JP2000512981A - aPL immunoreactive peptide, conjugate thereof and method of treatment for aPL antibody-mediated pathology - Google Patents

Abstract

  (57) [Summary] (A) Disclose aPL analogs that specifically bind to B cells to which the aPL epitope binds. Optimized analogs lacking the T cell epitope (s) are useful as conjugates for treating aPL antibody-mediated diseases. Conjugates comprising aPL analogs and non-immunogenic valency platform molecules are provided as novel non-immunogenic valency platform molecules and linkers. Disclosed are methods of preparing and identifying the analog, methods of treatment with the analog, methods and compositions for preparing conjugates of the analog, and diagnostic immunoassays for the aPL antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION   APL immunoreactive peptides, conjugates thereof and methods of treatment for aPL antibody-mediated pathology                               Cross-reference application   This application is a continuation-in-part of U.S. Ser. No. 08 / 660,092, filed Jun. 6, 1996. Application. U.S. application Ser. No. 08 / 660,092 is a U.S. application filed June 7, 1995. This is a continuation-in-part application of application No. 08 / 482,651.                                  Technical field   The present invention is in the field of immunology and for treating anti-phospholipid (aPL) antibody-mediated pathologies. And to compositions and methods for diagnosing. More specifically, the present invention provides Non-immunogenic valency platform molecules (valency platform mol) ecule) and immunospecific analogs of aPL binding epitopes and conjugates thereof And a method for producing the same. Optimized analogs are T cells Lack of epitope. In addition, the present invention relates to antiphospholipid antibodies in biological samples. It relates to a diagnostic assay for detecting the presence of the body and quantifying its amount. Departure Ming also randomized to identify immunospecific analogs of aPL-binding epitopes. The present invention relates to a method using a peptide library.                                 Background of the Invention   Antiphospholipid antibodies are used to treat systemic lupus erythematosus (SLE) and antiphospholipid antibody symptoms In autoimmune diseases such as the herd (APS), and Occurs. APS is associated with arterial or venous thrombosis, thrombocytopenia, and fetal death It is characterized by one or more clinical features, such as fetal loss. APS May be primary, or may be associated with other conditions, mainly SLE (PHOSPHOLIPID-B INDING ANTIBODIES (ed. By Harris et al., CRC Press, Boca Raton, FL, 1991); McNeil et al. , ADVANCES IN IMMUNOLOGY, Vol. 49, pp. 193-181 (ed. By Austen et al., Academic Press, San Diego, CA, 1991)). About 30-40% of patients with SLE have aPL, body 50% of patients who have SLE do not have SLE. 50% of these are other autoimmune rheumatic diseases May have a heterogeneous condition, or they may be subject to drug therapy, especially chlorpromazine May have been lost. 70 people with primary APS (PAPS) but no evidence of SLE In one study of 26 patients (26 men and 44 women), the following characteristics were observed: Detected: Deep vein thrombosis (DVD) in 31; Arterial occlusion in 31, especially seizures or transient Ischemia; 15 myocardial infarction; 24 recurrent fetal deaths; 32 thrombocytopenia (TCP); 1 0 positive Coombs test; 7 Evans syndrome; 32 antinuclear antibodies (ANA), but 29 <1: 160 in humans; and anti-mitochondrial antibodies (AMA) in about 24 (McNeil et al., Supra). . Although estimates vary, in about 5% of all seizure patients, aPL antibodies It is considered to be a factor.   Transient aPL antibodies, such as those detected in VDRL tests, are Happens in between. About 30% of patients with persistent aPL antibodies experience a thrombotic event I have. The presence of an aPL antibody indicates that one or more of thrombosis, TCP, and fetal death The group of patients presenting with a syndrome with characteristic features is defined in the SLE. This for the entire SLE The risk of the syndrome is approximately 25%; this risk is increased to 40% in the presence of aPL antibodies. Increases, and decreases to 15% in its absence. aPL antibodies are phospholipids in cell membranes Because they were thought to be Directly in vivo by interfering with the process of hemostasis that occurs in phospholipid membranes It has been inferred that it may have a significant pathogenic effect. AP in patients with PAPS The fact that L antibodies seem to be the only risk factors that exist is that these antibodies Further evidence that it has a direct pathogenic role. Mouse to human anti-cardi Induction of PAPS by immunization with oripin antibodies indicates that aPL antibodies are directly pathogenic Is the best evidence to date (Bakimer et al., 1992 J. Clin. Invest. 9: 1558-1563; Blank et al., 1991 Proc. Natl. Acad. Sci. 88: 3069-3073).   Measurement of aPL antibodies in a clinical setting is still an incomplete technique. Standard Commercial sets of antisera (APL Diagnostics, Inc., Louisville, KY) are available from various laboratories. Allows the generation of standard curves for comparison of assays performed in the laboratory. I However, the exact GPL and MPL (IgG and IgM anti-reagents, respectively, to evaluate a given serum) Unit of measurement of phospholipid antibodies), and classified as high, medium, or low titers Between the results obtained in these laboratories with respect to GPL and MPL levels There is a mismatch. Commercially available kits have large values assigned to commercially available standards. (Reber et al. (1995) Thrombosis and Haemostat. 73: 444-452). these APS, PAPS, and recurrent seizures and recurrent fetuses despite limitations In other PL antibody-mediated diseases, including death, the epitope recognized by the antibody is , Β_Two-Located in the fifth domain of GPI and β_Two-GPI binding to cardiolipin After, there is a general consensus of exposure to antibodies.   aPL antibody_Two-Glycoprotein (β_Two-GPI) and negatively charged phospholipids, such as Cardiolipin (McNeil et al., (1990) Proc. Natl. Acad. Sci. 87: 4120-4124; Galli Et al., (1990) Lancet I: 1544-1547) (hereinafter `` aPL immunogen '')) Recognition of the proto-complex is now generally accepted. β<sub>Two</sub>-GPI is It is found free and contains lipoprotein lipids (here, apolipoprotein H (also known as (apoH)) It is. It consists of five independent folding domains called Sushi, or other Short consensus repeat domain similar to similar domains in different proteins Consists of β<sub>Two</sub>-GPI binds to phospholipids, changes antigenicity and conformation (Wagenkneckt et al., (1991) Thromb. Haemostas. 69: 3 61-365; Jones et al., (1992) Proc. 5th Intl. Symp. Antiphospholipid Antibosies ( Abstract S5)). The fifth domain contains putative sites for lipid binding and aPL antibody binding (Hu nt J. and S. Krilis, (1994) J. Immunol. 152: 653-659; Lauer et al. (1993) Immuno. l.80: 22-28). The pathological mechanism of aPL is unknown (McNeil et al., supra). Hoton Which explanation begs for endothelial cell function or platelet involvement (Haselaar et al., (1990) Thromb . Haemostas. 63: 169-173). These explanations are based on vascular endothelial cell injury or platelet activation. After sensitization, exposure of anionic phospholipids to surfaces exposed to plasma or bilayer penetration Travel is β<sub>Two</sub>-Induces GPI binding and triggers aPL antibody formation I do.   The aPL antibody reduces prostacyclin formation (Vermylen, J . And J. Arnout (1992) J. Clin. Lab. Med. 120: 10-12); By direct interference with; or to inhibit the intrinsic blood clotting pathway<sub>Two</sub>-GPI Noh Power, Block platelet prothrombinase activity and ADP-mediated platelet aggregation (Arvieux et al., (1993) Thromb. Haemostas. 60: 336-341). It may be thrombinic.   A major new tool in medicinal chemistry to explore lead compounds is the widespread molecular Is the emergence of combinatorial libraries that offer a variety of Diversity of molecules Sex can arise from chemical synthesis or from biological systems (Scott., J.K. RATIONAL DRUG DES IGN (CRC Press, Weiner, DB and W.V. Williams eds., Boca, Raton, FL., 199 4); Moos et al., (1993) Ann. Reports Med. Chem. 28: 315-324). Table of filamentous phage Display of random peptides on the surface allows for the Epitope library containing hundreds of millions of clones to be cloned (Scott, J.K. and G.P.Smoth (1990) Science 249: 286-390; Cesarenl, G. (1992) FE BS Lett. 307: 66-70). Such a phage library was randomized Prepared by incorporating the oligonucleotide sequence into the phage genome . This phage genome is usually the pIII gene, which is a single gene on the surface of each phage. Encoding the peptide sequence of Continuous rounding of affinity purification and amplification After loading, these phages that bind to the antibody are grown in E. coli and bound. Peptides are identified by sequencing the corresponding coding region of viral DNA Is done. In most cases, subsequent studies have established the ability to bind antibodies, For the corresponding synthetic peptide. Phage-based libraries are used for discrete (Luzzago et al., (1993) Gene 128: 51-57; B aLass et al. (1993) Proc. Natl. Acad. Sci. 90: 10638-10642). Peptide-based drugs Potential plasma instability may be due to N-terminal blocking or (Powell, M.F. (1993) Ann. Rep orts Med. Chem. 28: 285-293).   At present, selective, immunospecific therapy for patients with high titers of aPL antibodies There is no. Often drugs like aspirin, steroids, and warfarin Use of the object has proven to be very inappropriate (PH0SPH0LIPID-BINDINGA NTIBODIES (ed. Harris et al., CRC Press, Boca Raton, FL, 1991); McNeil et al., Supra). (i) cannot activate T cells, while (ii) retain the ability to bind immune B cells. Synthetic mimetic peptides, characterized by the following, relax B cells in an antigen-specific manner. Used to consolidate. This technology is a co-pending U.S. patent application No. 08 / 118,055 (filed Sep. 8, 1993) and US Pat. No. 5,268,454 Which are incorporated herein by reference in their entirety. Patent application above As disclosed in and patents, B cell tolerance is defined as B cell anergy, Suppresses antibody production through deletion of clones after cross-linking to surface immunoglobulin Coupled to a multivalent, stable, non-immunogenic valency platform to Need to be administered.   The exact molecular nature of the target epitope recognized by aPL antibodies is unknown However, the use of peptides from the epitope library has led to the successful construction of a tolerogen. enable. B cell tolerogen for the treatment of human systemic lupus erythematosus-related nephritis Are also disclosed in commonly owned U.S. Patent Nos. 5,276,013 and 5,162,515, These are incorporated by reference herein in their entirety.                                 Disclosure of the invention   The present invention relates to PAPS, APS, and other such as recurrent seizures and recurrent fetal deaths. Significance Recognized by aPL Antibodies in Patients With APL Antibody-Mediated Diseases Analogs of different epitopes using random peptide phage libraries Finding a way to identify.   Thus, one aspect of the present invention is to optimize epitopes recognized by aPL antibodies. Random peptide phage licensing to identify peptide sequences that mimic An improved method of screening for bullies. This method comprises the following steps (A) library using methods modified from those known in the art. (B) (i) aPL antibody bound to protein G Incubating phage in microplate wells coated with body , (Ii) wash microplate wells to remove unbound phage (Iii) eluting the bound phage, and (iv) eluted phage. E. by infecting a microorganism such as E. coli and seeding on agar Screened from step (a) by counting the number of infected microorganisms H Very weak binding by micropanning the image (C) (i) coating the wells of the microplate with the aPL antibody (Ii) the best identified by microselection of step (b) in the coated wells Incubate the strongly binding clones and remove unbound phage. Washing off, (iii) goat anti-phage anti-enzyme bound in a colorimetric ELISA assay Phage capture by quantifying the number of phage bound to antibodies using the body Determine the strongest binding clone recovered in (b) by evaluation via ELISA And if clones that bind with several equal strengths are identified Add (d) phage ELISA with the most strongly binding phage capture ELISA clone Repeat of addition.   In this regard, the present invention provides a method for isolating a human from an aPL antibody-mediated disease. Method for identifying an analog of an epitope that specifically binds a selected aPL antibody The method comprises the following steps: (a) phage random peptide library Preparing (b) this line with an aPL antibody to identify an aPL mimetic epitope. Screening a library, the screening comprising: Screening this library by selection; (ii) organism selection in (i) Phage isolated by extraction are further screened by microselection And a fur containing the high affinity binding peptide of the aPL antibody recovered in (iii) (ii). Identifying the di by immunoassay.   The invention also provides a method for identifying and isolating peptides that bind to aPL antibodies. A method for bioselection of a gemrandom peptide library. Including (a) affinity-purified aPL antibody with Reacting with a phage having an insert; (b) random binding to aPL antibody A step of recovering the phage having the peptide insert; the steps of (c) and (b). Infecting the microorganism with the phage; and (d) an antibiotic to isolate the phage Culturing the infected microorganisms in the containing medium.   The present invention further identifies and identifies peptides with high binding affinity to aPL antibodies. Method for microselecting a phage random peptide library for isolation And the method comprises the following steps: (a) random peptide insert Isolating phage having the following by biological selection: (b) binding to protein G Microplate wells coated with the aPL antibody Incubating the phage; (c) to remove unbound phage Washing the microplate well; (d) eluting the bound phage And (e) infecting the microorganism with the phage recovered in (d); Culturing the infected microorganism in an antibiotic-containing medium to isolate the virus.   The invention also encompasses the above method wherein the immunoassay is a phage capture ELISA. The method comprises the following steps: (a) a microplate coated with an aPL antibody; Has random peptide inserts isolated by microselection in twells Incubating the binding phage; (b) washing away unbound phage (C) incubating the enzyme-labeled anti-phage antibody in the well (D) washing away unbound enzyme-labeled anti-phage antibody; (e) colorimetric substrate And (f) absorbance of the substrate to identify high affinity binding phage. The step of measuring the degree.   The present invention also encompasses the above method, and further provides for the high affinity binding phage. Performing a phage-capture ELISA assay, which includes the following steps: Including: (a) coating a uniform amount of phage on microplate wells; (b) Incubating the aPL antibody in the well; (c) washing away unbound antibody (E) incubating the enzyme-labeled anti-aPL antibody with the conjugated aPL antibody; f) washing away unbound enzyme-labeled anti-aPL antibody; (g) well of colorimetric substrate And (h) a substrate for measuring the relative binding affinity of the phage. Measuring the absorbance of the sample.   The invention also relates to the above, wherein the immunoassay is a colony blot immunoassay. This method comprises the following steps: (a) On the agar-containing culture medium On a nitrocellulose membrane with a random peptide insert. Culturing the microorganisms infected with the phage; (b) a second nitro on the agar-containing culture medium. By blotting the microorganisms on a rocellulose membrane, (a) Replication transcription of the cultured microorganism; (c) incubating the transcribed microorganism (D) lysing the microorganism; (e) digesting the microorganism with lysozyme; (f) blocking the membrane with a gelatin solution; (g) combining the membrane with an aPL antibody. Incubating; (h) washing away unbound aPL antibody; (i) enzyme Incubating the labeled anti-aPL antibody with the nitrocellulose membrane; ( j) washing away unbound enzyme-labeled anti-aPL antibody; (k) adding a colorimetric substrate Measuring the absorbance of the substrate to identify high affinity binding phage Process.   Specific binding of aPL antibodies isolated from humans with aPL antibody-mediated diseases Methods for assaying and ranking epitopes for affinity binding characteristics The method includes the following steps: (a) microtitration step Coating rate wells with cardiolipin; (b) binding to cardiolipin , And to prevent non-specific binding to the wells of the plate,<sub>Two</sub>-With GPI sources And adding mature bovine or human serum to the sample; Incubating the log and high titer aPL antibody solution; (d) aPL antibody / a Add the Nalog mixture to the wells of the microtitration plate, and add Incubating for a fixed period of time; (e) to wash away unbound aPL antibodies Washing the wells; (f) playing anti-human IgG conjugated to a label (eg, an enzyme) Adding to said wells and incubating for a predetermined time; (g) binding Washing the wells to wash away any anti-human IgG conjugate; Add the substrate for the coalescence and allow the substrate / labeling reaction to develop for a predetermined time. Step: (i) Finalize the substrate / labeling reaction to quantify the amount of aPL antibody bound to the wells. Measuring the product; (j) determining the affinity of the analog, if any, for the aPL antibody Calculating the rate of inhibition of the binding of the aPL antibody.   Another aspect of the invention determines the dissociation constant for a peptide that binds to an aPL antibody. Fluorescent polarization peptide binding assays. This assay is designed for use with aPL antibodies. Detects the direct binding of the peptide.   The invention can also be obtained from a subject suspected of having an aPL antibody-mediated disease. Diagnostic immunoassays for determining the presence of aPL antibodies in the body fluids Includes the following steps: an analog of an epitope that specifically binds to an aPL antibody; Contacting a sample of the body fluid, and whether the aPL antibody is present in the body fluid Is determined by methods well known in the art, and, if present, A step of quantifying the amount of the aPL antibody present in the body fluid. One such immunoassay B. The method includes the following steps: (a) Analyzing an epitope specifically binding to aPL antibody Coating the wells of the microtitration plate with a tubing; (b) binding Washing the wells to wash away any missing analogs; (c) a test sample of bodily fluids Adding to the wells and incubating for a predetermined time; (d) binding Washing the wells to remove untested sample; (e) the label is bound Anti-human IgG was added to the wells of the plate and incubated for a predetermined time. (F) washing the wells to wash away unbound anti-human IgG conjugate (G) adding a substrate for the labeled conjugate, and Developing the labeling reaction; (h) determining the presence of the anti-aPL antibody in the test sample Measuring the final product of the substrate / labeling reaction. If the immunoassay is quantitative Also included are diagnostic immunoassays as described above.   The phage ELISA assay consists of the following steps: (i) Microtitration Coating a uniform amount of the various clones onto the wells of the plate, and then (ii) Peptide insulin binding to aPL antibody most strongly by adding body to well Identifying the salt and developing the reaction with an enzyme-labeled anti-human IgG conjugate . Measured by phage ELISA, colony blot, or phage capture ELISA Runs displayed by phage with high binding affinity to aPL antibodies as shown Dam peptides represent analogs of the aPL-specific epitope. Then, these pages The peptides are synthesized and the binding strength is ranked using a competition assay.   Another aspect of the invention relates to aPLs that specifically bind to B cells to which the aPL epitope binds. Antibody binding analog. The optimized analog may comprise one or more T cells Deleting the epitope.   