMP) substrate solution (Sigma, Cat. No. P-5758), prepared from a stock solution of 0.13 M PPMP and 7.8 M 2-amino-2-methyl-l-propanol, adjusted to pH 10.15 with HC1, after a dilution of 1 :26 with deionized water. After approximately 30 minutes, the reaction was stopped with 50 ⁇ L of 0.2 M dibasic sodium phosphate (Mallinckrodt, Analytical Reagent) added per well.
  • PPMP phenolphthalein monophosphate
  • the optical density was read at 550 nm in a microplate autoreader (Bio-Tek Instruments, Winooski, VT, Model EL311).
  • the optical density of the odd-numbered control wells (blank, without cardiolipin (CL)) was subtracted from the optical density of the even- numbered wells.
  • the absorbance readings of the aPL standards were plotted using Graph Pad Prizm (Graph Pad Software, Inc., San Diego, CA) to generate the GPL (IgG phospholipid) standard curve.
  • the diluted 6501 test serum absorbance readings were used to calculate GPL scores based on the GPL standard curve.
  • the precipitate was washed 3 times with 25 mL of cold 0.96% NaCl using the RC3 centrifuge.
  • the pellet was dissolved in 1 mL of 2% (wt/vol) solution of n-octyl- ⁇ -D-glucopyranoside (Calbiochem, La Jolla, CA) in TBS and applied to a 0.6 mL protein A/cross-linked agarose (Repligen Corporation, Cambridge, MA) column which had been pre- washed with 15 times bed volume of 1 M acetic acid and equilibrated with 15 times bed volumes of TBS.
  • the antibody-protein A/agarose column was washed with 40 times bed volume of 2% octylglucopyranoside to remove lipids, followed by extensive washings with TBS until the optical density of the eluate at 280 nm approached the baseline.
  • the bound antibody was eluted with 1 M acetic acid.
  • One mL fractions were collected, neutralized immediately with 0.34 mL 3 M Tris (Bio-Rad, electrophoresis grade reagent) per fraction and kept in an ice bath.
  • the optical density of each fraction was determined at 280 nm in a spectrophotometer (Hewlett-Packard, 8452A Diode Array Spectrophotometer, Palo Alto, CA).
  • An affinity adsorbent containing ⁇ 2 -GPI was prepared using CNBr-activated agarose )Pharmacia, Inc., Piscataway, NJ) or Affi-Gel 10 (BioRad, Richmond, CA) in accordance with the manufacturer's instructions using purified ⁇ 2 -GPI obtained commercially (Perlmmune, Rockville, MD).
  • a 1 mL gel column containing the ⁇ 2 -GPI affinity adsorbent up to 100 ⁇ g of liposome-purified aPL IgG in 1 mL TBS was added. After 1 hour, the column was extensively washed with buffer to displace contaminants and any IgG that did not bind ⁇ 2 -GPI. The ⁇ 2 -GPI-binding IgG was displaced with 1 M HOAc and the fractions neutralized with Tris as described above. Fractions containing IgG were pooled, concentrated and subjected to buffer exchange as previously described.
  • Example 3 Construction of a p-III Library Vector Preparation fUSE 5 (Scott, J.K. and G. Smith, supra) was used as the vector for the construction of p-III libraries, and a variation of the method of Holmes, D.S. and M. Quigley (1981), Anal. Biochem. 144:193) was employed to generate the double- stranded replicative form (RF). Briefly, an 800 mL culture of E. coli K802, harboring fUSE 5, was grown in 2YT medium (Difco Labs, Ann Arbor, MI) containing 20 micrograms/mL tetracycline for 18 hours at 37 degrees with vigorous shaking. Cells were collected by centrifugation and resuspended in 75 mL STET.
  • 2YT medium Difco Labs, Ann Arbor, MI
  • STET consists of 8% sucrose in 50 mM Tris/HCi pH 8.0, 50 mM EDTA and contains 0.5% Triton X-100. Lysozyme, 10 mg/mL in STET, was added to a final concentration d ⁇ l mg/mL. After 5 minutes at RT, three equal aliquots were placed in a boiling water bath with occasional shaking for 3.5 minutes. The viscous slurry was centrifuged for 30 minutes at 18000 x G and an equal volume of isopropanol was added to the supernatant. The solution was cooled to -20°C and the nucleic acids were collected by centrifugation. The RF was isolated from a CsCl gradient as described by Sambrook et al MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2d ed., 1989).
  • the DNA for insertion was generated by the "gapped duplex" method described by Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382.
  • an oligonucleotide containing a degenerate region in the middle is surrounded by short constant regions on each end of the oligonucleotide.
  • Two shorter, complementary oligonucleotides are annealed to form a "gapped duplex" possessing overhangs that are complementary to the sticky ends produced by the restriction endonuclease used to digest the vector.
  • the longer degenerate oligonucleotide has the sequence:
  • EpiGS2 was designed to base pair to the 5' end of the degenerate oligonucleotide and has the sequence 5' GGGTCCAGCCCCGT 3 * .
  • EpiGS3 was designed to anneal to the 3' end of the degenerate oligonucleotide and has the sequence 5' CAGCCCCCGG 3'.
  • the three oligonucleotides When correctly annealed, the three oligonucleotides form a "gapped duplex" which, when inserted into fUSE 5 digested with Sfil, restores the reading frame of p-III with the random insert near the 5' end.
  • Oligonucleotides described above were prepared by excision from polyacrylamide gels. One nanomole of EpiGS2, EpiGS3, and 50 picomoles of the degenerate oligonucleotide were kinased separately in 66 microliter volumes. The three oligonucleotides were then pooled, NaCl was added to 50 mM and the mixture was heated to 65°C for 5 minutes followed by slow cooling to RT. The annealed oligonucleotides were then cooled on ice and used immediately in the ligation reaction with fUSE 5.
  • the ligation reaction consisted of 10 micrograms of fUSE 5 DNA digested to completion with Sfil, 30 ⁇ L of the "gapped duplex" solution and 1000 U of T4 ligase in a total volume of 450 microliters. The ligation was incubated for 18 hours at 16°C. The mixture was then phenol-chloroform extracted, precipitated with ethanol and the precipitate dissolved in 20 ⁇ L of water.
  • the ligated DNA was introduced into E. coli by electroporation (Dower et al. (1988) Nucleic Acid Res. 16:6127-6145). Frozen electrocompetent MCI 061 cells (0.1 mL) were mixed with 4 ⁇ L of ligated DNA in a cold 2 mm cuvette and subjected to 2.5 kV, 5.2 mS pulse by a BTX electroporation device (BTX Corp., San Diego, CA). Immediately after the pulse, 1 mL of SOC, a cell growth medium (see Dower et al, supra) was added. Five separate electroporations were carried out, pooled, and incubated at 37°C for 1 hour.