Yet another aspect of the present invention induces specific B cell tolerance to aPL immunogen Comprising a non-immunogenic valency platform molecule and (a) aPL Specifically binds to B cells to which the immunogen binds, and (b) one or more T cells APL antibody binding analogs that lack an epitope.BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows that the use of fish gelatin in place of mature bovine serum is based on a commercially available standard aPL antibody. Abolished all anti-cardiopyrine (ACA) activity in ELISA analysis of Is shown. This result indicates that β in determining the target epitope of ACA<sub>Two</sub>-Glycoprotein I (Β<sub>Two</sub>-GPI) by McNeil et al. (Supra) and Galli et al. (Supra). Supported.   FIG. 2 shows that analog 5A12 bound to the resin was identified as ACA-6501 immunospecifically. Binding to affinity-purified IgG.   FIG. 3 shows aPL antibody-binding analogs obtained by screening using ACA-6626. , But bound to the aPL antiserum but not to the standard serum.   FIG. 4 also shows that the ACA6501 / 5A12 analog bound immunospecifically to the ACA-6501 antiserum. , And cross-react with ACA-6626.   4 and 5 show ACA650 obtained by screening according to the method of the present invention. APL antibody-binding analogs of 1 / 5A12 and ACA6626 / 4D3 take precedence over screening antibodies Shows that a significant degree of cross-reaction was detected while binding specifically.   FIG. 6 shows a method for calculating a GPL value for the ACA-6501 aPL antibody.   FIG. 7 shows a comparison of the activity of affinity-isolated ACA-6501 with GPL standard serum. You.   FIG. 8 shows that the sequence diversity of the clones isolated by the four biological selections rapidly increased. Indicates a decrease.   FIG. 9 shows that all clones tested did not respond to normal IgG Nevertheless, in the phage capture ELISA using ACA-6635, three clones (3A12 , 3B3, 3A5) showed very strong immunospecific signals.   FIG. 10 shows that in the phage ELISA using ACA-6501, seven clones showed strong It shows that it showed a gunal.   FIG. 11 shows competition using ACA-6501 obtained with peptides 5A12, CB2, and 3B10. The results of the binding ELISA are shown. To inhibit by 50%, 0.08 μg CB2 and 0.5 μg 3B Despite the need for 10, 0.16 μg of peptide 5Al2 gave ACA6501 aPL anti- Tetravalent peptide ACA6501 / bound to the body of a polystyrene microplate well 3B Binding to 10 was inhibited by 50%.   FIG. 12 shows a comparison of the activity of the modified ACA6641 / 3G3 analogs.   FIG. 13 shows peptides 139, 142 in a competitive binding ELISA using the ACA-6501 aPL antibody. And 143 indicate that the 50% inhibition values were 6.9, 0.7 and 0.9 μg, respectively. .   FIG. 14 shows α-Me-Pro at positions 3, 9, and both 3 and 9 of peptide 3B10. Shows the replacement effect. The substitution effect of α-Me-Pro at position 9 is similar to the “native” peptide By comparison, the peptide activity was increased four-fold.   FIG. 15 shows that peptide 6641 / 3G3 (LJP688) has nine affinity-purified ACA antibodies. Indicates high cross-reactivity with the body.   FIGS. 16 and 17 show LJP685-MTU-DAB at 100 μM, 20 μM, and 4 μM, respectively. Using A-PEG conjugate (Compound 36) and LJP685-ATU-MTU-AHAB-TEG conjugate (Compound 35) After incubating spleen cells of mice immunized with LJP685-KLH for 2 hours each, Using spleen cells of mice immunized with JP685-KLH 10<sup>8</sup>Dependence of anti-685 antibody ABC at M Indicates a survival decrease.   FIG. 18 shows N of the nine structures elucidated for peptide 925 that are closest to the center of gravity. Figure 3 shows the MR structure, a rational description of the form of the peptide 925 molecule.   FIG. 19 shows the structure of peptide 925 (labeled 3G3 at the bottom of the figure) and the structure of peptide 5A12. Compare. Both peptides have turns at approximately the same position in the peptide sequence.   Figures 20A and 20B show that the drug delivery of the aPL analog is one small hydrophobic group and Indicates that it was experimentally identified as a charged group. As shown in FIG. The gem-dimethyl and amino groups of 925 can be tested for drug delivery of this peptide. Is identified. The length of the hydrocarbon linker that connects the drug transfer group to a particular backbone These linkers are identified in FIG. 20A as well as the distance separating the positions where they attach to the backbone.   FIG. 21 shows inhibition of β2GPI by a 6501-derived peptide using diluted 6501 serum.   Figure 22 shows that CB2<sup>*</sup>-F; Shows ACA-6701 titration of FITCGPCILLARDRCG.   Figure 23 shows CB2<sup>*</sup>-F: Indicates ACA-6501 titration of FITCGPCILLARDRCG.   Figure 24 shows CB2<sup>*</sup>-F: Shows complete ACA-6501 titration of FITCGPCILLARDRCG.   FIG. 25 shows CB2<sup>*</sup>CB2 from ACA-6701 using 1.04 equivalents of<sup>*</sup>Indicates a substitution for -F.   FIG. 26 shows CB2<sup>*</sup>CB2 from ACA6701 using<sup>*</sup>Figure 5 shows a cFP titration replacing -F.   FIG. 27 shows CB2 from ACA6701 using 3B10.<sup>*</sup>Figure 5 shows a cFP titration replacing -F.   FIG. 28 shows the dose response of (LJP685) 4 / MTU-AHAB-TEG conjugate for tolerance activity .   FIG. 29 shows the dose response of (LJP685) 4 / MTU-DABA-TEG conjugate for tolerance activity .   FIG. 30 shows the (LJP685) 4 / MTU-DABA-TEG conjugate tested in an in vitro model. Figure 3 shows a dose response of tolerant activity.   FIG. 31 shows the (LJP685) 4 / MTU-DABA-TEG conjugate tested in an in vitro model. Figure 3 shows a dose response of tolerant activity.   FIG. 32 shows (LJP685) 4 / MTU-AH comparing various routes of administration and dosage ranges. Fig. 4 shows the tolerance effect of the AB-TEG conjugate.                           Embodiments for implementing the present invention   A.Definition   As used herein, the term “aPL antibody” specifically binds to β2-GPI that mediates disease. Any antibody that matches.   As used herein, the term "B cell anergy" produces and secretes antibodies. Are intended to be non-responsive to B cells that require the help of T cells Lack of immature and / or mature B cell clones and / or Include the inability of B cells to produce antibodies.   “Non-responsive” means effective therapeutic reduction of humoral response to immunogen I do. Quantitatively, this decrease (as measured by a decrease in antibody production) is at least 50 %, Preferably at least 75%, and most preferably 100%.   "Antibody" means an antibody that is dependent on T cells.   As used herein, the term "immunogen" elicits a humoral immune response that includes aPL antibodies Means a substance. Immunogens use both B and T cell epitopes Have. The aPL immunogens involved in pathologies mediated by aPL antibodies are foreign (external to the individual). Incoming) immunogens, including, for example, therapeutic proteins, peptides and antibodies. Drugs that contain foreign biological materials that are foreign to any individual) or antibodies It may be an autoimmunogen such as that associated with mediated clotting (seizure).   The term “analog” of an immunogen refers to (a) an antibody to which the immunogen specifically binds. Differentially binding molecules, and (b) molecules lacking T cell epitopes. You. Analogs are usually fragments or derivatives of an immunogen and are therefore (Eg, the immunogen is a polypeptide and the analog is a Peptide)), but chemical similarity is not essential. Therefore, analog As long as it has the functional characteristics of (a) and (b) above, (Eg, the immunogen is a carbohydrate and the analog is polypep Is a tide). Analogs can be peptides, carbohydrates, lipids, polysaccharides, nucleic acids or It can be another biochemical. In addition, the chemical structure of either the immunogen or the analog Need not be defined for the present invention.   The term "analog" of an immunogen also includes the term "mimotope". The term “mimotope” refers to a component that competitively inhibits the binding of an antibody to an immunogen. Means child. To specifically bind antibodies, mimotopes are used to determine immunogen antigen It is thought to be similar to the group.   As used herein, "valency platform molecul" e) "indicates non-immune components containing sites that promote binding of a modest number of immunogen analogs. Means child.   As used herein, “non-immune” refers to a valence platform molecule. Used when the valence platform molecule is administered alone to an individual. Means not substantially eliciting an immune response.   As used herein, “individual” refers to a member of the mammalian species and includes humans, primates Animals, livestock such as cattle and sheep, sport animals such as horses, and dogs and And pets such as cats.   As used herein, "pharmacophore" refers to an antibody to an aPL analog It refers to the three-dimensional orientation and chemistry of the central group involved in binding to the target. B.aPL Of antibody-binding analogs of   aPL antibody-binding analogs are screened for candidate Does (a) specifically bind to aPL antibody and (b) lacks T cell epitopes Can be identified by determining. aPL antibody specific Binding is performed using routine immunoassays such as the ELISA assay described in the Examples below. Can be determined. Further, whether the T cell epitope is present or deficient, Similarly, it can be determined in the routine T cell activation assay described in the Examples. From this point An analog that "specifically binds" to serum antibodies may be capable of binding<sup>7</sup>M<sup>- 1</sup> Shows a moderate affinity for Whether the T cell epitope is present or deficient The tritiated thymidine incorporation method disclosed in U.S. Patent Application No. 08 / 118,055. This can be determined using the assay. The presence of T cell epitopes also It can be determined by measuring the secretion of fokine by methods well known in the art. Analogs that do not induce statistically significant thymidine incorporation over background , Are thought to be deficient in T cell epitopes. The amount of thymidine incorporation It is understood that it can vary with the immunogen. Typically about 2-3, more usually A stimulation index lower than about 1-2 indicates that the T cell epitope is missing. C.Preparation of conjugate   aPL antibody-binding analogs can be linked to non-immune valency platform molecules. Thus, the conjugate of the present invention is prepared. Preferred valence platform molecules are Several hours in vivo to be biologically stable, i.e. to increase treatment efficiency It has an elimination half-life of ~ days to months, and is formed in a synthetic single-strand of defined composition It is preferred that this be done. They typically have molecules ranging from about 200 to about 200,000 Quantity, usually about 200 to about 20,000. In the present invention, the valence Examples of home molecules include polyethylene glycol (PEG), poly-D-lysine, Polymers such as vinyl alcohol and polyvinylpyrrolidone . Preferred polymers are based on polyethylene glycol (PEG), from about 200 to about 8,8. It has a molecular weight of 000.   Other valency platform molecules suitable for use in the present invention are chemically defined A non-polymeric polymeric valency platform molecule, which No. 08 / 152,506, filed Nov. 15, 1993, which is hereby incorporated by reference. , Which is hereby incorporated by reference in its entirety. Features suitable for use in the present invention Preferred homogeneous chemically defined valency platform molecules are 2,2'-ethyl Les Derivatives of dioxydiethylamine (EDDA) and triethylene glycol (TEG) It is.   Further suitable valence platform molecules include tetraaminobenzene, pep Taamino beta cyclodextrin, tetraaminopentaerythritol, 1,4,8,11 -Tetraazacyclotetradecane (Cyclam), and 1,4,7,10-tetraazacyclod Decane (Cyclen) is included.   The binding of aPL antibody-conjugated analogs to valency platform molecules depends on various factors. But may typically be affected by one or more crosslinkers and analogs. And functional groups on the valency platform molecule.   Polypeptide analogs serve as sites for binding the analog to the carrier. Functional groups such as amino, carboxyl, or sulfhydryl groups And the amino acid side chain portion containing If the analog does not have these functional groups Residues with such functional groups can be added to the analog. Such residues are Introduced by solid phase synthesis or recombinant methods, both well known in the field of tide synthesis. obtain. In the case of carbohydrate or lipid analogs, functional amino groups and sulfhydrides The radical can be introduced by conventional synthesis. For example, primary amino groups are cyano hydrogenated Incorporated by a reaction using ethylenediamine in the presence of sodium boron, The sulfhydryl group, after reaction with cysteamine dihydrochloride, is It can be introduced by reduction with a sulfide reducing agent. Valence platform molecule Also have functional groups in a similar way if they do not already have the appropriate functional groups. Can be derivatized.   Hydrophilic linkers of various lengths can valence peptides or other bioactive molecules Useful for binding to platform molecules. Suitable linkers include Includes linear oligomers or polymers of lenglycol. Such a linker ー is R<sup>1</sup>S (CH<sub>Two</sub>CH<sub>Two</sub>O)<sub>n</sub>CH<sub>Two</sub>CH<sub>Two</sub>O (CH<sub>Two</sub>)<sub>m</sub>CO<sub>Two</sub>R<sup>Two</sup>And a linker of the form Where n = 0 to 200, m = 1 or 2, R<sup>1</sup>= H or a protecting group such as trityl, R<sup>Two</sup>= H or alk Or aryl, for example, 4-nitrophenyl ester. These resources Are molecules containing thiol-reactive groups such as haloacetyl, maleamide, etc. To the second molecule containing an amino group via an amide bond via a thioether. Congruence Useful to make These linkers are flexible in the order of attachment and That is, the thioether can be formed first or last.   Conjugates are usually prepared for administration by injection (eg, intraperitoneally, statically). Intravenous, intramuscular, subcutaneous). Thus, they typically include saline, Ringer's With pharmaceutically acceptable vehicles such as solutions, dextrose solutions, etc. Is done. The conjugate will typically comprise from about 0.01% to 10% by weight of the formulation. Join The body treats the individual with a “therapeutically effective amount”, ie, B cells against the immunogen involved. Prevent and ameliorate anergy-producing and antibody-mediated conditions in question Alternatively, an amount sufficient to achieve elimination is administered. Specific dosing regimen, ie dose , Timing, and number of repetitions will depend on the particular individual and that individual's medical history. Normal, Dosages may range from about 1 μg to about 100 mg conjugate / kg body weight, preferably from about 100 μg to about 10 mg. / kg body weight per week. Other suitable dosing regimens include daily, three times a week, or once a week Times, or once every two to four weeks, or once a month, or depending on the individual or disease. A less frequent schedule. Usually, depending on the turnover rate of B cells, Timed multiple doses achieve and / or maintain humoral anergy status May be needed to Such repeated administrations typically detect an increase in antibody titer. If known, give about 1 μg to about 10 mg / kg body weight or more every 30 to 60 days, or Or requires treatment at shorter intervals. Alternatively, in some pathologies, binding A continuous release formulation of the body may be suggested. In the field, to achieve sustained release Various formulations and devices are known.   Anti-helper T cell treatment can be administered with the conjugate. Such treatment is usually Drugs that suppress T cells, such as steroids or cyclosporine, are used. D.aPL Antigen properties of antibodies   Initial studies by the inventors on aPL antibodies were recognized by these antibodies. This was consistent with the publication of a report pursuing the nature of the antigenic site to be derived. Initially, this These antibodies recognize cardiolipin molecules in the same way as antibodies detected in the VDRL test It was considered. As shown in FIG. 1, anti-cardiolipin antibody (ACA) solid-phase ELISA When mature bovine serum was replaced with fish gelatin, it was obtained commercially as an ACA standard. All ACA activity of the antibody preparation was abolished. This finding was made by McNeil et al. ACA antibodies, as suggested by Galli and Galli et al. Rather than those that recognize serum determinants. This protein is Β by these authors<sub>Two</sub>-Shown to be a GPI. Therefore, "ACA" or " The term “anti-cardiolipin antibodies” is a misnomer in nature, but today these antibodies Is still used when referring to   Many autoimmune IgG aPL antibodies have β<sub>Two</sub>-Recognize determinants on GPI molecules. this These epitopes can only be expressed upon binding to cardiolipin.<sub>Two</sub>-Form or on GPI Can be exposed (neo-epitope). Or β<sub>Two</sub>-The epitope on GPI is β<sub>Two</sub>-GPI There may be one copy per molecule and may not have low affinity for aPL antibodies You. Β adjacent by binding to cardiolipin<sub>Two</sub>-These sites on the GPI molecule are 2 Only by lining up will a sufficient affinity be generated to maintain the antibody-antigen interaction I can do it.   Native β<sub>Two</sub>Of the aPL analog in the present invention that mimics the epitope of -GPI From magnetic resonance (NMR) analysis, β<sub>Two</sub>-More information on the structure of GPI epitopes Was. For example, the NM of two peptide aPL analogs that are highly cross-reactive with aPL antibodies From the comparison of R solution structures, both peptides were targeted at almost the same position in the peptide sequence. (See FIGS. 18 and 19). E. FIG.ACA ELISA   GPL assessment of clinical samples and study antibodies and β<sub>Two</sub>-GPI chromatography The need for large-scale testing in connection with purification by Lead to development, its function is very similar to the commercial kit, and the best reproduction Similar to a known good design (Redder, supra). Modified ACA ELISA also It has also been implemented. It bound directly to a specific microplate well β<sub>Two</sub>-Using GPI, aPL IgG is added to Direct binding in the absence of pins (Roubey et al. (1996) Arthritis & Rheumatism 39: 1606-1607; R.A.S. Roubey (1996) ArthritiS & Rheumatism 39: 1444-1454) Teru. F.aPL Purification of antibodies by immunoaffinity   For isolation of aPL antibodies, multilamellar, cardiolipin-containing dispersions (Liposo APL plasma (or blood), including cholesterol and dicetyl phosphate Incubate with Kiyoshi). Are these liposomes serum by centrifugation? And then pelletized. After washing, the liposome mixture is added to the 2% octyl glucoside interface. Crushed with an activator and applied to a protein A-agarose column. After thorough cleaning First, remove lipids, then remove non-IgG components, and protect the IgG aPL antibody with a mild acid. Eluted from in A, neutralized, buffer exchanged, and performed ACA ELISA assay. This APL antibody was concentrated 10,000-fold, and rabbit IgG anti-human β<sub>Two</sub>-GPI antiserum Β as shown by Western blotting with<sub>Two</sub>-GPI is completely mixed Do not enter. A further affinity purification step is the solid phase β<sub>Two</sub>-Affinity on GPI -Chromatography of the purified antibody. This second affinity The purification step is β<sub>Two</sub>-APL antibody that binds directly to GPI has stronger clinical relevance Recommended as a result of good knowledge. This also means that further contamination (especially β<sub>Two</sub>-GPI) Served to ensure a final preparation without any. G. FIG.Construction of a filamentous phage random peptide library   11 different fUSE5 filamentous phage random peptide libraries for p-III protein Li (5 copies of peptide p-III per phage) are constructed. These live Rally offers vast sequence morphology and structure to discover mimetic epitopes . The four libraries, named “x” libraries, are 8, 10, 12 and And a peptide insert 15 residues in length, containing both amino and carboxyl The terminal proline residue is adjacent. The purpose of these proline residues is Eliminating the contribution to secondary structure that can result from the active p-III protein, and Exposing the salt to the solvent. The "y '" library is 6,7,8,9,1 in length Includes cysteine-linked inserts that are 1 and 13 amino acids. "Y" library Lee excludes the absence of the 6 and 8 amino acid inserts of the "y '" library. This is the same as the "y '" library. "Y" and "y '" libraries -Mushroom These peptide inserts are circular and form amino acids to form more rigid structures. And carboxyls are flanked by cysteine residues at both ends. Proline residue These cysteine residues are for the same reasons as for the "x" library described in Incorporated outside the group. The "x", "y '" and "y" libraries are It is located 5 residues from the amino terminus of the active p-III protein. "Z" la The library consists of a random 8-amino acid located at the amino terminus of the p-III protein. Consists of inserts and does not contain adjacent proline or cysteine residues . The combination of the "x", "y '" and "z" libraries has approximately 100 million different Peptide inserts represent 11 different libraries each having one.   