  • BSA bovine serum albumin
  • a suspension of £ coli freshly grown in 2YT medium for about 5 hours with 250 rpm shaking at 37°C was spun at 1000 x g for 10 minutes at RT in 50 mL polypropylene tubes. Twenty mL of 80 M NaCl was added to the packed E. coli pellet and then incubated for 45 minutes at 37°C at 100 rpm. Following centrifugation as above, the starved E. coli pellet was suspended in 1 mL of 50 mM ammonium phosphate/80 mM NaCl and used later for phage amplification. Protein G-agarose beads were washed 2x in TBS BSA and 2 times in TBS/0.5% Tween-20 and stored at 4°C as a 50% suspension in TBS/Tween.
  • the acidic eluate supernatant was collected and an additional 100 ⁇ L of elution solution was added to the bead pellet and the procedure repeated.
  • the phage-containing eluates (representing the unamplified first round phage) were pooled (—400 ⁇ L) and placed in a sterile 17 x 100 mm polypropylene cell culture tube to which was added 50 ⁇ L 0.5 M NaCl followed by pH neutralization with 2.5 M Tris base (usually -25-35 ⁇ L).
  • An equal volume of the starved E. coli suspension was added immediately and then incubated for 10 minutes at 37°C at 100 rpm.
  • the mixture was then transferred to a 250 mL sterile culture flask containing 25 mL 2YT with 20 ⁇ g/mL tetracycline (Tet) and incubated overnight at 37°C at 250 rpm.
  • the suspension was centrifuged at 12,000 x g for 10 minutes in polycarbonate tubes and the pellet discarded. After heating the supernatant at 70°C for 30 minutes in polypropylene tubes, the material was spun again in polycarbonate tubes and the supernatant saved. To the supernatant, 1/4 volume of 20% (w/v) polyethylene glycol, molecular weight 8000 (PEG 8000) was added to precipitate phage. The solution was mixed by inversion 100 times and then incubated at 4° C for 2 hours.
  • PEG 8000 polyethylene glycol, molecular weight 8000
  • the phage-containing pellet was resuspended in ⁇ 0.5 mL of TBS/BSA and transferred to a 1.4 mL microfuge tube. After a 1 minute spin in a microfuge at 16,000 x g, the supernatant was transferred to a clean tube and labeled first round amplified phage.
  • Immulon type 2 plates were coated with protein G.
  • Protein G was prepared at 10 ⁇ g/mL in 0.1 M NaHCO 3 and 100 ⁇ L per well was added to the wells of microtitration plates and incubated overnight at 4°C. After discarding excess protein G solution from plates, each well was blocked with 250-300 ⁇ L 2YT for 1 hour at RT with agitation on an oscillating platform. Tris-buffered saline, pH 7.4/0.5% Tween 20 (TBS/Tween), was used with an automatic plate washer to wash the wells 4 times with 200 ⁇ L.
  • TBS/Tween Tris-buffered saline, pH 7.4/0.5% Tween 20
  • affACA-6501 or control normal IgG
  • 2YT was added to washed wells.
  • the plate was transferred to a cold room rotator near the end of a 1 hour incubation at RT on a rotating platform.
  • Phage to be tested by micropanning were obtained from the agar plates generated by biopanning. Each clone to be tested was transferred using sterile toothpicks to a separate well of a round-bottom 96-well microtitration plate (Corning, Corning, NY) containing 250 ⁇ L 2YT/Tet per well and cultured overnight at 37°C. Clone designations are based on the screening antibody, the biopanning round of origin, and the location of the clone in the overnight culture plate, e.g., ACA- 6501/3B10 refers to the clone isolated by ACA-6501 in the third round located in the well designated BIO on the microtitration plate. Following overnight incubation, phage cultures were centrifuged using a microtitration plate holder at 1300 x g for 10 minutes at RT. Supernatants constituted the source of "neat" phage.
  • the incubation of dilute phage with aPL antibody or control IgG was carried out for 2 hours at 4°C on a flat rotator. After 9 washes with TBS/Tween in an automated plate washer, the IgG-bound phage was eluted with 20 ⁇ L of 0.2 N HC1- glycine/0.1 % BS A, pH 2.2. The elution incubation continued for 10 minutes at RT, during which time a new Corning microtitration plate was prepared containing 20 ⁇ L of freshly starved E. coli per well and kept chilled.
  • colonies were semiquantitatively scored from 0 to 4+, with 0 symbolizing ⁇ 10 colonies; +/-, 10-20; 1+, 20-50; 2+, 50-70% confluent; 3+, 70-90% confluent; and 4+ representing >90% confluent colonies.
  • 10 [representing 81 third, 6 fourth, and 7 fifth round clones]
  • six clones had micropanning scores of zero, three scored 1+, 14 scored 2+, 62 scored 3+, and 9 scored 4+.
  • a survey of random clones from the plates representing the second through fifth rounds of biopanning was carried out by G-track DNA sequencing as described below.
  • Single-stranded viral DNA was isolated from cultures incubated overnight at 37°C.
  • 2YT/Tet either as 2 mL in tubes or 250 ⁇ L in microtitration plate round-bottom wells, was inoculated with individual phage from spread plates of previously grown cultures in microtitration plates.
  • the purification of phage by 20% PEG/2.5 M NaCl precipitation of culture supernatants as well as the isolation or release of virion DNA by phenol-chloroform extraction or by alkali denaturation was performed as described in Smith, G.P. and J.K. Scott, "Libraries of peptides and proteins displayed on filamentous phage" (1993) Meth.
  • Clones ACA-6635/3A12, 3B3, 3C8, 3A5, 3C9, and 3B7 were grown as 3 mL cultures.
  • Affinity purified ACA-6635 was diluted to 2.5 ⁇ g/mL in phosphate-buffered saline, pH 7.2, and 100 ⁇ L added to Immulon-2 microtitration plate wells. After 2 hours, the plates were washed 3 times witi TBS/Tween in an automated plate washer with no shaking. The plate was then blocked with 150 ⁇ L 0.1% BSA (globulin-free) in PBS per well. After 1 hour at 4°C, the plate was washed 3 times as previously described.
  • each supernatant was diluted 1: 10 in 0.1% BSA/PBS and 100 ⁇ L added to each of the wells coated with affinity purified ACA-6635 and then incubated for 2 hours at 4°C. Plates were then washed with TBS/Tween as before.
  • Horseradish peroxidase-conjugated sheep IgG anti-M13 phage antibody (Pharmacia, Inc., Piscataway, NJ) was diluted 1 :5,000 in 0.1 % BSA/PBS and 100 ⁇ L applied to each well. Following incubation for 1 hour at 4°C, the plate was washed 4 times as before.
  • microfuge tubes Four 1.5 mL microfuge tubes were numbered 1 to 4. The following reagents were mixed in the first microfuge tube (Brinkman Instruments, Westbury, NY): 30 ⁇ L of 5% BSA; 284 ⁇ L TBS; 8 ⁇ L of a stock solution of approximately 400-500 ⁇ g/mL of monomeric peptide (ACA-5A12 or -CB2 or -3B10 or scrambled -3B 10 as negative control) in TBS; and 8.2 ⁇ L of 1 : 10 diluted serum 6501 in 0.5% BSA-TBS.
  • the following reagents were mixed in the second microfuge tube: 30 ⁇ L of 5% BSA; 290 ⁇ L TBS; 2 ⁇ L of a stock solution of approximately 400- 500 ⁇ g/mL of monomeric peptide (ACA-5A12 or -CB2 or -3B10 or scrambled -3B10 as negative control) in TBS; and 8.2 ⁇ L of 1 :10 diluted serum 6501 in 0.5% BSA- TBS.
  • the following reagents were mixed in the third microfuge tube: 30 ⁇ L of 5% BSA, 287 ⁇ L of a 1 :10 dilution of approximately 400-500 ⁇ g/mL of monomeric peptides (5A12, CB2, 3B10, or scrambled sequence 3B10 control), and 8.2 ⁇ L of ACA-6501 serum previously diluted 1 :10 in 0.5% BSA-TBS.
  • the following reagents were mixed in the fourth Eppendorf microfuge tube: 60 ⁇ L 5% BSA; 584' ⁇ L TBS and 16.5 ⁇ L of 1 :10 diluted serum 6501 in 0.5% BSA-TBS. The blocked plate was washed 5 times with TBS.
  • the solution in the first microfuge tube was added to triplicate wells, 100 ⁇ L per well. Identical amounts of the solutions in the second, third and fourth microfuge tubes were also added to triplicate wells. An aliquot of 100 ⁇ L of the solution in the fourth microfuge tube was added to each of the three blocked blank wells of the microtitration plate. The plate was incubated for 1 hour at RT with agitation at 40 rpm in an orbital shaker (American Dade, Miami, FL, Rotator V) and then washed 5 times with TBS. An aliquot of 100 ⁇ L of 1 :1000 diluted goat- anti-human-lgG/alkaline phosphatase conjugate (Zymed, South San Francisco, CA, Cat. no.
  • the CL-coated, blocked plate was washed 5 times with TBS and then to each well was added ⁇ 2-GPI as 100 ⁇ L of 2.3% (v/v in PBS) IgG-depleted human serum (Sigma Chemical Co., St. Louis, MO) and incubated 2 hours at RT.
  • variable amounts of each of six peptides were mixed with 22 ⁇ L ACA-6501 serum diluted with 3% fish gelatin in 1 :1 TBS/PBS (final dilution of 1 :400) in a final volume of 220 ⁇ L using Eppendorf microcentrifuge tubes.
  • tube #1 were mixed 181.3 ⁇ L of 3% fish gelatin in TBS-PBS, 16.7 ⁇ L of peptide stock solution plus 22 ⁇ L of ACA-6501 serum diluted 40 times in 3% fish gelatin/TBS -PBS.
  • Stock solutions ranged from 450 - 800 ⁇ g/mL for peptides #951 (diserine non-cyclized negative control), #952 (a lot of LJP 690), and thioethers CCTE-3G3, CHTE-3G3, HCTE-3G3 and HHTE-3G3.
  • Tube #2 the following were added: 148 ⁇ L fish gelatin/TBS-PBS, 50 ⁇ L of peptide stock solution, plus 22 ⁇ L of 40 times diluted ACA- 6501 serum.
  • Tube #3 contained 48 ⁇ L fish gelatin/TBS-PBS, 50 ⁇ L of peptide stock solution, plus 22 ⁇ L of 40 times diluted ACA-6501 serum.
  • the control tube #4 received 396 ⁇ L fish gelatin/TBS-PBS and 44 ⁇ L of 40 times diluted ACA-6501 serum (no peptide). Each of the tubes incubated for approximately 1 hour at RT.
  • the CL/ ⁇ 2-GPI microtitration plate was washed 5 times with TBS and 100 ⁇ L aliquots in duplicate from Eppendorf tubes #1, 2, 3, and 4 containing the antibody-peptide (or no peptide mixtures) were added to the wells. A volume of 100 ⁇ L from tube #4 was added to the duplicate control wells containing no cardiolipin.
  • microtitration plate was incubated for 1 hour at RT with agitation at 40 rpm in an orbital shaker (American Scientific, Rotator V), washed 5 times with TBS and then 100 ⁇ L of 1 : 1000 goat anti- human IgG alkaline phosphatase conjugate (Zymed, Cat No. 62-8422) in 0.5% (w/v) BSA-TBS was added.
  • the microtitration plate was again washed 5 times with TBS and the colorimetric enzyme detection developed by adding 100 ⁇ L of PMPP solution (7.8 g phenolphthalein monophosphate plus 69.5 g of 2- amino-2-methyl-l-propanol in 100 mL water stock solution diluted 1 :26 with water). After 21 minutes, the reaction was stopped by adding 50 ⁇ L of 0.2 M Na 2 HPO 4 (Mallinckrodt) to each well. Absorbance at 550 nm was read in a microplate reader (Bio- Tek Instruments, Model EL 311). Absorbance vs. peptide added was plotted on Graph Pad Prism (Graph Pad Software, Inc.) as shown in Figure 12. The amount of peptide that inhibited ACA-6501 binding by 50%, the IC 50 , was calculated from the graph at the intersection of half-maximal absorbance with amount of peptide added.
  • variable amounts of each of four test peptides were mixed with 22 ⁇ L of ACA-6501 serum diluted with sample diluent in a final volume of 220 ⁇ L using Eppendorf microcentrifuge tubes. Specifically, in tube #1, 188 ⁇ L of sample diluent, 10 ⁇ L of peptide stock solution (2 mg - 4 mg/mL diluent), and 22 ⁇ L of AC-6501 serum diluted 1 :35 in sample diluent were added. To tube #2, 158 ⁇ L sample diluent, 40 ⁇ L peptide stock solution and 22 ⁇ L of 1 :35 diluted ACA-6501 serum were added.
  • Tube #3 contained 38 ⁇ L sample diluent, 160 ⁇ L peptide stock solution and 22 ⁇ L of 1 :35 diluted ACA-6501 serum.
  • the control tube, tube #4, contained 396 ⁇ L sample diluent and 44 ⁇ L of 1 :35 diluted ACA-6501 serum (no peptide). Each of the tubes was incubated for approximately 1 hours at room temperature.
  • the ⁇ 2 -GPI microplate was washed 5 times with TBS and 100 ⁇ L of the mixtures contained in Eppendorf tubes #1, 2, 3 and 4 was added to duplicate wells of the microplate. 100 ⁇ L of the contents of tube #4 was added to each of the duplicate control wells that were not coated with ⁇ 2 -GPI. The plate was incubated for 1 hour at room temperature, washed 5 times with TBS and 100 ⁇ L of 1:1000 goat anti-human IgG/AP conjugate (Zymed, Cat. No. 62-8422) diluted in sample diluent buffer was added.