These libraries are of a suitable length to give the desired length of insert. FUSE random oligonucleotide sequences using standard molecular biology techniques It is constructed by incorporating 5 p-III genes. Restriction endonuclease of fUSE5 DNA Following the lase treatment, the kinased excess provided as a gap duplex An oligonucleotide is added and ligated. The DNA was then subjected to E. coli using electroporation. co li and selected by culturing in a tetracycline-containing medium. This culture Phage (including peptide insert) from the ground is isolated from the supernatant, washed, and Resuspend in the buffer. A typical library is 7 × 10<sup>8</sup>8 x 1 independent clones 0<sup>12</sup>It was shown to have in transformation units / ml. H.Phage screening methodology   The essence of screening phage-displayed peptide libraries is billions of latent The ability to reduce the potential candidate phage to a relatively small number with outstanding properties. Former Screening protocols are described in Scott, J.K and G.P. Smith, (1990) Science 249. : 315-324, but facilitates selection of the best epitope for various aPL antibodies As such, it has been significantly modified. These techniques are useful for presenting the best sequences Screening is continued until a point is reached where a sufficient number of clones can be thoroughly investigated. Designed to make a larger stringency selection as it progresses . For some antibodies, the library is a very tight binding amino acid sequence Do not seem to have a high degree of stringency S When used for cleaning, specific clones disappear. On the contrary , Libraries often yield many clones showing good analogs of the antigen And the use of methods with a high degree of stringency is the best epitope Is required to identify This indicates that the epitope library screen Varying degrees of stringency to identify the best epitope from Assays with the following parameters have been developed. Here, stringent assays Shown in order of increasing sea: organism selection <micro selection <phage capture ELISA <fur Di ELISA = colony blot = peptide ELISA. (i) Biopanning   “Bioselection” refers to affinity-purified aPL antibodies and any peptide antibodies. The phage retaining the insert is mixed and then bound by antibody-specific recovery. This is a technology for capturing images. Phage confers tetracycline resistance on E. coli Therefore, E. coli is cultured in a medium containing tetracycline and isolated. Creature Repeated selection increases the number of immunospecific phage in the sample. Let Phages are always recovered at the end of the third through fifth selections, but they are Can represent only non-specifically bound sequences with low affinity for the antibody being screened . A method for further evaluating these phages (micro selection) is required. (ii) Micro selection   Estimating the relative strength of phage binding to aPL antibodies is described in “Microphones. Micro-selection is more than three times biological selection And uses the same antibody used in the bioselection method. The method is raw Dilution of phage obtained in the last round of product selection, and more than 50 by microselection Analysis of these clones. In micro selection, each clone is After amplification to a similar density, the microtiter cells were previously coated with a certain amount of antibody. Incubate phage diluted at the optimal single concentration in the wells of the dilution Is achieved by doing The optimal single concentration of phage is The concentration that is most likely to give a black selection score (0 to 4+) This is the concentration that allows maximum discrimination. It is a series of dilutions When the score is determined at each of the several phage concentrations obtained, Based on the behavior of micro-selection of 6 randomly selected clones. Antibodies and Incubation After the bait, the unbound phage is washed away and the amount of bound phage is It is an indicator of the affinity of the insert for the antibody. Reduce the amount of bound phage After elution with kana acid, neutralize and infect E. coli and determine. Infected E.coli The number was determined for each clone by plating the microorganisms on an agar medium containing tetracycline. It is quantified by determining the density of the colonies formed. (Iii) Phage-Capture ELISA   The phage capture ELISA test was performed on micro-tubes with relatively low stringency. Phage ELISA or Peptide E with selection assays and high stringency Developed as an intermediate level assay that bridges the LISA assay. Preliminary experiment Now, when preparing some antibodies, using microselection Is too high, but using phage or peptide ELISA Not shown. The limitations of the phage ELISA are shown below, with 5 copies of p-III Only was present in each phage, and coated the wells with multiple phages. Even so, the product of the insert is very small, and is inserted into the insert for detection. Antibodies with very high affinity for them are required. Using phage capture ELISA If the signal is amplified multiple times, the lower Facilitates the detection of affinity-stable interactions.   The phage capture ELISA consists of the following steps. Microtitration wells With the aPL antibody, and clone the phage clones as in the microselection assay. Add. Unbound phage is washed away and the amount of bound phage is Quantification is performed using an enzyme-conjugated goat antiserum that binds to the major coat protein. Phage Phage screened by capture ELISA reacts with many aPL antibodies, It shows a strong signal in a subsequent ELISA assay. Micro selection positive fur Since this is almost non-positive in the phage capture ELISA, Degree levels make peptide synthesis attempts more efficient. As a result, Peptides synthesized from positive phage obtained by the image capture ELISA It shows immunoreactivity. (iv) Phage ELISA   This phage selection method provides very strong binding between the insert and the screening antibody Need. Cover the wells of the microtitration plate directly with phage. Cover, add screening antibody and incubate. Wash to remove unbound antibody After removal, the addition of an anti-human IgG alkaline phosphatase conjugate Binds to aPL antibody bound to di. The aPL antibody can then be purified by methods well known in the art. Therefore, by adding a chromogenic substrate that reacts with alkaline phosphatase to the wells , Will be detected. (v) Colony blot   This assay uses E. coli infected with phage selected in bioselection. coli large rule Allows for colony screening in a large scale. This procedure is for immunoreactive clones. Is an alternative to phage ELISA for the identification of There is no need to culture each phage clone before. In this assay, 1 E. coli infected with the phage obtained from the Spread on a low cellulose (NC) membrane and place on an agar medium containing tetracycline. Culture overnight (Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7978-7982). Each These colonies result from infection with phage having the same sequence. This NC Transfer the blot onto the NC using the `` master '' to make several replicates, and Culture can be performed on a natural medium. Phage-infected colonies on blots chemically and enzymatically Following the destruction of Ronnie, commonly used Western blotting techniques (Ie, staining and immunoblotting), the phage was probed. Can be Incubate blocked blots with screening aPL antibodies Can also be After washing away unbound antibody, anti-human IgG horseradish The oxidase conjugate is added and allowed to bind to the phage-bound aPL antibody. Coloring By adding the substrate, an immunospecific Separate colonies on the master plate displaying phage can be located. (vi) Peptide ELISA   The most reactive phage peptide insert in the assays described so far After DNA sequencing, the corresponding peptide is converted to a standard known in the art. Prepared using classical Fmoc peptide chemistry. For peptide ELISA assays, For example, a peptide can be made as a branched tetravalent molecule, ie, each molecule is a tetravalent molecule. With copy insert. Microtitration plates with such molecules Cover the wells of the plate and expose the epitope to the solution to allow the antibodies to bind. 4 The multivalent peptide is due to incorporation of lysine as a branch point in the first two couplings. This method is based on the method used for multiple antigen peptides (MAPS) (Pos (1988) J. Biol. Chem. 263: 1719-1725). Glycine-serine-gly A spacer consisting of syn-serine was added to each arm after lysine and further Skeletal amino acids, carboxyl-terminal proline-glycine, And an insert containing alanine-glycine-proline at the amino terminus. This All amino acids used for the synthesis of are added at once using the standard Fmoc method.   These peptides are then coated in microtitration wells with the peptide. And assay for reactivity with the aPL antibody in a standard ELISA format. Assayed by ELISA performed. In practice, the peptide is usually Binds very tightly with the screening antibody and has some interaction with other aPL antibodies Shows cross-reactivity. To eliminate non-specifically bound peptides, use non-aPL antibodies Include body controls. (vii) Competitive binding peptide ELISA   Once ELISA-positive peptides have been identified, their relative binding to aPL antibodies Affinity is quantified and the two peptides have the same number in the serum of a given patient. Requires binding to antibody by peptide competition ELISA assay It is. In this assay, various monomeric peptides were used for microtitration. Compete with the tetravalent peptide coating the plate well. To perform the assay In addition, the peptide to be evaluated is converted into a monomer by standard Fmoc chemical synthesis. That is, synthesized without branching by lysine used in tetravalent peptide synthesis I do. The monomeric peptide is purified and dissolved at a known concentration. Micro titres Plate wells with a tetravalent peptide known to bind to the aPL antibody. Overturn. The dilution series of the monomeric peptide was added to a fixed dilution of the aPL antibody and incubated. Beate. For aPL antibody dilution, measure antibody titer against tetravalent peptide, and titer It is predetermined by choosing the dilution at which the slope of the curve becomes negative. Antibodies and antibodies After 1 hour incubation with the nomeric peptide, the antibody / peptide solution is Perform a standard colorimetric ELISA in addition to the titration wells. aPL antibody and tetravalent pep The concentration of each monomeric peptide that reduces binding to tide is determined by the colorimeter of each well. It is determined by plotting the readings at. 50% inhibition point is monomer pepti Used as a measure of the relative strength of the bond to the bond.   A variation on this assay is human beta_Two-Glycoprotein I / cardiolipin (β_Two-GPI / C L) using a microtitration plate coated in place of the tetravalent peptide And β_TwoPeptide for Binding of aPL Antibody to Epitope on -GPI / CL Test the inhibitory potency of the drug. This assay uses concentration-optimized IgG-depleted human serum To β_Two-Use as a GPI source. Assay using tetravalent peptide as plate substrate In a similar manner, several concentrations of the monomeric peptide are introduced together with the optimal concentration of the aPL antibody. Incubate. (β_Two-PL / peptide incubation on (-GPI / CL) plates After the application, the peptide concentration required for 50% inhibition of antibody binding was determined using a tetravalent peptide. Determined by the absorbance at half maximum of the value in the assay.   A further variation of this assay is the use of the monomeric peptide Nunc Maxisorp Micro Β coated directly on the wells of a titration plate_TwoOf aPL antibody against -GPI Test the ability to inhibit binding. In this variant, the use of cardiolipin Omitted, and instead of fish gelatin, reagent dilutions and blockers, skim milk Use / Tween. (viii) Fluorescence polarization peptide binding assay   This assay detects the direct binding of the peptide to the aPL antibody. aPL antibody Is β_Two-GPI (antigen), ELISA competitive inhibition assay_Two-To GPI Inhibition due to binding and inhibition due to peptide binding to aPL antibody Can be shown. Binding to the antibody is necessary for the peptide to function as a Toleragen. It is established that peptides can bind directly to aPL antibodies. And are mandatory. Using this assay, two aPL antibodies, ACA-6501 and ACA Determine the dissociation constant for the peptide that binds to -6701. ELISA assay Requires less antibody than light polarization assays, so high throughput screens ELISA-positive peptides, while useful for To determine whether they can bind directly to aPL antibodies Should be. (Ix) Assessing the contribution of amino acids to binding by substitution and deletion synthesis   The desired epitope for tolerance induction can be generated with as many aPL antibodies as possible. It should have as strong an interaction as possible, but should not include unnecessary residues. Create analogs of each peptide to infer minimal epitope composition Do: (i) analogs lacking certain residues (eg, carboxyl terminus and / or Or the backbone residue at the amino terminus is deleted), or (ii) the epitope library Amino acid substitution different from the sequence found in Lee's screen analog. These amino acid substitutions are naturally occurring (eg, for leucine). Soleucine) or unnatural (eg, α-methyl Lorin). The effects of these deletions and / or substitutions are as follows: As determined by peptide competition ELISA. (x) β_TwoOf aPL serum specificity by mutagenesis of the fifth domain of G-GPI   For a tolerogen to be generally effective, the majority of aPL antibodies in most patients It must combine. Several antibodies from different patients have Β which is thought to contain pitope_Two-Identical in the fifth domain of 84 amino acids of GPI It is important to decide whether to bind to a residue. Several antibodies bind to the same residue A single reactive with all antibodies, derived from the peptide structural data Mimotopes can be designed. In contrast, if the antibody binds to different residues, Specific tolerogens are required for each antibody. β_Two-In the 5th domain of GPI To identify whether important residues are involved in the aPL antibody binding residues of Site-directed mutagenesis was performed. Mutant β obtained_Two-Reaction of GPI with several aPL antibodies Assayed for gender. The results were uncertain. β_Two-GPI Fifth Domain and Guru A fusion protein containing Tathione S Transferase (GST) was obtained from A. Steinkassere r and expressed in E. coli (Steinkasserer et al. (1992) FEBS Lett. 313: 193 -197). In the ACA ELISA, this fusion protein is native β_Two-Good with GPI Was replaced. Amino acid substitutions are made using standard site-directed mutagenesis. I. Isolation of synthetic aPL epitope   GPL score of 151 (high titer) with recurrent seizures and fetal deaths, lupus and three major Antibody ACA-6501 obtained from a patient with a history of arterial valve transplantation was used for cardiolipin liposome. Purified by immunoaffinity in zyme, xy, xyz, xy'z, and Based on the specific pro / cys binding 7 with arginine immobilized at position 7. -mer library to screen antibodies from four different phage libraries Used in tanning. As shown in Table 1, phage subjected to microselection In this way, 36 sequences were obtained (from 140 tested). These show considerable homology And the conserved DR residues at positions 6 and 7 are particularly noticeable. Consensus sequence Is CLLLAPDRC. Despite this homology, after the phage ELISA colorimetric test, Only seven phages were positive. (See Table 2). Affinity purified ACA- Strain xy'z phage using 6626 (from a patient with high but lower titer than ACA-6501). Cleaning yielded five independent sequences that were tested in a phage ELISA. Th Neither was a colorimetric positive, but the ELISA immunoconjugate was developed by the chemiluminescent substrate. When tested, two were positive. The sequence motif associated with ACA-6626 is related But different from those recognized by ACA-6501 (see Table 3). Rival The body is cys-bonded (possibly circularized and tethered) over the open sequence before binding. Preferred epitope.               Table 1 ACA-6501 phage library sequenceACA-6501 phage library sequence                                  Table 2                       ACA-6501 colorimetric ELISA positive phage * Corresponds to consensus or average sequence.                                   Table 3                   ACA-6626xy'z phage library sequence   ACA-6644 was prepared using the pooled p-III plasmid following the method set forth herein. High titer aPL antibodies used to screen large libraries It is. The following sequence was found:                     ACA-6644 / CBe GILLNEFA                     ACA-6644 / CBd GILTIDNL                     ACA-6644 / CBf GILALDYV All of these sequences are "z" epitopes that delete phage backbone residues at the N-terminus Derived from library components. When synthesized as a peptide, the sequence It showed immunoreactivity with several ACA sera including A-6644 and ACA-6501. Analysis results , As shown in Table 4, undoubted homology with sequences already obtained using ACA-6501 Revealed.                                   Table 4   Two very different sources screened by these two aPL antibodies The homology of the aggregated sequences from the libraries of Suggests that it may mimic a major, possibly immunodominant, region of the antigen I do.   Screening of the p-III library with ACA-6701 revealed a high degree of internal Two homologous but different from those previously obtained with other aPL antibodies A unique sequence was obtained. The sequence is as shown below.             ACA-6701 / 3B1LSDPGYVRNIFH             ACA-6701 / 3E1LTDPRYTRDISNFTD   As a resin-bound peptide, the sequence showed strong immunoreactivity with its parental serum (ACA-6701). But was minimally cross-reactive with other aPL antibodies.   Of any p-III phage library using affinity-purified ACA antibody As a result of continuing screening, the nine affinity-purified Peptides showing significant cross-reactivity to all of the ACA antibodies obtained were found. this The capabilities of the peptide and the other four peptides are shown in Table 5. All 5 peptides Is just below the table. All are CL as described herein. / β_Two-Tested using a competitive binding peptide ACA ELISA using GPI plates.                 Table 5Percentage inhibition of ACA by peptide monomers   From subsequent tests, this peptide AC with the sequence of AGP-CLGVLGKLC-PG (LJP688) A-6641 / 3G3 was determined to be cross-reactive with many ACA antisera. this Chemical optimization of peptides includes shortening, systematic amino acid substitutions and non-disulfation. This was done by experiments on the cyclization of the amide.   Several peptides selected by screening arbitrary phage libraries The motif is shown below.                                   Table 6 J.Immunoreactivity of affinity-purified IgG aPL antibodies with aPL-related peptides   Obtained using affinity purified ACA-6501 and phage ELISA Phage sequences that have been identified as Synthesized on a recently developed solid support, this support of Rapp resin A hydrophilic polyethylene glycol that has a peptide density and is coupled before the coupling of the first amino acid Use recall spacers. Ideal for antibody binding experiments as a result of synthesis A resin-bound peptide was obtained. As shown in FIG. 2, peptide 5A12 (CLILAPDRC Sequence) outperformed the unrelated control peptide, but Did not bind significantly. Similar results were obtained with the other phage ELISA Was obtained using a sex peptide. As shown in the experiments, Affinity-purified ACA-6501 aPL antibody was detected by immunoconjugate color reaction . K.aPL Reactivity of serum antibodies with synthetic peptides.   