  • Microtitration plates (96-well, flat bottom polystyrene, Immulon-2, Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 ⁇ L/well for 1 hour at RT with tetrameric ACA-6501/3B10 peptide at 10 ⁇ g/mL in carbonate buffer, pH 9.6 (15 mM Na 2 CO 3 /35 mM NaHC0 3 ). After the liquid from the wells was removed, each well was blocked for 1 hour at RT with 200 ⁇ L 0.5% (wt/vol) BSA (globulin-free, cat. no. A7638, Sigma Chemical Co., St. Louis, MO) in TBS. Three wells on the plate were left uncoated by tetravalent peptide to serve as blank control wells.
  • BSA globalulin-free, cat. no. A7638, Sigma Chemical Co., St. Louis, MO
  • each soluble, monomer peptide to be tested was set up in three test tubes (Eppendorf micro test tubes, Brinkmann Instruments, Westbury, NY) each containing 30 ⁇ L 5% BSA/TBS, 8.2 ⁇ L ACA-6501 serum (at 1 :10 dilution with 0.5% BSA/TBS), plus a variable volume of the peptide/TBS stock and the necessary volume of TBS buffer to yield a final volume of 330 ⁇ L.
  • the 330 ⁇ L volume was sufficient to generate triplicate 100 ⁇ L samples for each peptide concentration that was tested for its ability to block ACA-6501 binding to the tetravalent peptide-coated plate.
  • the concentration of the stock solution was approximately 340-400 ⁇ g/mL TBS and aliquots of 19 ⁇ L, 75 ⁇ L, and 292 ⁇ L were removed to prepare the three peptide concentration tubes.
  • stock solution concentrations were 400-500 ⁇ g/mL in TBS. Aliquots from each stock solution of 1 ⁇ L, 4 ⁇ L, and 16 ⁇ L were removed to set up the three concentration tubes for each peptide.
  • a tube with a final volume of 660 ⁇ L was prepared containing 60 ⁇ L 5% BSA, 16.5 ⁇ L of a 1 : 10 dilution of ACA-6501 and 583.5 ⁇ L TBS, i.e., the same final concentrations (0.5% BSA and ACA-6501 serum at 1:400) as the 330 ⁇ L tubes but with twice the volume and without peptide.
  • the plate coated with tetravalent peptide was washed 5 times with TBS. From each 330 ⁇ L tube containing peptides 139, 142 and 143 at different concentrations, 100 ⁇ L was added to coated triplicate wells. Three 100 ⁇ L aliquots from the 660 ⁇ L control tube without peptide were each added to coated wells and three additional 100 ⁇ L aliquots were each added to uncoated blank wells. The plate was incubated at 40 rev/min on a rotary orbital shaker (Rotator V, American Dade, Miami, FL) for 1 hour at RT and then washed 5 times with TBS.
  • Rotator V American Dade, Miami, FL
  • Microtitration plates were coated with tetramer 3B10 peptide as described for Example 5. For each of the four peptides tested, three peptide concentrations were prepared in tubes. As in Example 5, these twelve tubes had final concentrations of 0.5%) BSA/TBS and ACA-6501 serum at a final dilution of 1:400 in a final volume of 330 ⁇ L. All peptide stock solutions were at 400-500 ⁇ g/mL TBS.
  • Example 7 Abbreviated Description of Screen with 6626 Antibody and the Corresponding Sequences
  • Affinity purified ACA-6626 (AffACA-6626) was isolated by affinity purification from 8 mL of ACA-6626 plasma as previously described.
  • AffACA-6626 (10 ⁇ g) was incubated with the epitope xy'z phage library consisting of a pool of all p- III component libraries in a final volume of 100 ⁇ L as previously described for ACA- 6501 biopanning. Following three rounds of biopanning, randomly selected phage from the second and third rounds were tested by micropanning. Only a few clones were weakly immunopositive at a 1 : 1000 dilution. An additional 4th round of biopanning was carried out.
  • Micropanning of 94 fourth round clones revealed 43 immunopositives, some at phage dilutions as high as 1 :100,000.
  • G-Tracking DNA sequencing of the 43 immunopositive clones carried out as previously described for ACA-6501 revealed 5 unique sequences. After conventional four base DNA sequencing, the translated amino acid sequences of Table 3 were obtained.
  • the epi xy z phage display library was screened using methods similar to those in Example 4 with ACA affinity purified antibody from patient number 6644.
  • a colony blot assay as described previously was employed as the final identification step prior to peptide synthesis. Approximately 150 colonies were plated on the original nitrocellulose membrane and assayed. Antibody from patient 6644 was used at a concentration of 1 ⁇ g/mL. Of the 150 colonies plated on the nitrocellular membrane and assayed, only 4 were strongly positive and 2 weakly positive in this screen. Sequencing of the inserts of the six positive phage selected by this screen revealed us that the inserts were all derived from the 8-mer library with a free ami no-terminus
  • Example 9 Summary of Phage Library Screen with ACA-6641.
  • AffACA-6641 was isolated from 4 mL of plasma taken from patient number 6641.
  • AffACA-6641 (10 ⁇ g) was incubated with the pooled p-III phage libraries in a final volume of 100 ⁇ L as described previously.
  • 45 clones from the 3rd and 4th rounds were tested by micropanning. Of the 45, 23 scored negative.
  • the 3rd round phage yielded two clones that scored 4+, two that scored 3+ and two that scored 2+. From the 4th round, one clone scored 4+, one scored 3+ and three scored 2+.
  • G-tracking DNA sequencing revealed six unique sequences. Only one, clone 3G3, was strongly positive in the phage-capture ELISA. Four base DNA sequencing gave the following translated peptide sequence:
  • Example 10 Peptide conjugation to non-immunogenic. multivalent carriers
  • Non-immunogenic multivalent platforms with amine groups are synthesized as shown in the following scheme.
  • PPN-peptide-CO H A peptide is synthesized with standard solid phase methods using FMOC chemistry on a Wang (p-alkoxybenzyl) resin, using trifluoroacetic acid (TFA) stable protecting groups (benzyl ester or cyclohexyl ester on carboxyl groups and carbobenzyloxy (CBZ) on amino groups). Amino acid residues are added sequentially to the amino terminus.
  • TFA trifluoroacetic acid
  • the peptide is removed from the resin with TFA to provide a peptide with one free carboxyl group at the carboxy terminus and all the other carboxyls and amines blocked.
  • the protected peptide is purified by reverse phase HPLC.