Using serum, it was shown that resin-bound peptides can immunospecifically bind to aPL. As a result, the ability to test aPL antibodies has greatly improved. As shown in FIG. 3, the peptide , Obtained from screening with aPL antiserum conjugated to ACA-6626, but No clear bond with Qing was observed. FIG. 3 also shows ACA6501-5 against aPL serum. Figure 4 shows the immunospecific binding behavior of the A12 peptide. Normal blood for peptide 5A12 No binding to Qing, no data shown. L.Synthesis against aPL antisera not a source of library screening antibodies Cross-reactivity of peptides   Two peptides were selected for the aPL serum test. One (5A12) is ACA-6501 Represents leaning and the other (4D3) represents ACA6626 screening. 4 and 5 As shown in the figure, each peptide is preferentially compared to the original serum of the screening antibody. Reacted. However, a significant degree of cross-reactivity was noted, especially for ACA-6501 and ACA-66. 26 and was detectable. 19 patient sera with low or no GPL score A survey of 13 pathological sera with moderate to high GPL was performed using 5A12 resin-bound peptide. This was performed using Only 2 of 19 control samples have low pepti Of 13 detectable peptides in 13 pathological samples, whereas Demonstrated binding. These results indicate that the synthetic peptide is not diagnostic in treating seizure patients. Or useful for prognostic assays.   As mentioned in Section I above, synthetic peptide 6641 / 3G3 has several high titers of A. Tested against CA antiserum. This peptide was tested as shown in FIG. It was found to cross-react with the majority of ACA antisera. 6641 / 3G3 has 10 ACA antibodies All showed dose-dependent cross-reactivity, and completely inhibited (> 80%) at 1.8 mg / ml. And these were specific for the ACA antibody, as shown in Table 7 below. .                     Table 7 Use cardiolipin / β2-GPI coated plate           AGP-CLGVLGKLC-PG (LJP688)           Cross-reactivity data for ACA serumM.New peptide synthesis methodology   New candidate sequences can be identified by aPL library screening. The resulting peptides needed to be tested for antibody binding. Known pep Quantitative binding studies, such as saturation binding assays, using Rapp resin instead of tide And an equilibrium measurement using radiolabeled aPL IgG.   The synthesis of peptides allows for the molecular resolution of mimotopes by selective synthesis. This involves modification of each amino acid along the chain to enhance antibody binding. Selective synthesis Reveals the relative importance of each amino acid in the sequence. If necessary If present, the selective substitution at a particular residue position will To maintain the reactivity of B cells while eliminating the proliferation reaction of cells Can be designed.   Peptide 6641 / 3G3 (LJP688) was used in many assays. In that assay Included: truncated, disulfide at both the N- and C-termini Substitutions, substitutions of amino acids at positions 2 to 8 with glycine and alanine, 2, 4, 5, and Substituted for branched aliphatic amino acids at positions 8 and 8, substituted for α-methyl amino acids Substitutions to amino acids that affect the conformation of the peptide, including substitutions, basicity at position 7 Substitutions for amino acids, substitutions for D-amino acids at positions 2 to 9, and 1 to 9 Substitute at position with N-α-methyl amino acid. The results are shown in Table 9 below. This Table 8 shows the relationship between the structure and activity of these substitutions and truncations.       Table 8Optimization of peptide 3G3                                   Table 9                   Structure-activity relationships of cross-reactive ACA epitopes N.Increases peptide immunoreactivity and increases resistance to protease attack Use of α-methyl amino acid substitutions and unnatural amino acids to confer   Proline residues have a special meaning because they affect the conformation of the polypeptide chain. I do. They are often present in reverse turns on the surface of globular proteins. Book In the phage epitope library of the invention, all the random peptide The insert is adjacent to the border proline. In addition, minos discovered using ACA-6501 Most of the tops are based on computer-based predictions, It has a third proline that can be present as part. The imitation of the β-turn is It can be used to increase the stability of the inverted-turn conformation in peptides. That Similar mimics include the proline analog (S) -α-methylproline (α-MePro). , Which, in addition to stabilizing the turn conformation, is resistant to protease degradation. Is given. Protease resistance is a promising design designed to work in plasma This is a desirable property for drugs. Peptide ACA-6501 / 3B10 AGPCLLLAPDRCPG (insert G Is shown in bold) is the consensus peptide. It has 35 other homologous Sequences characterized by the most widely occurring residue at each position, based on comparison with the sequence Having. Because of its typical characteristics, the sequence has many systematic modifications and deletions. Loss was made and activity was later assessed by aPL antibody binding. the most important One of the findings is the finding that proline at positions 3 and 9 is important for activity. Step Lorin-3 is derived from the phage backbone and is part of the random insert. Absent. The most dramatic effect was obtained by replacing the proline at position 9 with α-MePro. Was done. With this substitution, immunoreactivity was increased 6-fold.   Studies with substitution of α-methyl amino acids and N-α-methyl amino acids showed that peptide 66 This was done for 41 / 3G3 and the results are shown in Table 8 above.   In addition to the 20 naturally occurring amino acids and their homo and non-analogs Thus, several other classes of alpha amino acids can be utilized in the present invention. this Examples of other classes of these include d-amino acids, N^a-Alkylamino acids, α-alkylamino Acid, cyclic amino acids, chirrieric amino acids, and miscellane ous) amino acids. These unnatural amino acids are Modify the peptide to increase its resistance to proteolysis and / or Widely used to provide conformational constraints to improve biological activity (H ruby et al., (1990) Biochem. J 268: 249-262; Hruby and Bonner (1995) Methods in M olecular Biology 35: 201-240).   The most common N^a-Alkyl amino acids are N^a-Methyl amino acids (eg, N^a-Met Lucysteine (nC), N^a-Methylglycine (nG), N^a-Mesylleucine (nL), N^a-Met Luridine (nK), and N^a-Methylvaline (nV)). Examples of α-alkyl amino acids Include α-methylalanine (niA), α-aminoisobutyric acid (aiB), α-methylpro Phosphorus (mP), α-methylleucine (mL), α-methylvaline (mV), α-methyl α-amino Butyric acid (tv), dimethylglycine (deG), diphenylglycine (dpG), and Clohexylglycine (dcG) (Balararn (1992) Pure & Appl. Chem. 64: 1061-1066; Toniolo et al. (1993) Biopolymers 33: 1061-1072: Hinds et al. (1991) Med Che m. 34: 1777-1789).   Examples of cyclic amino acids include 1-amino-1-cyclopropanecarboxylic acid (cG), 1-amino No-1-cyclopentanecarboxylic acid (Ac5c), 1-amino-1-cyclohexanecarboxylic acid Acid (Ac6c), aminoindane (aminoin.dane) carboxylic acid (ind), tetrahydroiso Quinoline carboxylic acid (Tic), and pipecolic acid (Pip) (C. Toniolo (1990) Int'l. J. Peptide Protein Res. 35: 287-300; Burgess et al. Am. C hem. Soc. 117: 3808-3819). Examples of chimeric amino acids include penicillamine (Pe), cis Combination of thein, valine, 4R- and 4S-mercaptoproline (Mpt), homocysteine Combination of proline, proline, 4R- and 4S-hydroxyproline (hyP) Combinations of phosphorus and proline are mentioned. Examples of miscellaneous α-amino acids include basic amino acids. Amino acid analogs (e.g., ornithine (Or), N^ε-Methyl lysine (mK), 4-pyridylalanine (pyA), 4-piperidinoalanine (piA), and 4-aminophenyl Rualanine); acidic amino acid analogs (eg, citrulline (cit), An aromatic amino acid analog (eg, 1-naphthylalanine (1-N al), 2-naphthylalanine (2-Nal), phenylglycine (pG), 3,3-diphenylala Nin (dpA), 3- (2-thienyl) alanine (TE), and halophenylalanine (e.g., 2-fluorophenylalanine and 4-chlorophenylalanine); Amino acid analogs (eg, t-butylglycine (ie, tertiary leucine (tL)), 2-aminobutyric acid (Abu), cyclohexylalanine (Cy), 4-tetrahydropyrani Lualanine (tpA), 3,3-dicyclohexylalanine (dcA), and 3,4-dihydropropane Lorin).   In addition to alpha amino acids, others such as beta amino acids may also be used in the present invention. obtain. Examples of these other amino acids include 2-aminobenzoic acid (Abz), β-aminopro Panic acid (β-Apr), γ-aminobutyric acid (γ-Abu), and 6-aminohexanoic acid (ε-Ahx) Is mentioned. Carboxylic acids (eg, 4-chlorobutyric acid (By) and 3-chloropropio Acid (Pp) is also the first residue on the N-terminus in the synthesis of cyclic thioether peptides. Has been used as O.Preparation of cyclic thioether analogs   The mimetic peptide identified by the method of the invention contains a thioether substitution It can be further modified. Cyclic disulfide analog to cyclic thioether analog Modification increases the plasma half-life of the analog conjugate, and therefore lower doses Need quantity. Cyclic thioether analogs also include cyclic disulfide peptides Together with the problem of disulfide bond exchange that often occurs. in addition, Cyclic thioether analogs may also interfere with MHC class II presentation to T cells. Therefore, induction of anergy is promoted. Finally, the cyclic thioether analog , Thiol-dependent binding used in the formation of valence platform molecular conjugates Useful for combined reactions.   The four cyclic thioether analogs are referred to as alternatives, which are hereby incorporated by reference in their entirety. A person listed in a co-pending patent application related to the sharing of reference number 252312006600 Prepared by methodology. Rink^TMAmido 4-methylbenzohydrylamino resin or Prepares a full-length peptide analog of 6641 / 3G3 using either MBHA resin, Converted to chloropeptide, cleaved from solid support and then cyclized. Below 1 shows a scheme of an ether reaction. Suitable cyclic thioether analogs are shown below. Including analogs. Cyclic thioether reaction scheme IAlternatively, thioether analogs are prepared according to the following reaction scheme.                     Cyclic thioether reaction scheme 2  The following analogs are analogs made by the cyclic thioether reaction scheme 2. It is a typical example of the blog.  Representative cyclic thioether analogs were raised against the ACA6501 and ACA6701 ACA antibodies. Was tested for activity. The results are shown in Table 10 below.                                 Table 10                           I c₅₀Value (μg / 0.1mL)   LJP699 of HCTE analog, LJP of reference peptide of CLGVLGKLC, which is truncated LJP688 690, better than AGPCLGVLGKLCPG.   All articles, references, patents and patent applications cited herein are hereby incorporated by reference. In its entirety is incorporated by reference. The following examples illustrate the invention and its originality. It is intended to further demonstrate gender. These examples in no way limit the scope of the invention I am not doing it.                                   Example Example 1Measurement of anti-cardiolipin antibody (ACA) in serum 6501   Microtitration plate Immulon I (Dynatech Laboratories, Inc. , Chantilly, VA) with 30 μL ethanol per well In 50 μg of cardiolipin (Sigma Chemical, St. Louis, MO). The plate was dried at 4 ° C. overnight and 10% mature bovine serum (ABS) (Irvine Scientific Co., Santa Ana, CA) with 200 μL of phosphate buffered saline (ABS / PBS) at 2:00 Block at room temperature (RT). Plates are washed with Tris-buffered saline (TBS) for 5 minutes. Washed twice, after which 10 μL of test serum ACA-6501 and aPL standard were added. a PL reference material (APL Diagnostics, Inc., Louisville, Ky.) And diluted 1:50 with 10% ABS-PBS. Test serum ACA-6501, 1:50 From 1: 2000 serially diluted with 10% ABS-PBS and added to selected duplicate wells, 2 hours At room temperature. Wash plate 5 times with TBS, 1000% with 10% ABS-PBS Goat anti-human IgG / alkaline phosphatase conjugate (Zymed, South San  Francisco, CA, Cat. No. 628422), and incubate at room temperature for 1 hour. I did it. Again, the plate was washed 5 times with TBS and the assay was Add phthalein monophosphate (PPMP) substrate solution (Sigma, Cat. No. P-5758) It was done by doing. The substrate solution is diluted 1:26 with deionized water and then with hydrochloric acid. By adjusting to pH 10.15, 0.13M PPMP and 7.8M 2-amino-2-methyl-1-propyl Prepared from a stock solution of lopanol. After about 30 minutes, the reaction was allowed to proceed with 0.2 M dibasic phosphate Thorium (dibasic sodium phosphate) (Mallinckrodt, Analytical Reagent) 50 μL was added to each well to stop. The optical density is automatically read by the microplate. Taking Read at 550 nm on a oscilloscope (Bio-Tek Instruments, Winooski, VT, Model EL311) Was. Odd numbered control wells (blank, no cardiolipin (CL)) Optical density was subtracted from the optical density of even numbered wells. Absorption of aPL standard Using the Graph Pad Prizm (Graph Pad Software, Inc., San Diego, CA) Plotting was performed to create a standard curve of GPL (IgG phospholipid). Of diluted 6501 test serum The GPL score was calculated based on the GPL standard curve using the absorbance values.   In a modified ACA ELISA, load the Nunc Maxisorp microtitration plate After incubation for 1 hour at room temperature, 100 μL / mL β_Two-With GPI Coated. After this step, the microplate was washed with 2% skim milk and 0.4% (w / v) Twee. Blocked with PBS containing n80 for 2 hours at room temperature. Add skim milk / Tween to reagent diluent and And used as a blocking agent_Two-Before adding GPI As previously described for Dynatech microplates, first coated with β_TwoNunc microplates directly coated with -GPI were used for ACA IgG binding studies. Was. Example 2:Purified anti-cardiolipin antibody (ACA) from serum 6501   In a 25 mL round bottom flask (Kontes Scientific Co., Vineland, NJ), add 1.2 ml of Diolipine (Sigma Chemical, St. Louis, MO, # C-1649), 0.464 mL cholesterol (Sigma Diag., St. Louis, MO., # 965-25), 5 mg dicetyl pho 0.088 mL of a mixture of sulfate (Sigma Chemical, St. Louis, MO, D-2631) was added to Rotava Dry for about 5 minutes in p (Buchi, Switzerland). After removing the solvent, 0.96% (w / v) NaC Add 2 mL of l (J.T.Baker, Inc., Phillipsburg, NJ) Baker analyzed reagent) Using Vortex Genie Mixer (Scientific Industries, Inc., Bohemia, NY) And mixed for 1 minute. The liposome suspension was incubated at 37 degrees for 1 hour. So Of serum 6501 at 8 ° C. for 10 minutes in a Sorvall RT 6000 centrifuge (Dupont Co. Wilm. (ington, DE) at 600 × g. Add 4 mL of the supernatant to the prepared liposome suspension. The suspension was transferred to a 25 mL round bottom flask containing 1 mL. The mixture is swirled by Tekta tor (Scientific Products, McGraw Park, IL) while shaking at medium speed. Incubation was performed at 4 ° C. for 48 hours. Then, incubate for 2 hours at 37 ° C. Cubbed. Add 20 mL of cold TBS and mix the mixture with 50 mL of polycarbonate Transferred to a centrifuge tube (Nalge Co., Rochester, NY). And SS-34 rotor (Sorvall-D upont, Washington, DE) in a RC3 centrifuge for 15 minutes at 4 ° C at 27,000 xg And centrifuged. The precipitate is washed three times with 25 mL of cold 0.96% NaCl using an RC3 centrifuge. Washed. Pellets are pelleted with 2% (w / v) n-octyl-β-D-glycopyranoside (Calbioche m, Lajolla, CA) in 1 mL of a TBS solution and add 0.6 mL of protein A / cross-linking Galose (Repligen Corporation, Cambridge, Mass.) Column. The column is The plate was previously washed with 1 M acetic acid at 15 times the bed amount, and equilibrated with 15 times the amount of TBS. Antibody-Pro Remove the tein A / agarose column with 40 times the bed volume of 2% octane to remove lipids. Washed with tyl glucopyranoside. After that, the optical density at 280 nm becomes the baseline Washed thoroughly with TBS until reaching. Bound antibody was eluted with 1M acetic acid. 1mL Of each fraction, and 0.34 mL of 3M Tris (Bio-Rad, And then stored in an ice bath. The optical density of each Photometer (Hewlett-Packard, 8452A Diode ArraySpectrophotometer, Palo Alto, C A) was determined at 280 nm. Collect fractions containing antibodies and concentrate on Centricon-30 (Amicon Division, W.R. Grace & Co., Beverly, MA) Concentrated and washed 4 times with TBS according to protocol. Purified from 4mL serum 6501 The final yield of the antibody₂₈₀Aliquot of the concentrate at 280 nm It was determined from the value of the optical density. The average yield obtained is from antibody 750μ from 4mL serum 6501. g. The purified antibodies are tested for ACA activity and run on Laemmli SDS-PAGE. The purity was checked. CNBr-activated agarose (Pharmacia, Inc., P.C.) according to the manufacturer's instructions. iscataway, N) or Affi-Gel 10 (BioRad, Richmond, CA) using commercially available Made β_TwoΒ using GPI (Perlmmune, Rockville, MD)_Two-Affinity containing GPI The adsorbent was prepared. β_Two1mL TBS to 1mL gel column containing -GPI adsorbent Up to 100 μg of liposomal purified aPL IgG was added. After 1 hour, buffer with buffer. Thoroughly wash the ram and remove contaminants and β_Two-Replace any IgG that did not bind to GPI Was. β_Two-Replace GPI-bound IgG with fraction neutralized with IM HOAc and Tris as described above. Was. Fractions containing IgG were pooled, concentrated and buffered as previously described. The solution was exchanged. Example 3P-III Construction of library vector preparations   fUSE 5 (Scott, J.K. and G. Smith, supra) for the construction of the p-III library And Holmes, D.S. and M. Quigly (1981), Anal. Bioch em. 144: 193) was used to generate a double-stranded replicate form (RF). Easy theory To elucidate, 800 ml of E. coli K802 culture holding fUSE 5 was added to 2YT medium (Difco Lab s, Ann Arbor, MI), add 20 μg / mL tetracycline and shake vigorously at 37 ° C for 18 hours. The culture was performed with shaking. The cells were collected by centrifugation and resuspended in 75 ml STET. S TET is 50 mM Tris / HCl pH 8.0, 8% sucrose, 0.5% Triton X-100 and 50 mM EDTA. And Add lysozyme (10 mg / mL in STET) to a final concentration of 1 mg / mL did. After 5 minutes at room temperature, aliquot aliquots into boiling water with occasional stirring. For 3.5 minutes. The viscous slurry is centrifuged at l8000xG for 30 minutes. Was added with an equal volume of isopropanol. Cool solution to -20 ° C and centrifuge nucleic acids Collected by. RF, Sambrook et al., MOLECULAR CL0NINIG: A LAB0RAT0RY MANUA L (Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 2ded., 1989 It was isolated from a cesium chloride gradient as described in (2).Preparation of random insert   The DNA for insertion was prepared according to Cwirla et al. (1990) Proc. Natl. Acad. Scl. USA 87: 6378-6382 Produced by the "gapped duplex" method described. using this method Indicates that oligonucleotides containing a degenerate region in the center have short constant Surrounded by an area. Vector the two shorter complementary oligonucleotides Produced by the restriction endonuclease used to digest the protein Anilin to form a "gapped duplex" with complementary overhanging ends To In this case, the longer degenerate oligonucleotide has the following sequence: 5'GG GCTGGACCC (NNK) xCCGGGGGCTGCTG3 ', where N is A or C or G or T , K is G or T, and x is the number of codons in the random region. Ep iGS2 is designed to base pair at the 5 'end of a degenerate oligonucleotide, and Is 5'GGGTCCAGCCCCGT3 '. Similarly, EpiGS3 is a degenerate oligonucleotide H It is designed to anneal to the 3 'end of the peptide and has the sequence 5' CAGCCCCCGG3 '. When correctly annealed, the three oligonucleotides are "gap duplexes" To form When inserted into fUSE 5 digested with Sfi1, these were near the 5 'end. Retains p-III reading frame with random insert fragment.   The oligonucleotides described above were excised from polyacrylamide gel Prepared. 