  • the protected peptide (0.3 mmol) is dissolved in 1 mL of dimethylformamide (DMF), and to the solution is added 0.3 mmol of diisopropylcarbodiim.de and 0.3 mmol of 1-hydroxybenzotriazole hydrate (HOBT).
  • the solution is added to a solution of 0.025 mmol tetraamino platform, 2 in 1 mL of DMF.
  • the DMF is removed under vacuum to yield a crude fully protected conjugate 3.
  • the conjugate, 3_ is treated with hydrofluoric acid (HF) in the presence of anisole for 1 hour at 0° to give conjugate 4. Purification is accomplished by preparative reverse phase HPLC.
  • the following scheme shows the attachment of an amino group of a peptide to a carboxy group on a platform.
  • a peptide is synthesized with standard solid phase methods on an amide resin, which resulted in a carboxy terminal amide after cleavage from the resin, using TFA stable protecting groups (benzyl ester or cyclohexyl ester on carboxyl groups and CBZ on amino groups). Amino acid residues are added sequentially to the amino terminus using standard FMOC chemistry.
  • the peptide is removed from the resin with trifluoroacetic acid to provide a protected peptide with a free amine linker.
  • the protected peptide is purified by reverse phase HPLC.
  • pep cyclic non-disuffide peptide
  • DCC Dicyclohexyl carbodiimide (2.41 g, 1 1.7 mmol) was added to a 0 ⁇ C solution of 2.72 g (7.8 mmol) of 2 and 1.08 g (7.8 mmol) of p-nitrophenol in 41 mL of CH 2 C1 2 . The mixture was stirred for 16 hours allowing it to come to RT. The mixture was filtered to remove N,N-dicyclohexylurea (DCU), and the filtrate was concentrated. The residue was crystallized from hexane/CH 2 Cl 2 to give 3.17 g (86%) of 1Q as pale yellow crystals.
  • DCU N,N-dicyclohexylurea
  • the solid was placed under vacuum, then purged with N 2 , allowed to come to room temperature, placed in a 20° water bath, and irradiated from above at close range with a 500W sunlamp for 5 minutes.
  • the mixture was concentrated on the rotary evaporator and purified by silica gel chromatography (40 mm X 150 mm, toluene was used as eluent until UV active material finished eluting, 2% EtOAc/toluene (500 mL), 5% EtOAc/toluene (500 mL). Impure fractions were repurified. Pure fractions by TLC (R f 0.23, 5% EtOAc/toluene) were combined and concentrated to give 3.23 g (72%) of compound 14 as a waxy solid.
  • N-BOC-glycylproline-4-nitrophenyl ester compound 15: A solution of 3.0 g (11.6 mmol) of N-BOC-glycylproline and 1.93 g (13.9 mmol) of 4-nitrophenol in 82 mL of dry THF was cooled to 0°C and 3.34 g (16.2 mmol) of DCC was added. The mixture was stirred at 0°C for 1 hour, the ice bath was removed, and the mixture was stirred for 16 hours at room temperature. HO Ac (579 ⁇ L) was added to the mixture and it was allowed to stir for 30 minutes. The mixture was kept in the freezer for 30 minutes and filtered under vacuum.
  • the filtrate was concentrated and purified by silica gel chromatography (18 X 150 mm bed, 2.5% EtOAc/97.5% CH 2 Cl 2 /l%HOAc). Traces of acetic acid were removed by concentrating from dioxane several times on the rotary evaporator.
  • N-FMOC-S-t-butylthiocysteineamide N-FMOC-S-t-butylthiocysteineamide.
  • compound 16 A solution of 5.0 g (1 1.6 mmol) of FMOC-S-t-butylthiocysteine and 1.33 g (11.6 mmol) of N-hydroxysuccinimide in 115 mL of THF was cooled to 0°C. To the solution was added 3.58 g (17.37 mmol) of DCC. The mixture was stirred at 0°C for 1 hour and 42.9 mL of a solution of 1.6 g of (NH 4 )HCO 3 in 50 mL of water was added.
  • the mixture was stirred for 4.5 hours, allowing the ice bath to gradually warm to room temperature and concentrated on a rotary evaporator to remove THF and give an aqueous phase with white solid.
  • the mixture was stirred with 200 mL of CH 2 C1 2 until most of the solid dissolved, then was shaken with 100 mL of IN HC1 solution.
  • the CH 2 C1 2 layer was washed with 100 mL of saturated NaHC0 3 solution, dried (Na 2 S0 4 ), and filtered.
  • N-FMOC-L-Alanyl-L-2-methylproline N-FMOC-L-Alanyl-L-2-methylproline.
  • compound 1 A solution of 2-methylproline (Seebach et al (1983) J. Am. Chem. Soc. 105:5390-5398) (1.00 g, 4.76 mmol), 4.00 g (47.6 mmol) of NaHC0 3 , and 31 mg (0.23 mmol) of HOBT in 6.9 mL of DMF was cooled to 0°C and 3.18 g (6.66 mmol) of N-FMOC-L-alanine was added. The reaction was stirred for 1 hour at 0°C, then at room temperature for 18 hours.
  • N-FMOC-L-I isopropylcarbodiimide
  • the concentrated oil was dissolved in a minimal amount of DMF (approximately 1 mL) and added to the swelled resin followed by a solution of 266 mg (2.18 mmol) of DMAP dissolved in approximately 1 mL of DMF. The mixture was gently rocked for 1 hour and washed (2 X DMF, 2 X MeOH, 2 X DMF, 2 X MeOH). The resin was dried under vacuum to give 2.77 g (85%) and the substitution was determined by the Geisen test to be 0.540 mmol/g.
  • N-FMOC-linear peptide with t-butyl ester on aspartic acid and Pmc group on arginine and with thioether insert compound 20: This peptide was prepared by standard FMOC synthesis on N-FMOC-L-leucinyl-HMPB-MBHA resin. Three equivalents of amino acid, HOBT and (DIC) were used for each coupling step with the exception of the coupling step of compound 18. Two equivalents of compound 1_8_ were used with three equivalents of HOBT and diisopropylcarbodiimide. Each step was monitored by using 10 ⁇ L of bromophenol blue indicator.
  • the residue was triturated with 2 X 50 mL of Et 2 0 and the white opaque residue was treated with 5 mL of 92/3/2/3 TFA/anisole/EDT/Me 2 S for 1 hour.
  • the product was precipitated by adding the mixture to 40 mL of Et 2 O in a 50 mL polypropylene centrifuge tube. The precipitate was cooled to 0°C and centrifuged for 5 minutes at 2000 rpm. The supernatant was decanted and the pellet was washed with Et 2 O and recentrifuged. The pellet was dried and dissolved in 4 mL of 50/50 CH 3 CN/H 2 O.