1 nmol of EpiGS2 and EpiGS3 and 50 pmol of degenerate oligonucleotide Phosphorylation was performed separately in a volume of 6 μL. Then pool the three oligonucleotides NaCl to 50 mM and heat the mixture at 65 ° C. for 5 minutes, then to room temperature Cooled slowly. Cool the annealed oligonucleotide in ice and It was later used for ligation reaction with fUSE5. The ligation reaction was performed with 10 μg of complete digestion with Sfi1. Consists of fUSE5 DNA, 30 μL of “gap duplex” solution, and 1000 U T4 ligase The total volume was 450 μL. The ligation was incubated at 16 ° C. for 18 hours. Mixed The compound was then extracted with phenol / chloroform, precipitated with ethanol, and precipitated. The material was dissolved in 20 μL of water.Library formation and amplification   The ligated DNA was subjected to electroporation (Dower et al., (1988) Nucleic Acid R es. 16: 6127-6145). Frozen electrocompetent MC1 061 cells (0.1 mL), 4 μL of the ligated DNA were mixed in a 2 mm cold cuvette, and BTX 2.5 kV, 5.2 mS pulse by troporation apparatus (BTX Corp., San Diego, CA) Was added. Immediately after applying the pulse, 1 mL S0C of the cell growth medium (Dower et al., Supra) Was added. Perform 5 separate electroporations, pool and 1 at 37 ° C. Incubated for hours. At that time, remove the sample and generate colonies Dilutions were made to determine the total number of dishes. Remove the remaining mixed material to 20 µg / mL tetracycline. 2YT (1.6% peptone, 140, 1% yeast extract, 0.5% NaCl, Difco Labs, A nn Arbor, MI) and cultured at 37 ° C. for 18 hours with shaking at 275 rpm. Phage , Purified by performing two PEG / NaCl precipitations, containing 1% containing 0.02% sodium azide. Resuspended in .2 mL TBS. Phage particle number was determined by absorbance at 269 nm. fur Mix the titer with 10 μl of the phage dilution in 10 μl of starved E. coli. And to Decided. Example 4:aPL Screening p-III libraries with antibodies   Affinity-purified ACA-6501 (affACAA-6501, 10 μg; 7 μL of 1.44 mg / mL Stock solution) with 1.4 mL of silicone microfiber Transfer to X-tubes and add X, Y, and Z libraries (epi^xyz) [11μL + 8.5μ each L + 2 μL; total volume 21.5 μL, about 10^TenCloned] derived pooled phage To the final volume of 100 μL of TBS (pH 7.4) containing 0.5% bovine serum albumin (BSA). Incubated for 2 hours at warm. During this incubation, fresh starvation The final stage of the preparation of the native E. coli K-91 strain was performed. In 2YT medium, 37 ° C, 250 rpm E. cultured with shaking for 5 hours. coli suspension using a 50 ml polypropylene tube. Centrifuged at 000 × g for 10 minutes at room temperature. E. Packed with 20 ml of 80 mM Nacl. coli And incubated at 37 ° C for 45 minutes at 100 rpm. Following the centrifugation above Hunger. Suspend the E. coli pellet in 1 mL of 50 mM ammonium phosphate / 80 mM Nacl. It became cloudy and was later used for phage amplification. Protein G-agarose beads with TBS / BSA Wash twice, wash twice with TBS / 0.5% Tween-20, as a 50% suspension in TBS / Tween Stored at 4 ° C.   Then, 200 μl of the protein G-agarose bead suspension was added to ACA-6501 / Fur The mixture was added and incubated for another hour at room temperature. At this point, the mixture Cool, wash three times with cold TBS / Tween, and spin the precipitate by microcentrifugation in a cold room. Received. Wash the washed beads in advance with TBS / BSA and And transferred to a new microcentrifuge tube washed with TBS / Tween. 3 more TBS / Tween After washing with, the beads having the phage retaining bound ACA-6501 were Elution by inverting 1 l of 0.2N HCl / glycine, pH 2.1 for 10 minutes at room temperature Was. After centrifugation at 16000 xg, collect the supernatant of the acidic eluate, and Was added to the bead pellet and the procedure was repeated. After 10 minutes, lysis containing phage The effluent (pointing to the first phage that has not been amplified) is pooled (〜400 μl) and 17 × Transfer to a 100 mm sterile polystyrene cell culture tube and add 50 μl of 0.5 M Na Cl was added, followed by neutralization of the pH with 2.5 M Tris base (usually about 25-35 μl). Equal amount Starved E. Add the E. coli suspension immediately and incubate at 37 ° C and 100 rpm for 10 minutes. Was. The mixture contains 25 ml of 2YT medium containing 20 μg / ml tetracycline (Tet) Transfer to a sterile 250 ml culture flask and incubate at 37 ° C overnight at 250 rpm. Was.   To isolate amplified phage from overnight cultures, suspend the suspension Then, the mixture was centrifuged at 12,000 × g for 10 minutes. The pellet was discarded. Polypro After heat treating the supernatant in a pyrene tube at 70 ° C for 30 minutes, the material is again The mixture was centrifuged in a heat tube and the supernatant was saved. To precipitate the phage, 1/4 volume of 20% (w / v) polyethylene glycol (molecular weight 8000 (PEG 8000)) ) Was added. Mix the solution by inverting 100 times and incubate at 4 ° C for 2 hours. I was vate. After centrifugation at 35,000 xg for 30 minutes at 4 ° C, the pellet containing phage The pellet was resuspended in approximately 0.5 ml of TBS / BSA and transferred to a 1.4 ml microcentrifuge tube. 1 After centrifugation at 6000 xg for 1 minute in a microfuge, the supernatant is transferred to a clean tube. And labeled with phage amplified once.   Between 2, 3, and 4 bioselections, 75 μl of the amplified phage from the previous round Was incubated with 7 μl of affACA-6501 in a final volume of 100 μl. 5 For phage rounds, affACA-6501 was first diluted 1: 1000 and described in other rounds Treated as follows. All subsequent steps were performed as described in the first round. 5 The phage obtained in the second round of biological selection was used to determine the phage concentration in 2YT / Tet Titration was performed temporarily on the plate. Temporary titration of amplified phage Contains 1x10 in TBS / BSA or 2YT medium⁶Requires initial phage dilution. For all rounds, 10 μl of the diluted phage was fed to starved E. coli. 40μl of coil Both were incubated at 37 ° C. for 10 minutes without shaking. 950 μl of 2YT / After addition of diluted Tet (0.2 μg / ml), the mixture was shaken at 37 ° C. and 250 rpm for 30-45 minutes. Incubated. Phage solutions diluted 1:10, 1: 100, and 1: 1000 Agar plate containing 20 μg / ml tetracycline The spots were spotted on 2YT / diluted Tet usingMicro selection   Immulon type 2 plates were coated with protein G. 0.1M protein G NaHCO_ThreeMedium, 10 μg / ml, and add 100 μl per well to microtiter And incubated overnight at 4 ° C. Excess from plate After removing the complete Protein G solution, each well was treated with 250-300 μl of 2YT at room temperature. For 1 hour to block on a shaking table. Tris buffered saline (pH 7.4 / 0.5% Tween 20 (TBS / Tween)) with an automatic plate washer at 200 μl. The wells were washed four times. AffACA-6501 diluted to 2.5 μg / ml with 2YT (or 100 μl of standard IgG as a control) was added to the washing wells. Plate at room temperature After incubation for 1 hour on a turntable, the sample was transferred to a rotating device in a cold room.   The phage to be tested by microselection are transferred to the agar plate generated by microselection. Obtained from rate. Each clone to be tested is cloned using a sterile toothpick. Round bottom 96-well microtitration plate containing 250 μl per well of 2YT / Tet Rate (Corning, Corning, NY) into another well and incubated overnight at 37 ° C. K The name of the loan is the screening antibody, the number of selections of the organism of origin, and the Based on rate clone position. For example, ACA-6501 / 3B10 uses ACA-6501 At the third run, is the well named B10 of the microtitration plate? Clones isolated from the same. After overnight incubation, remove the phage culture 1300 xg for 10 minutes at room temperature using an icrotitration plate holder Heart isolated. The supernatant is the "net" phage source.   First micro selection, 1:10 to 1:10⁶6 clones tested at a dilution of Limited to The results of these experimental clones were To derive an appropriate single dilution that gives sufficient results to the source plate clone It was used for. Randomly select from 3rd, 4th, and 5th biological selection and culture Plates representing clones isolated are placed in microtiter plates in a final volume of 250 μl Dilute 1: 100,000 from the “net” solution using 2YT in the titration plate. I shouted. 100 μl from the final plate at the desired dilution was prepared as described above. Was added to a plate containing the isolated protein G-conjugated ACA-6501 and normal IgG. Diluted Phage with aPL antibody or control IgG at 4 ° C for 2 hours on a horizontal rotator. Incubated. After 9 washes with TBS / Tween in an automatic plate washer The IgG-bound phages were eluted with 20 μl of 0.2N HCl-glycine / 0.1% BSA, pH 2.2. Was. The incubation of the eluate was continued for 10 minutes at room temperature, during which time the freshly starved E. coli c a new Corning microtitration plate containing 20 μl of oli Prepared and cooled. Phage elute 140 μl of 29 mM Tris to neutralize pH Solution and then add 20 μl phage suspension from each well to Starved E. Transferred to corresponding wells of plate containing E. coli. 10 minutes at 37 ° C After the incubation, add 200μ of 2YT / diluted Tet and incubate at 37 ° C for 30 minutes. I did it. Using a multichannel dispenser, 10 μl from each well Spotted on a large 2YT / Tet agar plate. However, the last micro title 8x12 well pattern and placement from the solution plate Was. After drying the spot for 30 minutes, the plate was incubated overnight at 37 ° C . The next day, colonies were evaluated semi-quantitatively from 0 to 4+, with 0 representing <10 colonies. +/- is 10-20; 1+ is 20-50; 2+ is 50-70% confluent; 3+ is 70-90% confluent. Luent; 4+ represents> 90% confluent colonies. 1:10^FiveTested at a dilution of 9 Of the four clones [81 clones from the third round, 6 clones from the fourth round, 7 clones from the fifth round] 6 clones have a microselection score of 0, 3 are 1+, 14 are 2+, 62 Was 3+ and 9 were 4+. Nothing from the plate representing the second to fifth biological selection Surveys of the clones made were performed by G-track DNA sequencing described below. 4 times Bioselection of the eyes resulted in a dramatic decrease in sequence diversity (Figure 8). Therefore, the second plate is 1: 3 × 10^FiveUsing phage at a dilution of Then, micro selection was performed. At this time, clones from earlier (second time) including. A total of 94 clones were tested, which was 1 × 10^FiveHigh score at dilution of 29 [third to 26, fourth to one, and fifth to two], and 2 Includes 65 clones from the round (not previously tested). 1: 3 × 10^Fiveof Of the 94 clones tested at dilution, 26 score 0,11 +/- and 10 Were 1+, 13 were 2+, 34 were 3+, and 4+ were none.G- Track DNA sequencing   Single-stranded viral DNA was isolated from cultures incubated overnight at 37 ° C. Culture To prepare nutrients, use 2YT / Tet (2 ml in tubes or microtiter Microtiter wells in a round bottom well of the plate Individual cultures from a spread plate of a culture previously cultured on an isolation plate Inoculated. Purification of phage by 20% PEG / 2.5M NaCl precipitation of culture supernatant, Of virion DNA by phenol-chloroform extraction or alkali denaturation The isolation or release procedure is described in Smith, G. et al. P. and J.K. Scott, "Peptide and protein libraries in filamentous phage "(1993) Meth. Enzymol. 217: 228-257, and microtitration plates. For phage in a plate, see Haas, S.J. and G.P. Smith G. P., "Fibrous Rapid Sequencing of Viral DNA "(1993) BioTechniques 15: 422-431 Followed the description. Sanger et al. (Sanger et al. "DNA sequencing with chain termination inhibitors" (1970) Proc. Natl. Acad. Sci. 74: 5463-5467). The sequencing technique was performed using a commercially available Sequenase kit (U.S. Biochemical / Amersham, Arlington).  Heights, IL). For phage DNA in tube culture, use Smith and And Scott (supra), and phage D from microtitration plates NA was performed as described by Haas and Smith (supra). ddCTP stop mixture only Electrophoresis on 7% polyacrylamide / urea sequencing gel, using autoradio G tracking indicated by a single base (G) by using chromatography Alternatively, an array pattern was obtained. Standard gel electrophoresis and autoradiograph (Sambrook et al., Supra).   1: 1 × 10 from ACA-6501 library screen^FiveTest with phage dilution G-tracking of 76 clones from the micro selection plate Eleven different sequences were identified, but 1: 3 × 10^FiveSecond time tested with phage dilution 64 clones from the microselection plate identified 30 different sequences Was done. Conventional DNA sequencing using all four dideoxynucleotide triphosphates Applied to phage clones with the highest microselection score and unique sequence. And 12 sequences shown in Table 1 for the xyz library.Phage capture ELISA   Clones, ACA-6635 / 3A12, 3B3, 3C8, 3A5, 3C9, and 3B7, in 3 ml culture And cultured. Affinity-purified ACA-6635 is converted to phosphate-buffered physiological diet. Dilute to 2.5 μg / ml with saline, pH 7.2, and add 100 μl Immulon-2 microtiterase Plate wells. After 2 hours, play the plate automatically without shaking And washed three times with TBS / Tween using a washing machine. The plate was then applied to the wells. The cells were blocked with 150 μl of 0.1% BSA (without globulin) in PBS. At 4 ° C After one hour, the plates were washed three times as described above. Add each phage culture to 17 After centrifugation at 000 xg for 3 minutes, each supernatant was diluted 1:10 with 0.1% BSA / PBS And 100 μl was added to each well coated with affinity-purified ACA-6635 . Then, it was incubated at 4 ° C. for 2 hours. Then, plate as described above Washed with TBS / Tween. Horseradish peroxidase-conjugated sheep IgG anti-M13 fur Diantibody (Pharmacia, Inc., Piscataway, NJ) was diluted 1: 5000 with 0.1% BSA / PBS. , 100 μl was added to each well. Incubate at 4 ° C for 1 hour After that, the plate was washed four times as described above. Adjust according to manufacturer's instructions 100 μl of the prepared substrate was added to each well. After 19 minutes, 4 of each well Automatic microplate absorbance reader (Biotek, Winooski) , VT). Tested from ACA-6635 phage library screen 7 clones with high microselection score and negative phage ELISA score (See below). As shown in FIG. 9, the seven clones tested Three of them (3A12, 3B3, and 3A5) were very immunospecific in phage capture ELISA Signal.Phage ELISA   35 clones of 3 ml culture (some phage library screens) Previously isolated with ACA-6501 affinity affinity purified) and one FUSE2 phage clone (control lacking peptide insert) Was used). After centrifugation at 17000 xg for 3 minutes, Buffer (approximately 2 × 10¹¹(Adjusted to particles / ml) Iteration plates (Falcon, Becton-Dickinson Labware, Lincoln Park, N Y) and incubated overnight at 4 ° C. Washed 4 times with TBS 7.4 Thereafter, the plate was blocked with 125 μl of 0.5% BSA / TBS for 1 hour at room temperature. 4 more After washing once with TBS, 100 μl of affACA-65 previously diluted to 2.5 μg / ml with TBS / BSA 01 was added to each well and incubated at 37 ° C. for 1 hour. Four more After washing, 100 μl of anti-human IgG diluted 1: 1000 in TBS / 0.5% Tween was added. Was added to each well. After 1 hour at room temperature, add enzyme substrate and incubate for 2 hours Continued. 50 μl of 0.2 M Na to stop the reaction_TwoHPO_FourAfter adding The absorbance at 550 nm was measured with a rate absorbance reader. As shown in FIG. 10, A Using CA-6501, 7 clones showed significant signal by phage ELISA Was done. : 5A12, 3B6, 3E7, 3B10 (same sequence as 2D7), 2G11, 2H1, 2H2. These The arrangement of the loans is shown in Table 2.Peptide ELISA   Stock solution of tetrameric peptide in dimethylformamide with standard protocol Was diluted 1000-fold to 10 μg / ml with a carbonate buffer at pH 9.5. Each micro Fill the titration plate wells with 100 μl of the diluted peptide solution at 4 ° C. After overnight coating, block with buffered albumin. Peptide-coated microphone Rotate the titration plate with several dilutions of aPL blood, including 1:50. Incubate for 1-2 hours at room temperature in clear. After washing, peptide-bound human IgG Was determined by enzyme-linked anti-human IgG according to standard ELISA procedures.Competitive binding peptide ELISA   (A) Immulon II plate (Dynatech Laboratories, Inc., Chantilly VA) web Except for three wells that serve as blank controls, mM sodium bicarbonate (Fisher Scientific, Pittsburgh, PA, reagent grade) 10 μg of the tetravalent peptide ACA 6501 / 3B10 in 50 mM sodium carbonate (pH 9.5) 100 μl of the solution at room temperature for at least 1 hour. The liquid is then Except 0.5% (wt / vol) BSA in TBS (Sigma Chemical, St. Louis, MO, # A7638) Was added at 200 μl per well (blank for blocking) Wells). And incubated at room temperature for at least 1 hour. 4 pieces 1.5 to 4 microcentrifuge tubes were numbered 1 to 4. Place the following reagent in the first microcentrifuge tube ( (Brinkman Instruments, Westbury, NY): 30 μl 5% BSA; 284 μl T BS; about 400-500 μg / ml monomer peptide (ACA-5A12 or -CB2 or -3B in TBS 10, including scrambled-3B10) as a negative control 8 μl of stock solution; and 8.2 μl of serum 6501 at a 1:10 dilution in 0.5% BSA-TBS. Less than Were mixed in a second microcentrifuge tube: 30 μl of 5% BSA; 290 μl of TBS; 400-500 μg / ml monomeric peptide (ACA-5A12 or -CB2 or -3B10, or negative 2 μl of a stock solution containing the disrupted -3B10) as a control for 8.2 μl serum 6501 at a 1:10 dilution in 5% BSA-TBS. Use the following reagents in a microcentrifuge # 3 Mix in tube: 30 μl of 5% BSA; about 400-500 μg / ml of monomeric peptide (5A12 or Or -CB2 or -3B10) or of the randomized sequence-3B10) as a control 287 μl of 1:10 dilution; 8.10 of ACA-6501 serum solution previously diluted 1:10 with 0.5% BSA-TBS. 2 μl. The following reagents were mixed in a No. 4 Eppendorf microfuge: 60μ of 5% BSA; 584 μl of TBS; 16.5 μl of serum 6501 diluted 1:10 with 0.5% BSA-TBS. Blocked The washed plate was washed 5 times with TBS. Add 1 microcentrifuge tube solution per well 00 μl was added in triplicate. Repeat the same amount of solution in microcentrifuge tubes # 2, # 3 and # 3 in triplicate. Of wells. Aliquot 100 μl aliquot of the solution in a fourth microfuge tube Three blocked blank wells of a clot titration plate Added. In plate shaker (American Dade, Miami, Fl, Rotator V) For 1 hour at room temperature with shaking at 40 rpm, then washed 5 times with TBS. 0. Goat anti-human IgG / alkaline phosphatase conjugate diluted 1: 1000 in 5% BSA-TBS 100 μl aliquots of the body (Zymed, South San Francisco, CA, Cat. No. 62-8422) And incubated for 1 hour at room temperature. Then wash the plate 5 times with TBS After that, add 100 μl of the diluted PPMP substrate solution as described in Example 1. Thus, the assay was performed. After 20 minutes, 50 μl per well of 0.2 M Na_TwoHPO_Four(Malli nckrodt, St. The reaction was stopped by adding Louis, MO., Reagent grade). Optical density was read at 550 nm in a microplate reader (Bio-Tek Instrument s, Winooski, VT, Model EL311). 550nm light for amount of peptide per well Academic density using Graph Pad Prizm (Graph Pad Software, Inc., San Diego) Painted. Peptides required to inhibit serum 6501 binding to tetravalent 3B10 by 50% The amount of C was calculated from the graph.Card (Dynatech Laboratories, Inc., Chantilly, VA) in ethanol. 30 μl of a solution containing oripin (CL, 50 μg; Sigma Chemical Co., St. Louis, MO) Wells were coated. Two control wells contained 30 μl of ethanol. I just met. After overnight evaporation at 4 ° C., each well was filled with 5% (w / v) Blocked with 200 μl of PBS containing fish gelatin for 2 hours at room temperature. CL coated, blown The washed plates were washed 5 times with TBS and each well was filled with 2.3% (v / v PBS) IgG. Β with 100 μl of depleted human serum (Sigma Chemical Co., St. Louis, MO)_Two-GPI In addition, it was incubated at room temperature for 2 hours.   During incubation, 22 μl diluted in 1: 1 TBS / PBS containing 3% fish gelatin ACA6501 serum (final dilution 1: 400) and various amounts of each of the six peptides were added to Eppendorf. Mix in a final volume of 220 μl using a microcentrifuge tube. In particular, tube number 1 181.3 μl of 3% fish gelatin TBS-PBS solution, 16.7 μl of peptide stock solution and 22 μl of ACA6501 serum diluted 40-fold with 3% fish gelatin / TBS-PBS was mixed. Pepti # 951 (a diselin acyclic negative control), # 952 (a single blot of LJP690), For thioether CCTE-3G3, CHTE-3G3, HCTE-3G3, and HHTE-3G3, The solution was in the range of 450-800 μg / ml. For the second tube, add: 148 μl of fish gelatin / TBS-PBS, 50 μl of peptide stock solution, ACA6501 serum 22 μl of a 40-fold diluted solution of In the third tube, 48 µl of fish gelatin / TBS-P BS, 50 μl of peptide stock solution, and 22 μl of ACA6501 serum diluted 40-fold 1 was added. No.4 control tube contains 396μl of fish gelatin / TBS-PBS 44 μl of a 40-fold diluted solution of ACA6501 serum (without peptide) was added. So Each tube was incubated at room temperature for about 1 hour.   