  • 26 peptide (U£_-_25) LJP 685, also referred to as compound 21, was treated with the PNP ester of 3- tritylmercaptopropionic acid and the resulting product was detritylated to give compound 22, the peptide with the free thiol linker. Reaction of an excess of compound 22 with valency platform molecule 12 produced tetravalent conjugate 25. Treatment of compound 21 with the longer linker, compound 21 (see reaction scheme below), followed by detritylation, gave compound 24. Compound 24 reacted with valency platform molecule 12 to give the tetraconjugate 26_. Both conjugation reactions appeared very clean by HPLC.
  • the thiobenzoate ester, compound 28, was prepared from compound 22.
  • Compound 28 was converted to compound 22 in portions.
  • the thiobenzoate was removed by ethanolysis and the resulting thiol was tritylated.
  • the ethyl ester was then hydrolyzed to give compound 22.
  • the nitrophenyl phosphate (PNP) ester, compound 30. was prepared from compound 22.
  • Aminotrioxoudecanoicacid ethylester, compound 21, was prepared by treatment of compound 27 with sodium azide and reduction to the amine.
  • Amine 31 was acylated with compound 2Q to provide compound 32. Hydrolysis of compound 32 was achieved by treatment with sodium hydroxide to give a free carboxylic acid.
  • the intermediate carboxylic acid was condensed with p-nitrophenol to give para-nitrophenyl (PNP) ester, compound 21- Linker 33. was attached to the peptide and the trityl group removed to give compound 34 which was used to produce an MTU-ATU-AHAB-TEG conjugate.
  • PNP para-nitrophenyl
  • Example 15 Svnthesis of a (LJP685yMT ⁇ -ATU-AHAB-TEG conjugate- compound 35
  • Tetravalent conjugate 3_5_ was prepared as shown below.
  • the peptide with linkers attached, compound 24 was dissolved in He sparged, pH 8.5, 200 mM borate buffer.
  • To the mixture was added 0.3 mol equivalents of platform compound 12. The mixture was stirred for 1 hour and the product was purified by HPLC.
  • Tritiated thymidine uptake by peptide-stimulated T cells was monitored in 96- well round bottom plates.
  • a single-cell suspension of draining lymph node cells (mice) or isolated peripheral blood lymphocytes (human), 5 x 10 5 were mixed with between 1 and 30 ⁇ g of peptide in a final volume of 150 ⁇ L per well and incubated for 5 days at 37°C in 5% C0 2 . At that point, 1 micro curie of labeled thymidine was added and incubated for an additional 15-24 hours.
  • the harvested cells were collected on filters and counted by liquid scintillation spectrometry.
  • mice Eight groups, each containing five C57B1/6 mice, were primed with 10 ⁇ g/mouse of a conjugate of LJP685-KLH on alum plus B. pertussis vaccine as an adjuvant. After three weeks, spleen were harvested and single cells suspensions were prepared, washed three times with balanced salt solution and resuspended in complete RPMI-1640 medium at a concentration equivalent to one spleen/1.5 mL of medium.
  • the cell suspension was divided into aliquots of 2.5 mL /petri dish and incubated for 2 hours at 37°C with (LJP685) 4 -DABA-PEG, compound 36, and (LJP685) 4 -TEG, compound 21, in concentrations of 100 ⁇ M, 20 ⁇ M and 4 ⁇ M.
  • One group of cells was incubated without toleragen and acted as the positive control.
  • the cells were then washed with large volumes of balanced salt solution and resuspended in 2.5 mL of balanced salt solution.
  • the cells were then injected into 650R irradiated syngeneic recipient mice in such a manner that all of the cells from a given treatment group were divided evenly into five recipients.
  • mice All of the recipient mice, including the positive controls, were then given a booster immunization of 10 ⁇ g of LJP 685-KLH in saline, intraperitoneally. Seven days after the booster immunization, the mice were bled and their sera tested for the presence of anti-LJP 685 antibody.
  • the resulting cyclic peptides are 5 A 12 (GPCLILAPDRCG) AND CB2 (GPCILLARDRCG).
  • the major difference between the peptides was the substitution of arginine for proline at position 8 which resulted in much less dispersion in the ID 1 H NMR spectrum of CB2 which was consistent with 5A12 being a more rigid peptide.
  • the arginine substitution also produces a 0.55 kcal/mol stabilization of the ionized aspartyl carboxy group as reflected in pKa values.
  • a structural analysis was carried out on the more ordered 5A12 peptide.
  • NMR data coupled with distance geometry calculations were used to determine the three dimensional structure of peptide 925 (CLGVLAKLC), a truncated version of peptide 3G3 (AGPCLGVLGKLCPG) with alanine substituted for glycine in position 6 of the 925 peptide.
  • CLGVLAKLC three dimensional structure of peptide 925
  • AGPCLGVLGKLCPG truncated version of peptide 3G3
  • the structure of peptide 925 was determined in water at pH 3.8 and at 25 °C.
  • An ensemble of nine structures were calculated all of which were consistent with the NMR data.
  • the RMSD for all non-hydrogen atoms was 2.45 a *
  • Figure 18 displays the structure closest to the centroid of the ensemble and, therefore, is a reasonable representation of the shape of the peptide 925 molecule.
  • Figure 19 compares the structure of peptide 925 (labeled at the bottom of the figure as 3G3) with the structure of peptide 5A12. Both peptides have turns at approximately the same positions in the peptide sequence.
  • the pharmacophore of the peptides has been tentatively identified as a small hydrophobic group and a positively charged group.
  • the gem-dimethyl and amino groups of peptide 925 are tentatively identified as the pharmacophore of this peptide as shown in Figure 20.
  • the hydrocarbon linkers that tether the pharmacophore groups to some scaffold have the lengths specified in Figure 20 and the points at which these linkers are attached to the scaffold are separated by the distance specified. Finally, the dihedral angle defining the relative orientation of the two linkers was determined to be 22°.
  • Example 21 Svnthesis of the Tetravalent Platform IA/DABA/ATE 46 Bis-N-Ct-butoxycarbonvn-diaminobenzoic acid, comnound 40: A solution of 7.18 g (32.9 mmol of di-t-butyldicarbonate in 5.5 mL of MeOH was slowly added to a solution of 2.5 g (16.4 mmol) of 3,5-diaminobenzoic acid and 2.76 g (32.9 mmol) of NaHCO 3 in 44.5 mL of H 2 0 and 22.5 mL of MeOH, and the mixture was stirred at room temperature for 24 h.
  • N-hydroxysuccinimidyl ester of compound 40 N-hydroxysuccinimidyl ester of compound 40.
  • compound 41 Dicyclohexylcarbodiimide (3.34 g, 16.2 mmol) was added to a solution of 3.8 g (10.8 mmol) of compound 40 . and 1.24 g (10.8 mmol) of N-hydroxysuccinimide in 55 mL of EtOAc which had been cooled to 0°, and the resulting mixture was stirred for 18 h allowing to come to room temperature. To the mixture was added 0.55 mL of acetic acid. The mixture was stirred for 30 min and placed in the freezer for 2 h. The mixture was filtered to remove solids, and the filtrate was concentrated to give 5.80 g of pink foamy solid. Purification by silica gel chromatography (60/40/1 hexane/EtOAc/HOAc) gave 4.30 g (89%) of compound 41 as a slightly pink solid.
  • Diamino-TEG his-trifluoroacetate salt, compound 44 300 mg (0.57 mmol) of compound 43 . was dissolved in 3.5 g of CH 2 C1 2 , and 3.5 mL of trifluoroacetic acid was added. The mixture was stirred for 3 h at room temperature, and the solution was concentrated to give 398 mg of crude compound 44 which was used directly in the next step.
  • Example 22 Svnthesis of the Tetravalent Platform BA/PABA/DT/TEG. 51. N-(t-butoxycarbonyl)PABA. compound 47: A solution was prepared of 3.0 g (21.9 mmol) of p- aminobenzoic acid in 60 mL of H 2 O. Na 2 C0 3 (2.16 g, 25.7 mmol) was added slowly followed by 30 mL of MeOH. When all solids were dissolved, a solution of 4.77 g (21.9 mmol) of di-t- butyldicarbonate in 10 mL of MeOH was added and the mixture was stirred at room temperature for 18 h.
  • N-rt-butoxycarhonvnPABA N-hvdroxvsuccinimidyl ester compound 48 DCC (2.61 g, 12.6 mmol) was added to a 0° solution of 2.0 g (8.43 mmol) of compound 42 and 0.97 g (8.43 mmol) of N-hydroxysuccinimide in 50 mL of EtOAc. The ice bath was removed, the mixture was stirred for 16 h at room temperature, and 0.5 mL of acetic acid was added. The mixture was stirred for an additional 30 min, placed in the freezer for 1.5 h, filtered, and concentrated to give 3.75 g of crude 4£. Purification by silica gel chromatography (50/50 hexane/EtOAc) gave 2.53 g (90%) of compound 48 as a white solid.
  • Compound 50 A solution of 135 uL (0.66 mmol) of triethyleneglycol bis-chloroformate in 0.3 mL of THF was added to a 0° solution of 855 mg (1.58 mmol) of compound 42 and 275 uL (1.58 mmol) of diisopropylethylamine in 13 mL of THF. The cloudy mixture cleared when the ice bath was removed. An additional 70 uL of diisopropyethylamine was added to maintain a basic pH. The mixture was stirred at room temperature for a total of 3 h and partitioned between 25 mL of H 2 0 and 25 mL of EtOAc.
  • Compound 60 Compound 58 ( 1 mg, 0.48 mmol) was dissolved in 3 mL of 30% HBr/HOAc and the resulting mixture was stirred at room temperature for lh at which time 5 mL of Et 2 O was added. The mixture was placed in the freezer for 1 h and centrifuged. The resulting pellet was washed with Et 2 0 and dried to give the tetrahydrobromide salt 52 which was dissolved in 1 mL of H 2 O. To the mixture is added 49 mg (0.58 mmol) of NaHCO 3 and 3 mL of dioxane. More NaHCO 3 is added, if needed, to make the mixture basic.
  • Triethy leneglycol ditosylate ( 1.0 g, 2.18 mmol) was added to a solution of 725 mg (4.36 mmol) of ethyl 4-hydroxybenzoate and 723 mg (5.23 mmol) of K 2 C0 3 , and the mixture was refluxed for 16 h. The mixture was concentrated, and the residue was partitioned between 20 mL of water and 3 X 20 mL of Et 2 O. The combined organic layers were washed with 2 X 40 mL of sat NaHCO 3 solution, 40 mL of sat NaCl solution.
  • Compound 65 Compound 64 is dissolved in acetone containing 2.2 equivalents of LiOH and the mixture is stirred for 3 h (until complete as evidenced by TLC). The mixture is acidified with acetic acid and concentrated, and the residue is purified by silica gel chromatography to give 65- Compound 66: Compound 66 is prepared similarly to the method of preparing compound 56 in example 23. Compound 65 is used instead of N-BOC-iminodiacetic acid, and compound 52 is used instead of mono-CBZ-piperazine. Purification is accomplished using silica gel chromatography.
  • Compound 68 Compound 66 is converted to compound 8. in essentially the same manner as described for the conversion of 5£ to 6 ⁇ in Example 23. Purification is accomplished using silica gel chromatography.
  • Compound 69 Compound 51 is hydrolyzed with LiOH in essentially the same manner as described for the hydrolysis of 64 in example 25 with the exception that 4.4 equivalents of LiOH is used.
  • Compound 70 The tetra-acid, compound 62, is converted to compound 7_0_ in essentially the same manner as described for the conversion of pyromellitic acid to 6_L in example 24 with the exception that 62 is used instead of pyromellitic acid.
  • Compound 72 is converted to compound 22 in essentially the same manner as described for the conversion of 5 to 60 in Example 23. Purification is accomplished using silica gel chromatography.
  • a solution is prepaicd of four equivalents of compound 24 and five equivalents of Cs 2 CO 3 in DMF.
  • One equivalent of BMP/TEG, platform compound 55, is added to the mixture which is then stirred for 1 h.
  • Pep can be LJP685 or other relevant peptide.
  • Pep can be LJP685 or othei lelevant peptide.
  • the crude material was purified on a preparative HPLC eluted at 10 mL/min with a linear gradient from 30 to 55% B over 40 minutes ⁇ heie A was 0 1 % ( ⁇ ' ⁇ ) TFA in ILO and B was 0 085% (v/v) l ⁇ tsj
  • the cFP assay provides binding constants foi peptides that lack the FITC group and it consumes less antibody, on the order ol 10 ⁇ g
  • the cFP assay is modified from that reported in PanVeia Applications Guide (1994) PanVera Corporation such that it consumes 50-fold less antibody B ⁇ efly, antibody (ACA 6701) is mixed with trace FITC labeled peptide (CB2*-F) and enough time is allowed for equilibrium to be reached This was 1 hour for ACA 6701 and CB2*-F.
  • Equation 2 ⁇ (mP) -
  • Equation 4 P L is the same as in Equation 1.
  • 1 is the concentration of unlabeled peptide competitor, and K,' is the apparent dissociation constant for that peptide. Values for these parameters were obtained by fitting cFP titration data to the above equation. The true dissociation constant for I is obtained from Equation 4.
  • Equation 4 K, '/( 1.0 + R/K D )
  • R and K D are defined as in Equation 1 .
  • the R/K D ratio is obtained from the values of P H ' (from Equation 3) and P H and P L (from Equation 1) and using
  • Equation 5 can be used to determine aPL antibody concentrations once the titration defined by Equation 1 is performed as a "standard curve.”
  • this method provides a means of standardizing all aPL antibody stock solution concentrations and of analyzing their binding activity/stability over time using only 5-10 ⁇ g of antibody per cFP assay.