Wash the CL / β2-GPI microtitration plate 5 times with TBS, No. 1, 2, 3, 4 Eppendor containing tide (or mixture without peptide) Duplicate f-tubes were added to wells in 100 μl aliquots. 4th tube Aliquots of 100 μl from the kit were used for duplicate control without cardiolipin. Added to Luwell. Rotate the microtitration plate at room temperature (American Scientific, Rotator V) for 1 hour while shaking at 40 rpm Was added. Then, after washing 5 times with TBS, 1: 1000 with 0.5% (w / v) BSA-TBS. Diluted goat anti-human IgG alkaline phosphatase conjugate (Zymed, Cat No. 62-8422) 100 μl was added. Incubate for 1 hour at room temperature, then microtitration The plate was washed again 5 times with TBS and 100 μl of PMPP solution (diluted 1:26 with water, 7.8 g phenolphthalein monophosphate and 69.5 g 2-amino in 100 ml water (Stock solution of 2-methyl-1-propanol) was added to perform colorimetric enzyme detection. Two After 1 minute, 50 μl of 0.2 M Na_TwoHPO_FourAdd (Mallinckrodt) to each well To stop the reaction. Measure the absorbance at 550 nm using a microplate reader (Bio-Tek In struments, Model EL 311). The absorbance for the added peptide is As shown in FIG. 12, it was drawn by Graph Pad Prism (Graph Pad Software, Inc.). ACA Amount of peptide that inhibits the binding of 6501 by 50% (IC₅₀) Indicates the amount and maximum of the added peptide Calculated from the graph as the intersection of half the absorbance value. 100 μL / well of purified human P2 glycoprotein I (PerImmune, Ro ckville, MD). Two control wells received 100 μL PBS only I got it. After a 2 hour incubation at room temperature, the liquid was removed. Coating Plate with 2% (w / v) skim milk powder (Carnation, Glendale, CA) and 0.4% (w / v) 2 hours with 200 μL / well of PBS containing Tween 80 (Calbiochem, San Diego, CA) Blocked at room temperature. A blocking solution was also used as a reagent diluent.   During the second hour of incubation, varying amounts of each of the four test peptides Was diluted with 22 μL of ACA-650 diluted with the sample diluent in a 220 μL fmal volume. One serum was mixed with an Eppendorf microcentrifuge tube. For details, Tube #I contains 188 μL of sample diluent and 10 μL of peptide stock solution (2 mg-4 mg / mL dilution), and 22 μL of AC-650 diluted 1:35 in sample diluent. One serum was added. Tube # 2 contains 158 μL of sample diluent and 40 μL of peptide Dstock solution and 22 μL of 1:35 diluted ACA-6501 serum were added. Tube # 3 Contains 381 iL of sample diluent, 160 μL of peptide stock solution, and 22 μL of 1:35 dilution A CA-6501 serum (sertim) was included. Control tube, tube 94, 396 μL of sample diluent and 44 μL of 1:35 diluted ACA-6501 serum (no peptide) Was contained. Each of the tubes was incubated at room temperature for about 1 hour.   β_Two-Wash GPI microplate 5 times with TBS, and eppendorf tube 100 μL of the mixture contained in 91, 2, 3, and 4 was added to duplicate microplates. Added into wells. Transfer the contents of 100 μL of tube # 4 to β_Two-Not coated with GPI Was added to each of the duplicate control wells. Incubate the plate for 1 hour at room temperature. 100 μl, washed 5 times with TBS and diluted 1: 1000 in sample diluent L goat anti-human IgG / A-P conjugate (Zymed, Cat. No. 62-8422) was added. At room temperature After 1 hour incubation, the plate is again washed 5 times with TBS and Measure enzyme detection using 100 μL of PPMP solution (100 mL of water stock solution diluted 1:26 with water). 7.8 g of phenolphthalein monophosphate and 69.5 g of 2-amino-2- in the liquid (Methyl-1-propanol). After 22 minutes, the reaction , 50 μL per well of 0.2 M Na_TwoHPO_Four(Mallinckrodt) Stopped. A_550nmTo a microplate reader (Bio-Tek Instruments, Model EL) 311) read. The absorbance versus the added peptide was determined by Graph Pad Prism (Graph Pad Softw are, Inc.) as shown in FIG. A pair that inhibited ACA-6501 binding by 50% The amount of peptide (IC-50) was determined by crossing the amount of peptide added with the absorbance at half the maximum. Calculated from the graph in points. Example 5:Peptide 3B10 shortening experiment and the resulting peptide competition ELISA result   Microtitration plate (96 well, flat bottom polystyrene, Immulon -2 Dynatech Laboratories, Inc., Chantilly, VA) with carbonate buffer, pH 9.6 (15mM Na_TwoCO_Three/ 35mM NaHCO_Three) In 10 μg / ml tetramer ACA-6501 / 3B10 peptide 100 μl / well for 1 hour at room temperature. After removing the liquid from the well, it Each well was filled with 0.5% (wt / vol) BSA (globulin-free, cat. No. A7638, Sigma C Blocked with 200 μl of TBS containing (Chemical Co., St. Louis, MO) for 1 hour at room temperature. Was. Three wells of the plate are used as blank control wells. Therefore, it was not coated with the tetravalent peptide.   During the blocking step, each soluble monomeric peptide to be tested is Three test tubes (Eppendorf micro test tubes, Brinkmann Instruments, W estbury, NY). Each test tube contains 30 μl of 5% BSA / TBS, 8.2 μl of ACA-6501 serum (diluted 1:10 with 0.5% BSA / TBS) and various volumes of pepti Volume / TBS buffer solution to the required volume so that the final volume is 330 μl. including. This 330 μl volume is transferred to a plate coated with ACA-6501 with a tetravalent peptide. For each peptide concentration tested for its ability to block binding The volume was sufficient to make triplicate 100 μl samples. Short amino terminus However, for peptide 139, which lacks the contribution of the normally tested ala-gly backbone, The concentration of the check solution is about 340-400 μg / ml TBS, and the concentration of three peptide concentrations Aliquots of 19 μl, 75 μl and 292 μl were taken to prepare tubes . Peptide 42 (only the N-terminal ala was deleted) and Peptide 143 (not truncated) ), The concentration of the stock solution was 400-500 μg / ml TBS. It To prepare three concentrations for each peptide, 1 Aliquots of μl, 4 μl and 16 μl were removed. Peptide monomer control For tubing (lacking peptide), tubing with a final volume of 660 μl A tube was prepared containing the following: 60 μl of 5% BSA, 16.5 μl of a 1:10 dilution. ACA-6501 at a grade, and 583.5 μl TBS, i.e. the same as a 330 μl tube Final concentration (0.5% BSA and 1: 400 ACVA-6501 serum), but twice the volume, Does not include peptides.   After blocking incubation, plate coated with tetravalent peptide And washed 5 times. 330 μl containing peptides 139, 142 and 143 at different concentrations From each tube, 100 μl was added to triplicate wells coated. Contains no peptide Three 100 μl aliquots from a new 660 μl control tube were coated Add to each well and three additional 100 μl aliquots Each blank well was added. The plate is rotated by a rotary shaker (Rotat or V, American Dade, Miami, FL) for 1 hour at room temperature at 40 rev / min. Bait and then washed 5 times with TBS. Goat anti-chicken diluted 1: 1000 in BSA / TBS G / alkaline phosphatase conjugate (Cat. No. 62-8422, Zymed, South San Fr. ancisco, CA) was added to the microplate at 100 μl per well. At room temperature After a one hour incubation, the plates were washed again five times with TBS. newly Color development occurred after the addition of 100 μl / well of the prepared diluted PPMP substrate solution. PP MP (phenolphthalein monophosphate, cat. No. P-5758, Sigma Chemical  Co., St. Louis, MO) diluted solution with PPMP stock solution (0.13M PPMP, 7.8M amino-2-me) Chill-1-propanol adjusted to pH 10.15 with HCl) Prepared by making. After 30 minutes, 50 μl / well of 0.2 M Na_TwoHPO_Three(reagen t grade, Mallinckrodt, St. (Louis, MO) to stop the reaction. Was. 550 nm with microplate reader (Bio-Tek Instruments, Winooski, VT) Absorbance measurements were taken at room temperature and A to the added peptide per well_{550 nm} Using Graph Pad Prizm (Graph Pad Software, Inc., San Diego, CA) Plotted. As shown in FIG. 11, the horizontal line represents the competition for monomeric peptides. It was drawn to correspond to the half-maximal binding reaction obtained in the absence. 50% inhibition The amount of peptide required is plotted by the intersection of this line plotting each peptide tested. Can be read from the point. The 50% inhibition values are shown in FIG. The result is a deletion of the N-terminal Ala. Had no negative results, but further deletion of Gly achieved 50% inhibition Shows that the concentration required to increase the concentration was increased about 8-fold. Example 6:Substitution of α-methylproline for 3B10 and resulting ELISA   The peptide ACA-6501 / 3B10 and the prolines at positions 3 and 9 were replaced with α-Me-Pro Testing of the analog was performed using the methodology set forth in Example 5. Peptide 3B10 , 726 (substitution at position 3 with α-Me-Pro), 727 (substitution at position 9 with α-Me-Pro) , 728 (substituted with α-Me-Pro at both the 3-position and the 9-position) are referred to as soluble monomer peptides. And a microtitration plate coated with the tetrameric peptide 3B10 and Tested in competitive binding ELISA using ACA-6501 serum.   Microtitration plates were prepared as described in Example 5 for tetramer 3B10 Coated with peptide. For each of the four peptides tested, three peptides Tide concentrations were prepared in tubes. As in Example 5, these 12 tubes In a final volume of 330 μl, 0.5% BSA / TBS, and 1: 400 final dilution. It had a final concentration of ACA-6501 serum at a grade. All peptide stock solutions are 400- 500 μg / ml TBS. 30 μl of 0.5% BSA / TBS and 8.2 μl of ACA-6501 serum ( Tubes containing 1:10 diluted with 0.5% BSA / TBS) containing 4 peptides Add 1 μl, 4 μl, or 16 μl aliquots from each of the stock solutions Was. Further, an appropriate volume of TBS was added so that the final volume became 330 μl. Competition Run control tube without peptide to a final volume of 660 μl Prepared as described in Example 5.   After blocking incubation of the tetravalent peptide coated plate, Tubes for each peptide concentration of the four peptides, as well as peptides 3 tubes from the blank control tube and the blank control tube A 100 μl aliquot was tested as described in Example 5. Microtitre The plate ELISA procedure and data operation were performed as described in Example 5. Was. As shown in FIG. 13, peptide 727 in which position 9 was substituted with α-Me-Pro was unmodified Better than peptide 3B10, or both analogs with altered proline (peptide 728) It was very active. Peptide 726 substituted at position 3 loses activity as a result of the substitution. Was. Example 7:6626 Simple screening of antibodies and their corresponding sequences Description   Affinity purified ACA-6626 (AffACA-6626) was used as described previously for 8 It was isolated from ml of ACA-6626 plasma by affinity purification. AffACA-6626 (10μ g), as described above for ACA-6501 bioselection, with all p- Epitope xy'z phage library consisting of a pool of III component libraries Incubated together. After three organism selections, the second and third samples Randomly selected phage were tested by microselection. A few clones Only weak but immunopositive at a dilution of 1: 1000. 4th creature Made a selection. 43 immunopositive by microselection of 94 fourth clones And some were positive even at phage dilutions as high as 1: 100,000. Was. G-tracking of 43 immunopositive clones as described above for ACA-6501 DNA sequencing revealed five unique sequences. Conventional After four-base DNA sequencing, the translated amino acid sequences in Table 3 were obtained. Example 8:Identification of ACA-specific sequences from patient 6644   epi^xy'zThe phage display library was prepared using ACA Affinity Screening was carried out in the same manner as in Example 4 using the purified antibody. Pe Prior to peptide synthesis, the colony blot assay described above should be used as a final identification step. I was used. Play about 150 colonies on the original nitrocellulose membrane And assayed. Antibody from patient 6644 was used at a concentration of 1 μg / ml. 150 rollers plated and assayed on a nitrocellulose membrane Of the knees, only four were strong positives in this screen and two Weakly positive. Of the 6 positive phages selected by this screening, By sequencing the inserts, the inserts are free of the following free amino termini (epi^z) Derived from an 8mer library that: Example 9:ACA-6641 Of phage library screening using DNA   AffACA-6641 was isolated from 4 ml of plasma taken from patient number 6641. I mentioned earlier In a final volume of 100 μl, p-III phage plasmids pooled with AffACA-6641 (10 μg) Incubated with Ibrary. 3rd and 4th time after 4 organism selections 45 clones from were tested by microselection. 23 out of 45 are shaded Gender. In the third phage, two clones scored 4+ and two A score of 3+ and two scored 2+. From the fourth time, one click Loans have a score of 4+, one has a score of 3+, and three have a score of 2+ so there were. G-tracking DNA sequencing revealed six unique sequences. K Only one of the loans, 3G3, was strongly positive in the phage capture ELISA. 4 base DNA sequence From the sequence determination, the following translated peptide sequence was obtained.                                 CLGVLGKLC. Example 10:Peptide binding to non-immunogenic polyvalent carriers   Several tetravalent platforms for the development of B cell tolerogens are co-owned And co-pending US patent application Ser. No. 08 / 118,055, filed Sep. 8, 1993 No. 08 / 152,506 (filed on Nov. 15, 1993); U.S. Pat. Nos. 5,268,454; No. 5,276,013 and US Pat. No. 5,162,515, which are incorporated herein by reference in their entirety. Developed as described in APL antibody library for epitope library Binds candidate peptides selected by cleaning, and provides immunological Tested for changes in chemical behavior.   A non-immunogenic multivalent platform with amine groups is shown in the following scheme Combine as follows.Amine on platform-carboxyl on peptide Chemical compound 2: 8.0 g (5.7 mmol) in 50 ml of absolute ethanol1Solution and 35 ml The solution of clohexene was placed under nitrogen and 500 mg of 10% Pd on carbon was added. The mixture was refluxed for 2 hours with stirring. Allow the mixture to pass through celite as it cools. Peptide protected with a free carboxyl group (PHN-peptide-CO ₂ H   The peptide can be conjugated to a trifluoroacetic acid (TFA) stable protecting group (a benzyl group on the carboxyl group). Ester or cyclohexyl ester and carbobenzyloxy on amino group (CBZ)) using FMOC chemistry in Wang (p-alkoxybenzyl) resin , Synthesized by standard solid phase methods. Amino acid residues are added sequentially to the amino terminus. Pep The tide is removed from the resin with TFA and one free carboxyl is added to the carboxyl terminus. To provide a peptide having a group, and all other carboxyl groups and amines To block. The protected peptide is purified by reverse phase HPLC.   Peptide-platform binding, 4   Dissolve protected peptide (0.3 mmol) in 1 ml of dimethylformamide (DMF) And 0.3 mmol of diisopropylcarbodiimide and 0.3 mmol of Add 1-hydroxybenzotriazole hydrate (HOBT). Add this solution to 1 ml DMF 0.025 mmol tetraamino platform in2Add the solution. After completion, Fully protected conjugate made of3Remove DMF under vacuum to obtain. Union3 By reacting with hydrofluoric acid (HF) for 1 hour at 0 ° C in the presence of anisole. Coalescence4Get. Purification is achieved by preparative reverse phase HPLC.   The following scheme illustrates the peptide's amino acid to carboxyl group Shows the bond of the ノ group. Compound 5-4 platforms with carboxylic acid groups   861 mg (1.0 mmol) of succinic anhydride (1.0 g, 10 mmol)2And 252 mg (3.0 mmol) NaHCO_ThreeIncluding 1/1 dioxane / H_TwoO to a 20 ml solution, and the mixture is allowed to Stir for 16 hours. The mixture is acidified with 1N HCl and concentrated. Add the concentrate Purified by Ricagel chromatography5I will provide a.   Peptide protected with free amine (H ₂ N-peptide-CONH ₂ )   Peptides are synthesized on amide resins using standard solid-phase methods. This is TFA Stability protecting group (benzyl ester or cyclohexyl ester on carboxyl group) Carboxyl-terminal amino acid after cleavage from the resin using Get Amino acid residues are added sequentially to the amino terminus using standard FMOC chemistry You. The peptide is removed from the resin using trifluoroacetic acid and free amine linker A protected peptide having the formula Protected peptides, reverse phase HPLC To purify.   Peptide-platform conjugate, 7   In 1 ml DMF, 0.05 mmol of free amine protected peptide (H_TwoN-peptide-CO NH_Two), 0.1 mmol diisopropylethylamine, and 0.01 mmol5Solution containing Is prepared. BOP reagent (benzotriazol-1-yloxy-tris (dimethylamino) Phosphonium hexafluorophosphate) (0.1 mmol) is added and the mixture is analyzed Stir until the reaction is complete, as evidenced by HPLC. Peptide protecting groups Removal by treatment with HF at 0 ° C in the presence of anisole to remove the protecting groups Of the combined7Get. Compound7Is purified by preparative reverse phase HPLC.   The following scheme describes how to add a mercapto group linker to the amino terminus of a peptide. And then how the peptide / linker is tetrabromoacetylated Compound bound to rat form13Or show.Haloacetyl on platform and sulfhydryl on peptide Compound 9   Concentrated sulfuric acid (100 μl) was mixed with 4.48 g (17.2 mmol) of triphenylmethanol and 1.62 g ( 15.3 mmol, 1.3 ml) of 3-mercaptopropionic acid in 60 ml of 35 ml of EtOAc Added to the solution. The mixture is stirred at 60 ° C. for 10 minutes, cooled to room temperature (RT) and placed on ice for 1 hour placed. The resulting white solid was collected by filtration to give 4.52 g (75%).9I gotCompound 10   2.72 g (7.8 mmol) of dicyclohexylcarbodiimide (DCC) (2.41 g, 11.7 mmol) )of9And 41 ml of CH at 0 ° C. containing 1.08 g (7.8 mmol) of p-nitrophenol_TwoCl_Two Added to the solution. The mixture was stirred for 16 hours and returned to room temperature. The mixture is filtered to N, N -Dicyclohexylurea (DCU) was removed and the filtrate was concentrated. Hexane / C H_TwoCl_TwoCrystallized from 3.17 g (86%) as pale yellow crystalsTenI gotCompound 11-Cyclic Thioether Having a Mercaptopropionyl Linker peptide   Cyclic thioether peptide (one sulfur to CH_TwoSubstituted cyclic disulfide Water containing sodium chloride and dioxane Trophenyl esterTenTested. It is appropriate if the peptide contains lysine Must be blocked. The resulting modified peptide is reacted with trifluoroacetic acid. Thiol linker modified peptide11I gotCompound 13-cyclic thioether peptide with bromoacetylation platform Union   0.10 mmol thiol-modified peptide11He-sprayed 100 mM sodium borate containing 9/1 MeOH / H_TwoBromoacetylation of 0.025 as 40 mg / ml in O Platform12Was added. Allow the solution to complete binding as evidenced by HPLC. Until N_TwoStir under a nitrogen atmosphere. The conjugate was purified by reverse phase HPLC. Example 12:LJP685 Synthesis of γ-bromo-N-BOC-α-aminobutyric acid t-butyl ester, compound 14:40ml dry T 4.03 g (13.3 mmol) of N-BOC-glutamic acid-α-t-butyl ester in HF and 1.61 m l (1.48 g, 14.6 mmol) of a solution containing N-methylmorpholine_TwoUp to 15 ° C in an atmosphere And cooled. To this mixture, add isobutyl chloroformate (1.73 ml, 1.82 g, 13.3 mm ol) was added dropwise. The mixture is stirred for 10 minutes and 2.03 g (16.0 mmo) in 8 ml of THF l) A solution containing N-hydroxy-2-mercaptopyridine was added, and a further 2.23 ml Et_ThreeN (1.62 g, 16.0 mmol) was added. Cover the mixture with foil to block the light, and Stirred at room temperature for 1 hour. Filter the mixture and avoid exposing the filtrate to light Carefully concentrated on a rotary evaporator. Concentrate 20 ml BrCCl_ThreeDissolved in Allowed to cool, and the solution was cooled to -70 ° C. Place the solid under vacuum, then N_TwoIn pa And returned to room temperature and placed in a 20 ° C. water bath. Then a 500W solar lamp from a short distance above For 5 minutes. The mixture is concentrated on a rotary evaporator and Chromatography (40mm x 150mm, eluting toluene until UV active substance elutes) Used as an agent. 