  • CB2 is a much more flexible peptide and it has an arginine in this position.
  • a more flexible peptide like CB2 may be more cross-reactive because it may more readily adjust its shape to fit a given antibody binding site.
  • the implications of drug rigidification on binding affinity are discussed in Koehler et al. , p. 251 , GUIDEBOOK ON MOLECULAR MODELING IN DRUG DESIGN (Academic Press, N. Cohen, ed., 1996).
  • mice Two different conjugates containing the same peptide were tested for their ability to induce antigen specific tolerance in vivo. Briefly, mice were immunized with the peptide conjugated to the immunogenic carrier Keyhole Limpet Hemocyanin (KLH) to generate peptide-specific memory B cells. Three weeks later, groups of 5 mice per group were treated with various doses of the test conjugates, one group of mice was not treated and acted as the control. Five days later, all of the mice, including the control group, were boosted with the peptide conjugated to KLH and seven days later all of the mice were bled and their sera assayed for anti-peptide antibodies using a modified Farr assay. The Antigen Binding Capacity (ABC) was calculated for each individual serum sample according to the method described in G.
  • KLH Keyhole Limpet Hemocyanin
  • mice primed with the peptide conjugated to KLH were harvested and incubated in complete RPMI-1640 medium for 2 hours at 37°C with various doses of the test conjugates.
  • One group of cells was incubated without toleragen and acted as the positive control.
  • the cells were washed, transferred to irradiated syngeneic recipients and boosted with the peptide conjugated to KLH.
  • mice were primed with LJP-685-KLH on alum plus pertussis as an adjuvant. Three weeks later, the mice were treated with a range of doses of the (LJP685) 4 /MTU- AHAB-TEG conjugate. One group was not treated and acted as a control group. Five days later, all of the mice, including the control group, were boosted with 10 ⁇ g LJP685-KLH and seven days later the mice were bled. Their sera were analyzed for anti-LJP685 antibodies by a modified Fair assay as described above.
  • mice were primed with LJP685-KLH on alum plus pertussis. Three weeks later, the mice were treated with 5, 10 or 50 nmoles of the (LJP685) 4 /MTU-DABA- TEG conjugate. One group was not treated and acted as a control group. Five days later, all of the mice, including the control group, were boosted with 10 ⁇ g of LJP685- KLH and seven days later the mice were bled. Their sera were analyzed for anti- LJP685 antibodies by a modified Fair assay. The results as shown in Figure 29 demonstrate that (LJP685) 4 /MTU-DABA-TEG conjugate induces tolerance in the in vivo model with an ED 50 of 5 nmoles.
  • Spleen cells from mice primed 3 weeks earlier with LJP685-KLH were harvested and incubated in complete RPMI-1640 medium for 2 hours at 37°C with 4,
  • mice Seven days later, the mice were bled and their sera were analyzed for anti-LJP685 antibodies by a modified Farr assay.
  • the results as shown in Figure 30 clearly illustrate that the (L.1P685) 4 /MTU-A11AB-TEG conjugate can induce tolerance when tested in the in vitro model achieving an IC 50 of ⁇ 4 ⁇ M.
  • mice were primed with LJP685-KLH on alum plus pertussis. Three weeks later, the mice were divided into 5 groups of five mice per group. On day 1, one group was treated with a bolus of saline and another group was treated with a bolus containing 50 nMoles of the (LJP685) 4 /MTU-AHAB-TEG conjugate. The three remaining groups were implanted with osmotic pumps. In one group, the pumps were filled with saline and delivered at 1 ⁇ L/hour for 3 days. The two remaining groups received pumps filled with the (LJP685) 4 /MTU-AHAB-TEG conjugate (50 nMoles).
  • One group received pumps that deliver at 1 ⁇ L/hour for three days and the other received pumps that deliver at 0.5 ⁇ L/hour for seven days.
  • the pumps that deliver for three days were surgically removed.
  • all of the mice, including the control group were boosted with 10 ⁇ g of LJP685-KLH.
  • the pumps that deliver for seven days were surgically removed.
  • all of the mice were bled. Their sera were analyzed for anti-LJP685 antibodies by a modified Farr assay.
  • mice are primed with peptide-KLII on alum plus pertussis. Three weeks later, the mice are treated with the (LJP-peptide) 4 /MTU-BMP-TEG conjugate, one group is not treated and acts as the control group. Five days later, all of the mice, including the control group, are boosted with 10 ⁇ g of peptide-KLH and seven days later the mice are bled. Their sera ai e zed for anii-peptidc antibodies by a modified Farr assay. The results show that the (L.ll'-peptide) MTU-BMP-TEG conjugate induces UI

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Abstract

Analogues de l'anticorps antiphospholipide (aPL) qui (a) se lient spécifiquement aux lymphocytes B auxquels un épitope d'aPL se lie. Des analogues optimisés qui sont exempts du ou des épitopes de lymphocytes T sont utiles comme conjugués pour traiter des maladies dépendantes de l'anticorps aPL. Des conjugués comprenant des analogues d'aPL et des molécules à plate-forme de valence non immunogènes sont décrits, ainsi que de nouvelles molécules à plate-forme de valence non immunogènes et des séquences de liaison, des procédés de préparation et d'identification desdits analogues, de procédés de traitement au moyen desdits analogues, des procédés et des compositions destinés à la préparation de conjugués desdits analogues, et des dosages immunologiques et diagnostiques pour les anticorps aPL.
EP97933138A 1996-06-06 1997-06-06 PEPTIDES IMMUNOREACTIFS PAR RAPPORT AUX aPL, LEURS CONJUGUES ET PROCEDES DE TRAITEMENT DES PATHOLOGIES DEPENDANTES DE L'ANTICORPS aPL Withdrawn EP0954531A1 (fr)

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US08/660,092 US6207160B1 (en) 1995-06-07 1996-06-06 aPL immunoreactive peptides, conjugates thereof and methods of treatment for aPL antibody-mediated pathologies
US660092 1996-06-06
US76050896A 1996-12-05 1996-12-05
US760508 1996-12-05
PCT/US1997/010075 WO1997046251A1 (fr) 1996-06-06 1997-06-06 PEPTIDES IMMUNOREACTIFS PAR RAPPORT AUX aPL, LEURS CONJUGUES ET PROCEDES DE TRAITEMENT DES PATHOLOGIES DEPENDANTES DE L'ANTICORPS aPL

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JP2000512981A (ja) 2000-10-03
NO985636L (no) 1999-02-08
KR20000016414A (ko) 2000-03-25
CA2256449A1 (fr) 1997-12-11
AU3640497A (en) 1998-01-05
NO985636D0 (no) 1998-12-03
AU734638B2 (en) 2001-06-21
WO1997046251A1 (fr) 1997-12-11

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