2% EtOAc / toluene (500 ml), 5% EtOAc / toluene (500 · l)) Therefore, it was purified. The crude fraction was purified again. TLC (R_f0.23,5% EtOAc / tolue The purified fractions were combined and concentrated to give 3.23 g (72%) of an oily solid. Compound14I gotN-BOC- Glycylproline-4-nitrophenyl ester, compound 15 : 82 ml of dry THF 3.0 g (11.6 mmol) of N-BOC-glycylproline and 1.93 g (13.9 mmol) of 4-nitro The solution containing rophenol 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 at room temperature for 16 hours. Was. HOAc (579 μl) was added to the mixture and stirred for 30 minutes. Freezer the mixture And then vacuum filtered. Concentrate the filtrate and filter on silica gel Tomography (18 x 150mm bed, 2.5% EtOAc / 97.5% CH_TwoCl_Two/ 1% HOAc) And purified. A small amount of acetic acid is concentrated from dioxane several times with a rotary evaporator. It was removed by shrinking. Concentrate 3/1 hexane-Et_TwoO was added and milled, resulting in white The colored solid was collected by filtration to give 4.1 g (90%) of compound 15 as a white solid; T N-FMOC-St- Butylthiocysteinamide, compound 16: 5.0g (in 115ml THF) 11.6 mmol) FMOC-S-t-butylthiocysteine and 1.33 g (11.6 mmol) N-hydrido The solution containing roxysuccinimide was cooled to 0 ° C. 3.58 g (17.37 mmol) of D in solution Added CC. The mixture was stirred at 0 ° C. for 1 hour and 1.6 g (NH_Four) HCO_ThreeIncluding 42.9 ml of an aqueous solution was added. The mixture was stirred for 4.5 hours and slowly brought to room temperature from an ice bath. Concentrate on the return rotary evaporator to remove the THF and leave the aqueous phase with a white solid Occurred. 200 ml of CH in this mixture_TwoCl_TwoAnd stir until the solids are almost completely dissolved And added 100 ml of 1N HCl and shaken. CH_TwoCl_TwoLayers are washed with 100 ml of saturated NaHCO_ThreeWash with solution , (Na_TwoSO_Four) And filtered. Bring the filtrate to a boil on a hot plate and add CH_TwoCl_TwoF 4.36 g (87%) of a white solid compound crystallized from 300 ml of xan16Get Compound 17: 2.91 g (6.75 mmol) of compound16In 33.8 mL of dioxane containing water (16. 9 mL) and the solution was flushed with nitrogen for 5-10 minutes. This mixture is nitrogen Keep under atmosphere and add 1.13 g of NaHCO_Three(13.5 mmol), Then 1.77 mL of PBu_Three(1.44 g, 7.09 mmol) was added. Let this mixture at room temperature for 1 hour Stir, 1 × 100 mL 1N NaCl and 2 × 100 mL CH_TwoCl_TwoAnd distributed between. CH_TwoCl_TwoLayers Combine, concentrate, and partially dissolve the resulting white solid in 33.8 mL of dioxane. I understand. Water (9 mL) was added and purged with nitrogen for 5-10 minutes. Mixture in nitrogen atmosphere Hold at 2.17g K_TwoCO_Three(16.9 mmol) and then 2.63 g (8.1 mmol) of the compound object14Was added. The mixture was stirred for 16 hours, and 1 × 100 mL of 1N aqueous HCl was added and 2 × 1 00 mL of 10% MeOH / CH_TwoCl_TwoAnd distributed between. CH_TwoCl_TwoCombine layers, NaSO_FourDry Dry, filter, and concentrate to give a semi-solid residue. Silica gel chromatograph E (1/3 CH_ThreeCN / CH_TwoCl_Two) To give 3.2 g (80%) of compound 17 as a white solid Obtained as; TLC R_f0.27, 1/3 CH_ThreeCN / CH_TwoCl_Two. Further purification of analytical samples Performed by recrystallization from Xan-EtOAc; mp 104-106.5 ° C .; Compound 18: 20/1/1 TFA / H_Two1.27 g of O / mercaptoethanol solution (23.2 mL) 2.11 mmol) compound17Was added. The mixture was stirred at room temperature for 1 hour and concentrated to about 3 mL volume. Shrunk. The product was precipitated by the addition of 50 mL of ether. 2 additional solids formed Washed twice with ether and dried under reduced pressure to give 812 mg of solid. 10. 6 mL dioxane and 10.6 mL H_TwoSuspended in O. NaHCO_Three(355 mg, 4.22 mmol) Added to this mixture, then 1.33 g (3.38 mmol) of the compound1510.5mL Geo containing A solution of xane was added. The mixture is stirred at room temperature for 20 hours and 100 mL of 1N HCl and 3 x 100 mL CH_TwoCl_TwoAnd distributed between. Combined CH_TwoCl_TwoDry the layer (Na_Two SO_Four), Filtered and concentrated. Purification by silica gel chromatography (3 5 x 150 mm, step gradient, 5/95/1 to 10/95/1 MeOH / CH_TwoCL_Two/ HOAc (1L)). Pure Concentrate the fractions and triturate the residue with ether to give 881 mg (60%) of the compound.18I got TLC R_f0.59, 10/90/1 MeOH / CH_TwoCl_Two/ HOAc; melting point 116-117.5 ° C; N-FMOC-L- Alanyl-L-2-methylproline, compound 19: 2-methylproline (Seebac h et al. (1983) Am. Chem. Soc. 105: 5390-5398) (1.00 g, 54.75 mmol), 4.00 g (47.6 mmol) of NaHCO_ThreeAnd 6.9 mL DMF containing 31 mg (0.23 mmol) HOBT The solution was cooled to 0 ° C. and 3.18 g (6.66 mmol) of N-FMOC-L-alanine was added. . The reaction was stirred at 0 ° C. for 1 hour, then at room temperature for 18 hours. Mixture with 50 mL of EtOAc And 3 × 50 mL of 1N HCl. Dry the EtOAc layer (MgSO 4_Four), Filtered, And concentrated. Silica gel chromatography (step gradient 45/55/1 EtOAc / hex) SAN / HOAc ~ 47/53/1 EtOAc / Hexane / HOAc ~ 50/50/1 EtOAc / Hexane / HOAc) 1.72 g (86%) of the compound19Was obtained as a white solid. The product Concentrated several times from dioxane to remove traces of acetic acid: mp 59-60 ° C; N-FMOC-L- Leucinyl-HMPB-MBHA resin： 22.5mL CH containing N-FMOC-L-leucine_TwoCl_Two And a few drops of DMF were prepared and cooled to 0 ° C. 1.71 mL in this solution (1.38 g, 10.9 mmol) of diisopropylcarbodiimide (DIC) and The mixture was stirred at 0 ° C. for 20 minutes. The mixture was concentrated to an oil; in the meantime 2.5 g (0.87 mmol / g, 2.18 mmol) of HMPB-MBHA resin (Nova Bioch) em) to swell the resin. Dissolve concentrated oil in the minimum amount of DMF (about 1 mL) And added to the swollen resin, followed by 266 mg (2.18 mmol) of DMAP in about 1 mL of DM Dissolved in F. The mixture was gently shaken for 1 hour and washed (2 × DMF, 2 × MeO H, 2xDMF, 2xMeOH). The resin was dried under reduced pressure to give 2.77 g (85%), and The substitution amount was determined to be 0.540 mmol / g by Geisen test.T-butyl ester in aspartic acid and Pmc group in arginine, and An N-FMOC-linear peptide with an ether insertion, Compound20: This peptide Prepared by standard FMOC synthesis on N-FMOC-L-leucinyl-HMPB-MBHA resin. Was. Compound18Three equivalents of amino acid, HOBT, and (DI C) was used for each coupling step. Compound18Equal to 3 equivalents of HOBT And diisopropylcarbodiimide. Repeat each step with 10 μL Monitored by using lomophenol blue indicator. Reaction completion The ninhydrin test (1 mg, 1 drop of pyridine, and 5% ninhydrin With 1 drop of EtOH containing and 1 drop of 80% phenolin EtOH at 100 ° C. for 2 minutes Then, the beads whose reaction is not completed change to blue). Therefore 1.13 mg (0.613 mmol) of resin was used to prepare the peptide. Last mosquito After the coupling step, CH containing 1% trifluoroacetic acid_TwoCl_Two2 minutes with 15 mL of solution To achieve cutting from the resin. After 2 minutes, add 30 mL of 10% pyridine Under pressure. This is repeated 10 times, and HPLC (C18 , Gradient, 60/40 / 0.1 CH_ThreeCH / H_TwoO / TFA ~ 90/10 / 0.1, CH_ThreeCN / H_TwoO / TFA 210mn, 1mL / min , 4.6 mm x 250 mm column), combine the filtrate containing the peptide, And concentrated. The concentrate was dissolved in 40 mL of a 10% HOAc solution and purified by HPLC to give 0.1%. 528g (49%) peptide20I gotConversion of peptide 20 to cyclic peptide 21 (removal of FMOC group, cyclization and removal of protecting group) ):   10 mL CH containing 99 μL DBU_Three96 mg (0.052 mmol) of pep CN solution (5 mL) Tide20Was added. The solution was stirred for 1 hour and concentrated to a residue. This residue To 2 × 10 mL of Et_TwoTrituration with O gave a white solid. Add the solid to 100 mL CH_ThreeCN and 0.1M CH containing diphenylphosphoryl azide (DPPA)_Three312mL of CN solution (0.312m mol). The mixture was stirred for 20 hours and concentrated on a rotary evaporator. Was. Residue is 2 x 50 mL Et_TwoTriturated with O, and the opaque white residue was A / anisole / EDT / Me_TwoTreated with 5 mL of S for 1 hour. Transfer the product to 50 mL polypropylene In a centrifuge tube, mix the mixture with 40 mL Et._TwoPrecipitated by adding to O. Sinking The deposit was cooled to 0 ° C. and centrifuged at 2000 rpm for 5 minutes. Decant the supernatant Et pellets_TwoWashed with O and centrifuged again. Dry the pellet, 4 mL 50/50 CH_ThreeCN / H_TwoDissolved in O. This mixture is mixed with 36 mL of H_TwoDiluted with O / 0.1% TFA, Filter and HPLC (1 "C₁₈Column, gradient, 10/90/0/1 CH_ThreeCN / H_TwoO / TFA ~ 35/65/0 .1, CH_ThreeCN / H_TwoO / TFA 230 nm). Pure as confirmed by HPLC Lyophilized fractions and 52mg (90%) of compoundtwenty oneI got Example 13: Synthesis of conjugate of LJP 685   LJP 685 (compoundtwenty oneAlso known as) 3-trityl mercaptopropionic acid Treatment with NP ester and detritylation of the resulting product to free thiol Compound that is a peptide having an anchortwenty twoI got Valence platform molecule12 Excess compound withtwenty twoReaction, the tetravalent conjugatetwenty fiveOccurred. Compoundtwenty oneLonger than Linker (compound33(See reaction scheme below)), followed by detritization Compoundtwenty fourI got Compoundtwenty fourIs a valence platform molecule12And react And tetravalent conjugate26Occurred. HPLC shows both binding reactions very clearly Was.LJP 685 Attachment of MTU linker to compound 24, synthesis of compound 24: 15mg (0.013mmol) compoundTwo 1 With 29.5 mg of the compoundtwenty three0.5 containing 17.5 μL of diisopropylethylamine Added to 160 μL of DMF solution in mL. The mixture was stirred for 2 hours, Et._TwoPrecipitated by O I let it. The precipitate is dried and 650 μL of 1/1 / 0.056 / 0.040 TFT / CH_TwoCl_Two/ Thiophenol / Me_TwoDissolved in S and the solution was left for 1 hour. Et_TwoThe precipitation with O was performed by HPLC (C_{1 8} , 15-45% CH_ThreeCN / H_TwoO / H_TwoO, 0.1% TFA)twenty fourArose . The fraction containing the pure product is lyophilized to give 8.6 mg of compoundtwenty fourAs a white solid Obtained.LJP 685-MTU-AHAB-TEG , Compound 26: 8.6mg (6.3 × 10^-6mol) compoundtwenty fourIncluding He Was added to a 630 μL solution of 200 mM borate buffer, pH 8.5, into which1240mg / mL 40 μL of 9/1 MeOH / H containing liquid_TwoO was added. The mixture is stirred for 24 hours, 1 mL of 10% HOAc / H_TwoO solution was added. HPLC (C₁₈, Gradient 25-55% CH_Three CN / H_TwoO / water, 0.1% TFA) to give 8.3 mg of compound26(LJP 685-MTU-AHAB-TEG ) Got. Example 14:Fabrication of extended SH linker   Thiobenzoic acid ester (compound28) The compound27Prepared from Compound28The part Compound29Was converted to The thiobenzoic acid is removed by ethanolysis and the resulting The thiol obtained was tritylated. Then the ethyl ester is hydrolyzed to give the compound29 I got Nitrophenyl phosphate (PNP) ester (compound30) The compound29 Prepared from Aminotrioxodecanoic acid ethyl ester (compound31) Compound27Was prepared by treatment with sodium azide and reduction to the amine. A Amine compound 3-1 is acylated with compound 3-032Offered. Compound32Hydrolysis of And treatment with sodium hydroxide to give the free carboxylic acid. Intermediate Condensation of carboxylic acid with p-nitrophenol to give para-nitrophenyl (PNP) Te (Compound33) Got. Linker33To the peptide and the trityl group Remove and compound34I got This was used to generate the MTU-ATU-AHAB-TEG conjugate. Example 15:(LJP685) 4 / MTU-ATU-AHAB-TEG Synthesis of conjugate (compound 35)   Tetravalent conjugate35Was prepared as shown below. Pep with attached linker Tide (compound34) Was dissolved in a borate buffer (pH 8.5, 200 niM) into which He was introduced. To this mixture, 0.3 molar equivalent of platform compound 1-2 was added. This mixture The material was stirred for 1 hour and the product was purified by HPLC.Example 16:(LJP685) ₄ / DABA-PEG Synthesis of conjugate (compound 36)   8.5 Compound IA-DABA-PEG in borate buffertwenty fourCombined with processing36I got Example 17:T cell assay for activation   Incorporation of tritiated thymidine by peptide-stimulated T cells was demonstrated in 96 cells. Monitored in well round bottom plate. 5 × 10^FiveDraining lymph node cells (mouse ) Or isolated single cell suspension of peripheral blood lymphocytes (human) Mix with 1 to 30 jig of peptide in a final volume of 150 μl and add 5% CO_Two37 in Incubated for 5 days at ° C. At this point, 1 microcurie of labeled Thymidine was added and incubated for another 15-24 hours. Collected cells Was collected on a filter and counted on a liquid scintillation spectrometer. Example 18:In vitro induction of resistance   Eight groups, each containing five C57B1 / 6 mice, were alum and adjuvant B as a fund. About 10 μg / mouse of LJP685-KLH Priming was performed with the combination. Three weeks later, spleens are harvested and single cell suspensions are prepared. Prepared, washed three times with equilibrated salt solution, and correspond to one spleen per 1.5 mL medium. And resuspended in complete RPMI-1640 medium. 2.5 mL of cell suspension per Petri dish Aliquot into aliquots, and (LJP685)_Four-DABA-PEG (Compound 36) and (LJP685)_Four- TEG (compound35) With concentrations of 100 μM, 20 μM, and 4 μM, respectively. Incubated for 2 hours at ° C. Incubate one group of cells without tolerogen And served as a positive control. The cells are then transferred to a large volume of Washed with liquid and resuspended in 2.5 mL of balanced salt solution. The cells are then 650R in a manner that divides all cells from each treatment group into 5 recipients each Irradiated syngeneic recipient mice were injected. Then, including all positive controls All recipient mice were injected intraperitoneally with 10 μg of LJP 685-KLH in saline. Booster immunizations were performed. Mice were bled 7 days after the boost and their sera were raised The presence of the LJP 685 antibody was tested. Processing with both conjugates is shown in FIGS. 16 and 1 A significant dose-dependent reduction of the anti-LJP 685 antibody, as shown in FIG. this HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volume 2, Cellular Immunology (We ir, D.M., Blackwell Scientific Publications, Palo Alto, CA, 1986) Ive rson, G.M, `` Assay for invivo adoptive immune responses '', Antig en Bi It is measured by nding Capacity (ABC). Example 19:5A12 Of NMR solution structure of CB2, CB2 and 3G3   For phage library screening using the methodology described in the previous example. The two more isolated peptides were subjected to NMR analysis. Original peptide is last 2 With proline in the second position, but with two-dimensional (2D) two-quantum filter correlation spectroscopy ( DQF-COSY) NMR data derived from cis and trans isomers at this position This amino acid was removed to indicate the presence of three different structures. Proline Removal is in the range of 50-100 nM for binding to ACA antibodies._DResulting in a peptide having And the number of peaks in the fingerprint region of the DQF-COSY spectrum It is. The resulting cyclic peptides were 5A12 (GPCLILAPDRCG) and CB2 (GPCILLARDRCG ). The major difference between these peptides is that the arginine proline at position 8 Was replaced by This means that the variance in the 1D 1H NMR spectrum of CB2 is much smaller. This was consistent with 5A12 being a more robust peptide. Arginine substitution Also, ionized aspartyl carboxyl groups as reflected in pKa values Of 0.55 kcal / mol. Structural analysis for higher order 5A12 peptides went. Deuterium exchange and temperature coefficient data show no evidence of hydrogen bonding, but Nuclear Overhauser Effect (NOE), rotating overhaul Effect (Rotating Overhauser Effect, ROE) and coupling data Consistent with one structure. Distance geometry calculations yielded a family of 50 structures. Up Rank 15 is the root mean of the average for all atoms at 2.1 ± 0.2 Angstroms It had a squared deviation (RMSD). We believe that this peptide can be used at both ends of the molecule, Having an oval shape with a disulfide and a turn at proline 8 which is cis It was determined. Twisting is also present in the LIL region of the backbone. Finally, only the residues are positive Glycine at the carboxy terminus is very flexible because it has a sexual intraresidue NOE . This is the first determined peptide that mimics the ACA epitope on β2-GPI Shows the structure information of   NMR data combined with distance geometry calculation was converted to peptide 925 (CLGVLAKLC) and 925 peptide. Truncated peptide 3G3 (AGP CLGVLGK) in which alanine is substituted with glycine at position 6 LCPG) was used to determine the three-dimensional structure. The structure of peptide 925 was And 25 ° C in water. Assemblies of nine structures were calculated, all of which were NMR data. Coincided with RMSD for all non-hydrogen atoms is centered on each structure It was 2.45 ± 0.36 angstroms when compared to. Figure 18 shows the center of gravity of the assembly. Shown is the closest structure, that is, a structure that reasonably represents the shape of the peptide 925 molecule. FIG. Compares the structure of peptide 925 (labeled as 3G3 below the figure) with the structure of peptide 5A12 Compare. Both peptides have turns at approximately the same position in the peptide sequence.   Peptide drug carriers are roughly seen as small hydrophobic and positively charged groups. The answer is correct. As shown in FIG. 20, the gem-dimethyl group and the The amino and amino groups are largely reminiscent of the drug delivery group of this peptide. I Hydrocarbon linkers anchoring drug delivery groups to several scaffolds are identified in Figure 20 The point at which these linkers are attached to the backbone at a specified distance Separated. Finally, the dihedral angle that defines the relative orientation of the two linkers is 22 °. I decided to. Example 20:TEG Synthesis of carbamate linker 2- [2- (2-tert- Butylthioethoxy) ethoxy] ethanol, compound 38: In the ice bath Cooled 2- [2- (2-chloroethoxy) ethoxy] ethanol (11.0 g, 65.24 mmol) and To a mixture of tert-butylthiol (7.35 mL, 65.23 mmol) [5.40] Undec-7-ene (DBU, 9.75 mL, 65.23 mmol) was added slowly. Anti The reaction mixture was warmed to room temperature and stirred overnight. The mixture is then washed with ethyl acetate. And filtered. The crude product in the filtrate was filtered using a filter eluted with ethyl acetate. 2- [2- (2-tert- Butylthioethoxy) ethoxy] ethyl p-nitrophenyl carbonate Salt, compound 39 : Compound 38 (2.0 g, 8.98 mmol) and p-nitro in 5 mL of dry THF cooled in an ice bath To a solution of rophenol chloroformate (1.81 g, 8.98 mmol) in 1 mL of dry THF Of dry pyridine (0.73 mL, 8.98 mmol) was added slowly. White precipitate immediately Generated. After stirring at room temperature for 15 minutes, the reaction mixture was diluted with 10 mL of ether and And filtered. The filtrate is concentrated and used directly for peptide synthesis without further purification. Used. Example 21:Synthesis of tetravalent platform 1A / DABA / ATEG, 46 Bis-N- (t-butoxycarbonyl) -diaminobenzoic acid, compound 40 : 5.5mL in MeOH Of 7.18 g (32.9 mmol) of di-t-butyl dicarbonate in 44.5 mL of H_TwoO and 2 2.5 g (16.4 mmol) of 3,5-diaminobenzoic acid and 2.76 g (32.9 mmol) in 2.5 mL of MeOH NaHCO_ThreeWas slowly added and the mixture was stirred at room temperature for 24 hours. Mixed The mixture was cooled to 0 ° C., and 6.53 g of citric acid was added, and the mixture was extracted with EtOAc. Extracted. Dry the combined EtOAc layers (MgSO 4_Four), Filtered and concentrated. The residue 40 mL Et_TwoDissolved in O and the solution was filtered through Celite. Et_TwoO layer with 40 mL HC Extracted twice with l. Et_TwoDry the O layer (MgSO_Four), Filter, and concentrate to give a pink 3.81 g (66%) as a foamy solid40I gotN-hydroxysuccinimidyl ester of compound 40, compound 41:   55 mL of dicyclohexylcarbodiimide (3.34 g, 16.2 mmol) cooled to 0 ° C 3.8 g (10.8 mmol) of compound in EtOAc40And 1.24 g (10.8 mmol) of N-hydroxy Add to the solution of succinimide and allow the resulting mixture to rise to room temperature. The mixture was stirred for 18 hours. The mixture was added to 0.55 mL of acetic acid. Stir the mixture for 30 minutes And placed in the freezer for 2 hours. The mixture is filtered to remove solids and The filtrate was concentrated to give 5.80 g of a pink foamy solid. Silica gel chromatography Purification by Raffy (60/40/1 hexane / EtOAc / HOAc) yielded 4.30 g (89%) Compound41Was obtained as a pale pink solid.Mono-N- (t-butoxycarbonyl) -ethylenediamine, compound 42 ： 15mL CH_TwoC l_TwoA solution of 1.5 g (25.0 mmol) of ethylenediamine in was cooled to 0 ° C. and 1.82 g ( A solution of 8.33 mmol) of di-t-butyl dicarbonate was slowly added to the mixture. Mixed The mixture was stirred at room temperature for 18 hours and filtered, and the filtrate was concentrated. Silica gel chroma Tomography (90/10/1 CH_TwoCl_Two/ MeOH / HOAc) to give 0.98 g (67%) Compound42Was obtained as an oil.Bis-N- (t-butoxycarbonyl) -amino-TEG, compound 43 : 6 mL CH_Two750 in Cl mg (4.25 mmol) of compound42And 345 uL (337 mg, 4.25 mmol) in pyridine Add 5uL (559mg, 2.02mmol) triethylene glycol bis-chloroformate did. The mixture was stirred for 3.5 hours and the mixture was added to 35 mL CH_TwoCl_TwoAnd 35mL of 1N HC l. CH_TwoCl_TwoH layer_TwoWash with O and dry (Na_TwoSO_Four), Filtered, And concentrated, 1.14 g of crude compound43Which was used directly in the next step.Diamino-TEG bis-trifluoroacetate, compound 44 ： 300mg (0.57mmol) compound43 3.5g CH_TwoCl_TwoAnd 3.5 mL of trifluoroacetic acid was added. mixture Was stirred at room temperature for 3 hours, and the solution was concentrated to give 398 mg of crude compound.44Get this Was used directly in the next step.Compound 45 : 567 mg of compound in 6 mL of dioxane41Of 398 mg of crude, compound object44(About 316 mg, 0.57 mmol) and 193 mg (2.30 mmol) of NaHCO_ThreeWas added to the solution. Mixed The mixture was stirred for 3 hours and acidified with 1N HCl, and 20 mL of 1N HCl and 30 mL With EtOAc. Mix the combined EtOAc layers with saturated NAHCO_ThreeWash with solution and dry (M gSO_Four), Filtered and concentrated to give 490 mg (86%) of crude compound45The white foamy solid Get as. Purification by silica gel chromatography (EtOAC) gave 276 mg (4 8%) compound45Was obtained as a white foamy solid.IA / DABA / ATEG , Compound 46 : 100 mg (0.1 mmol) of compound45Prepare a solution of 1 mL of trifluoroacetic acid was added and the mixture was stirred for 1 hour at room temperature. Mixed The mixture was concentrated under reduced pressure. Et the residue_TwoPowder with O and dry under reduced pressure, white A crystalline solid was obtained. Dissolve the solid in 1 mL DMF and add 104 uL (77 mg, 0.6 mmol) of diisopropane Ripylethylamine is added, the mixture is cooled to 0 ° C. and 212 mg (0.6 mmol) Iodoacetic anhydride was added. The ice bath was removed and the mixture was stirred at room temperature for 2 hours Was. Cool the mixture to 0 ° C. and add 1N H_TwoSO_FourAcidify with 10 mL of 1N H_TwoSO_FourWhen 6x20mL 8/2 CH_TwoCl_Two/ MeOH solution. Dry the combined organic layers (Mg SO_Four), Filtered and concentrated to give 330 mg of an orange oil. Preparative HPLC (25 cm × 22.4mm C₁₈, Gradient: 30% B-40% B 0-40 minutes, A = H_TwoO / 0.1% TFA, B = CH_ThreeCN / 0.1 % TFA, 12 mL / min) to give 19 mg of compound46Was obtained as a white solid. Example 22:Synthesis of tetravalent platform BA / PABA / DT / TEG, 51. N- (t-butoxycarbonyl) PABA, compound 47 ： 60mL of H_Two3.0 g (21.9 mmol) of p in O A solution of -aminobenzoic acid was prepared. Na_TwoCO_Three(2.16 g, 25.7 mmol), followed by 30 mL of MeO H was added slowly. If all solids dissolved, 4.77 g (21.9 m mol) of di-t-butyl dicarbonate and the mixture is left at room temperature for 18 hours. While stirring. 4.92 g (25.6 mmol) citric acid are added to the mixture and the resulting turbidity is obtained. 200 mL of H_TwoPartitioned between O and 200 mL of EtOAc. 200 mL of EtOAc layer 0.1 N HCl and 200 mL H_TwoWash continuously with O and dry (Na_TwoSO_Four), Filter, and And concentrate to give 3.0 g (58%) of the compound.47Was obtained as a white solid.N- (t-butoxycarbonyl) PABA N-hydroxysuccinimidyl ester Compound 48 : DCC (2.61 g, 12.6 mmol) with 2.0 g (8.43 mmol) of compound in 50 mL of EtOAc47 And 0.97 g (8.43 mmol) of a 0 ° C. solution of N-hydroxysuccinimide. The ice bath was removed, the mixture was stirred at room temperature for 16 hours, and 0.5 mL of acetic acid was added. The mixture was stirred for an additional 30 minutes, placed in a freezer for 1.5 hours, filtered and concentrated. 3.75 g crude48I got Silica gel chromatography (50/50 hexane / Purification by EtOAc) gave 2.53 g (90%) of the compound48Was obtained as a white solid.Compound 49 ： 50mL CH_TwoCl_Two3.00 g (8.97 mmol) of the compound in48Solution for 30 minutes. Or 30uL CH cooled in an ice bath_TwoCl_Two485 uL (4.49 mmol) of diethylene tria It was dropped into the min solution. The mixture was stirred at 5-7 ° C for 30 minutes, then at room temperature for 16 hours. 200 ml of milky mixture_TwoPlace in a separatory funnel with O, and add H_TwoO Adjust the pH of the layer to 10 and add the mixture to 200 mL of 4/1 CH_ThreeExtracted with Cl / MeOH. Organic Dry the layer (Na_TwoSO_Four) And concentrate to give 0.971 g (40%) of the compound49I got This is Pure enough to be used in the steps below. The aqueous layer is 100mL of 4 / 1CH_ThreeSix extractions with Cl / MeOH Issued. Combine the organic layers and dry (Na_TwoSO_Four) And concentrate, another 1.08 g (44%) Compound49I got This required further purification. Silica gel for further purification Chromatography (gradient, 80 / 20-70 / 30 CH_ThreeCl / MeOH).Compound 50 : 135 uL (0.66 mmol) of triethylene glycol bis-chloro in 0.3 mL of THF A solution of roformate was added to 855 mg (1.58 mmol) of the compound in 13 mL of THF.49And 275uL ( 1.mmol) to a 0 ° C. solution of diisopropylethylamine. Ice bath removed In that case, the cloudy mixture became clear. Additional 70 uL to maintain basic pH Of diisopropylethylamine was added. The mixture was stirred at room temperature for a total of 3 hours. 25 mL of H_TwoPartitioned between O and 25 mL of EtOAc. Extract the aqueous layer with a second 25 mL of EtOAc And the combined EtOAc layers were dried (Na_TwoCO_Three), Filtered and concentrated to 0.986 g crude50I got Silica gel chromatography (80/4/16 CH_ThreeCl / dioxane / Isopropanol) yielded 516 mg (61%) of the compound.50Is a white solid I got it.BA / PABA / DT / TEG Preparation of compound 51 : Compound 50 (487 mg, 8.79 mmol) was added to 9 mL of CH 2 Cl 2 And 5 mL of trifluoroacetic acid. The mixture is stirred for 1.5 hours and concentrated. Shrunk. The residue was treated with 7 mL of Et._TwoTriturate with O and dissolve 5 mL MeOH and 1 mL 48% HBr Dissolved in the liquid. The mixture was concentrated and placed under reduced pressure until dry. Generate 10 mL of HBr salt_TwoDissolved in O and 191 mg (2.27 mmol) of NaHCO_ThreeWas added. 10m A solution of 591 mg (2.27 mmol) of bromoacetic anhydride in L of dioxane was added to the mixture . An additional 2 mL of dioxane was used for rinsing. Maintain basic pH More NaHCO as needed to_ThreeWas added. The mixture was stirred at room temperature for 2 hours. 1N H_TwoSO_FourAnd acidified. The mixture was extracted with 3.times.25 mL of EtOAc. Mix EtO Dry the Ac layer (Na_TwoSO_Four), Filtered and concentrated to give 773 mg of an oil. Purification Kagel chromatography (gradient, 90 / 10-85 / 15 CH_ThreeCl / MeOH), 401 mg (77%)51Was obtained as a white solid.Example 23:Synthesis of tetravalent platform BMP / TEG, 55. Dimethyl-5-hydroxyisophthalate, compound 52 : 2.00 g (11 mm) in 30 mL MeOH ol) of 5-hydroxyisophthalic acid and 0.5 mL of concentrated HCl were refluxed for 5 hours. The mixture was concentrated and the resulting residue was dissolved in 100 mL of EtOAc. EtOAc solution, 50m L 5% NaHCO_ThreeWashing twice with the solution and twice with 50 mL of saturated NaCl solution, Dry (Na_TwoCO_Three), Filtered and concentrated to give 2.09 g (90%)52Is a white crystalline solid I got it.Compound 53 : 546 mg (1.19 mmol) of triethylene glycol ditosylate was added to 11 mL of C H_Three500 mg (2.38 mmol) compound in CN52And 395 mg (2.86 mmol) of K_TwoCO_ThreeIn suspension And mix the mixture with N_TwoRefluxed under for 16 hours. Concentrate the mixture and remove the residue 25 mL CHCl_ThreeAnd 25 mL of H_TwoWashed with O and 25 mL of saturated NaCl solution. CHCl_ThreeDry the layer (Na_TwoSO_Four), Filtered and concentrated. Silica gel chromatography Purification by feed (gradient, 50/50 hexane / EtOAc to 100% EtOAc) gave 93 mg (41% )of Compound53I gotCompound 54 : 93 mg (0.18 mmol) of compound in 4 mL of THF531.82 mL (1.82 mm ol) LiBHEt_ThreeWhile a 1M solution of_TwoStir under atmosphere. 18:00 And add a 10% solution of HOAc in water until the pH is acidic. A liquid was obtained. The mixture was concentrated under reduced pressure, and the residue was dissolved in 10 mL of water. mixture Was extracted with 3 × 10 mL of EtOAc, and the combined EtOAc layers were dried (Na_TwoSO_Four) And filter And concentrated. Purify the silica gel chromatography (gradient, 90 / 10-85 / 15 CH_ThreeCl / MeOH) to achieve 20 mg (27%)54Was obtained as a white solid.Compound 55 ： 5mL of Et_TwoO and 20 mg (0.048 mmol) in 1 mL of THF549.6 uL (0.1 mmol) PBr_ThreeIs added. The mixture is stirred for 3 hours and partitioned with EtOAc . Dry the EtOAc layer (Na_TwoSO_Four), Filter and concentrate, and filter the residue over silica gel. Chromatography (CH_Three(Cl / MeOH) to give the compound55Get. Example 24:Synthesis of tetravalent platform BA / PIZ / IDA / TEG, 60 Compound 56 : 1.02 g (4.37 mmol) of N- (t-butoxy) in 50 mL of dry THF cooled to 0 ° C Carbonyl) -iminodiacetic acid (US Pat. No. 5,552,391 (Chemically-Defined Non- Compounds in Polymeric Valency Platform Molecules and Conjugates Thereof) To a solution of 5) and 1.01 g (8.75 mmol) of N-hydroxysuccinimide, 2.26 g (10. 94 mmol) of dicyclohexylcarbodiimide was added. The mixture was stirred for 16 hours And gradually warmed to room temperature. And 2.22 g (10.1 mmol) of mono-CBZ pi in 25 mL of THF A solution of perazine followed by 1.22 mL (887 mg, 8.75 mmol) of Et_ThreeN was added to the mixture. The mixture was stirred at room temperature for 7 hours and filtered. The filtrate is concentrated and the residue is Dissolve in tOAc and add 2 x 125 mL of 1N HCL, 125 mL of saturated NaHCO_ThreeShake with solution And dry (MgSO 4_FourFiltration and concentration) gave 2.39 g of a sticky solid Was. Silica gel chromatography (95/5 CH_TwoCl_Two/ MeOH) to give 1.85 g (66 %)of56I gotCompound 57 ： 10mL CH_TwoCl_Two1.74 g (2.74 mmol) of the compound in5610 mL of trif to a solution of Add the fluoroacetic acid, andThatThe mixture was stirred at room temperature for 3 hours. Thicken the mixture And compress the residue with 5 mL CH_TwoCl_TwoDissolved into. The mixture is cooled to 0 ° C and 100 mL of saturated NaHCO_ThreeWas added. Next, the mixture was divided into four 100 mL portions of CH._TwoCl_TwoExtract with did. CH_TwoCl_TwoThe layers are combined, dried (MgSO_Four), Filtered and concentrated to a sticky hygroscopic 1.46 g (99%) as a functional solid57I got This was used directly in the next step.Compound 58 : 0.7 g (1.3 mmol) of compound at 0 ° C57Solution and 226uL (168mg, 1.30mm ol) to 4 mL of CH to diisopropylethylamine_TwoCl_Two127uL of triethylene glycol in Add biscmoroforinate and bring the mixture to room temperature For 3 hours. Mixture with 80 mL CH_TwoCl_TwoAnd 80 mL of 1N HCl.CH ₂ Cl ₂ The layer was washed with two 80 mL portions of water, dried (MgSO_Four), Filtered and concentrated 736 mg (93%) of compound as a crystalline solid upon shrinkage58I gotCompound 60 :Compound58(61 mg, 0.48 mmol) was dissolved in 3 mL of 30% HBr / HOAc, and The resulting mixture was stirred at room temperature for 1 hour, during which time 5 mL of Et_TwoO was added. mixture The material was placed in the freezer for 1 hour and centrifuged. The resulting pellet is added to Et_TwoUse O Wash, and dry, tetralaydrobromide salt59 I got 1 mL of H_TwoDissolved in O. 49 mg (0.58 mmol) of NaHCO to this mixture_Three And 3 mL of dioxane are added. If necessary, additional NaHCO_ThreeAdd and mix Make things basic. The mixture was cooled to 0 ° C. and 748 mg (2.89 mmol) of anhydrous Add lomoacetic acid. The mixture is stirred for 2 hours and 20 mL of 1N H_TwoSO_FourAnd 20mL of 80/20 C H_TwoCl_TwoPartition between / MeOH. Dry the organic layer (Na_TwoSO_Four), Filter and concentrate Crude60Get. This is subjected to silica gel chromatography (CH_TwoCl_Two/ MeOH) Made60Get. Example 25:Synthesis of tetravalent platform tetrakis-BAIPIZ / PMA, 63 Compound 61 : 640 mg (2.52 mmol) pyromellitic acid and 1.16 g (10.1 mmol) in 50 mL THF ) To a 0 ° C solution of N-hydroxysuccinimide in an amount of 2.6 g (12.6 mmol) of dicyclohexene. Add silcarbodiimide and allow the mixture to stir for 16 hours to room temperature. Warmed up. A solution of 2.5 g (11.3 mmol) of mono-CBZ-piperazine in 25 mL of THF was added to the mixture. And then 1.4 mL (1.02 g, 10.1 mmol) of Et_ThreeN was added. The mixture is filtered and The filtrate was concentrated. The residue was partitioned between 100 mL of EtOAc and 2 × 100 mL of 1N HCl, and The EtOAc layer to 100 mL of saturated NaHCO_Three, 100 mL H_TwoO, 100 mL of saturated NaHCO_ThreeWash with Clean, dry (MgSO 4_Four), Filtered and concentrated. Purification by silica gel chromatography Graphic (97.5 / 2.5 CH_TwoCl_Two/ MeOH) and 1.78 g as a white crystalline solid ( 66%)63I gotCompound 63 :Compound61Is the one described for the conversion of 58 to 60 in Example 23 Compounds essentially the same way63Is converted to Purification by silica gel chromatography Achieved using luffy. Example 26:Synthesis of tetravalent platform BA / PIZ / IDA / HB / TEG, 68 Compound 64 : 725 mg (4.36 mg) of 2-tosylated triethylene glycol (1.0 g, 2.18 mmol) mmol) of ethyl 4-hydroxybenzoate and 723 mg (5.23 mmol) of K_TwoCO_ThreeAdd to the solution Was added and the mixture was refluxed for 16 hours. Concentrate the mixture and remove the residue with 20 mL of water And 3 x 20 mL Et_TwoPartitioned with O. Combine the organic layers with 2 x 40 mL of saturated NaHCO_Threesolution Washed with 40 mL (in L) of saturated NaCl solution. Et water layer_TwoWash with O Et_TwoThe O layer is dried (MgSO_Four), Filtered and concentrated. Silica gel chromatography Purification by chromatography (70/30 hexane / EtOAc) gave 902 mg as a crystalline solid ( 93%)64Have.Compound 65 :Compound64Was dissolved in acetone containing 2.2 equivalents of LiOH, and mixed. Stir the material for 3 hours (until completely dissolved as evidenced by TLC). mixture The product was acidified with acetic acid and concentrated, and the residue was chromatographed on silica gel. Purified by65Get.Compound 66 :Compound66With the compound in Example 2356Prepared in the same manner as in the preparation of Compound65Is used in place of N-BOC-iminodiacetic acid, and the compound57The mono-CBZ-P Use in place of perazine. Purification achieved using silica gel chromatography You.Compound 68 :Compound66Of Example 2358From60Those who described conversion to Compounds essentially the same way68Convert to Purification by silica gel chromatography Achieved using fi Example 27:Synthesis of tetravalent platform BA / PIZ / HIP / TEG, 72 Compound 69 :Compound53Is the same as in Example 25 except that 4.4 equivalents of LiOH are used.64of Hydrolysis using LiOH essentially in the same way as described for hydrolysis Understand.Compound 70 : Tetra-acid, compound69To69Is used in place of pyromellitic acid Except that from pyromellitic acid in Example 2461Those who described conversion to Compounds essentially the same way70Convert toCompound 72 :Compound70In Example 2358From60Those who described conversion to Compounds essentially the same way72Convert to Purification by silica gel chromatography Achieved using fiExample 28:Synthesis of conjugates of haloacetylated tetravalent compounds (LJP685) ₄ / MTU / A / DABA / ATEG Synthesis of binding compound 73 :   21 mg (15.5 umol) of compound in 200 mM borate buffer pH 8.5 sprayed with heliumtwenty fourso A solution was prepared. To the solution, 3.25 mg (2.6 umol) of IA / DABA / dissolved in 396 uL of MeOH A second solution consisting of ATEG, compound 46 was added. A precipitate forms and 1 mL of M eOH was added to all solids and dissolved. The mixture is stirred at room temperature for 18 hours and then 9/1 H_TwoO / HOAc was added. Mixture 50ML H_TwoO / CH_ThreeDilute with CN, HPLC Loaded on the system. Purification was performed by preparative HPLC (25 cm x 2.24 mm C₁₈, Gradient: 35% B to 45 0-40 minutes to% B, A = H_TwoO / 0.1% TFA B = CH_Three(CN / 0.1% TFA, 12mL / min) 10.8 mg (67%) of the compound as a solid after lyophilization73I got (LJP685) ₄ / MTU Synthesis of conjugates, compounds 74, 75, 76, 77, and 78:   These conjugates are used as platform compounds46The right platform for Conjugate by substitution with compound73To the same reaction conditions as above for the synthesis of Each platform compound51,60,63,68,and72Prepared from Pep can be LJP685 or other related peptides.Example 29:Conjugate tetravalent platform 55, binding compound 79, (LJP685) ₄ / MP / TEG Success :   4 equivalents of compound in DMFtwenty fourAnd 5 equivalents of Cs_TwoC_ThreePrepare a solution of Platform Compound551 equivalent of BMP / TEG is added to the mixture and then stirred for 1 hour . Mix 80/20/10 H_TwoO / CH_ThreeDiluted with CN / HOAc and loaded onto a preparative HPLC column . Purification was performed by preparative HPLC (C₁₈, A = H_TwoO / 0.1% TFA, B = CH_Three(CN / 0.1% TFA) Compound after lyophilization79Is generated. Pep is LJP685 or other related peptides possible.Example 30:Conjugate tetravalent platform Tetrakis-BMB, binding compound 80, (LJP685 ₄ ) Synthesis of MTU / Tetrakis-MB :   4 equivalents of compound 24 and 5 equivalents of Cs in DMF_TwoC_ThreePrepare the solution with. 1 equivalent of te Trakisbromomethylbenzene is added to the mixture, and the mixture is then Stir. Mix 80/20/10 H_TwoO / CH_ThreeDilute with CN / HOAc and load onto a preparative HPLC column. Loaded. Purification was performed by preparative HPLC (C₁₈, A = H_TwoO / 0.1% TFA, B = CH_Three(CN / 0.1% TFA) Achieved by lyophilization of the compound80Get. Pep is LJP685 or other related pep It can be a tide.Example 30:FITC-GPCILLARDRCG (CB2 ^* ) Fluorescence Polarized Peptide Binding Assay Synthesis   20 mg of sodium carbonate (Na_TwoCO_Three, PH about 10.5) in 20 mL of ACN / water (1: 1) Disulfide peptide GPCILLARDRCG (20.0 mg, 14.4 μmol) and fluorescein A solution of isothiocyanate (FITC) (5.6 mg, 14.4 μmol) was stirred at room temperature . The reaction was monitored by analytical HPLC. After consuming the fluorescent labeling reagent, the crude Preparative H eluting the quality with a linear gradient from 30% to 55% of B over 40 min at 10 mL / min Purified by PLC. Where A is H_Two0.1% (v / v) TFA in O and B is 0.085% (in CAN) v / v) TFA. FITC peptide was obtained as a light yellow powder after lyophilization (3.7 mg, 15% generation): MS (ESI): m / e (M + 1) calculated value C₇₃H₁₀₂N₁₉O_{twenty one}S_Three: 1661, observation Value: 1661.Direct binding fluorescence polarization binding assay (dbFP)   The methodology is described in the Pan Vera Application Guide (1994), Pan Vera Corporation. It is. Briefly, trace amounts of fluorescein isothiocyanate (FITC) -labeled peptides C (CB2^*-F) with an antibody (ACA 6501 or 6701) and attach Are plotted against antibody concentration. Polarized light, Pan Vera Beacon equipment Was measured. The data was fitted to equation 1.Here, Y is a Y-axis value (millipolarization unit, mP), and R is a total concentration of the antibody receptor. Yes, P_LIs the polarization for free FITC-labeled peptide (F), and P_HIs connected to R This is the polarization for the combined F (FR). K_DIs the dissociation for F from the FR conjugate It is a constant (mutual coupling constant). For these expressions that should be legal, F << R That must be accurate. CB2^*-F binding to ACA-6701 and ACA-6501 antibodies The titrations are shown in FIGS. 22 and 23, respectively. As shown in Figure 23 In FIG. 24, complete titration was not obtained using ACA-6501. Above titration is 241nM K_Dshowed that. 6701 to CB2^*Replace -F (See Figure 25), a slight excess of CB2 in antibody 6701^*(GPCILLARDRCG ) By adding CB2^*About -F, K_off= 0.0184 seconds^-1Dissociation rate constant (t_1/2 = 38 seconds). 256nM K_DAssuming that this is K_on= 3.6 × 10^Four M^-1Second^-1(After correction for antibody bivalentity). Follow CB2 binding to ACA-6701^*-F states that these two molecules diffuse together. It is limited to only those who do.Competitive fluorescence polarization assay (cFP)   The dbFP assay described above provides the binding constant of the FITC-labeled peptide and 0.5 mg Requires an order of purified antibody. The cFP assay binds peptides lacking the FITC group. Provides a combined constant, which consumes less antibody, on the order of 10 μg I do. The cFP assay is a Pan-like method as it consumes 50 times less antibody. Vera Applications Guide (1994) Assay reported in Pan Vera Corporation Modified from b. Briefly, the antibody (ACA 6701) was converted to a trace amount of FITC-labeled peptide (CB2^*- Mix with F) and leave for sufficient time until equilibrium is achieved. This is ACA 67 01 and CB2^*It was one hour for -F. Then, increasing concentrations of the unlabeled to be tested Peptide (CB2^*Or 3B10) was added to the tube. After each addition, equilibrium is achieved It is necessary to select the concentration of (F) so that F << R. The measured polarization (P_H) Is P_LEnough to be significantly higher (see Figure 22). It only has to be high. This Δ (mP) value is important to ensure reliable results , Should have an mP value of 20 or more.                       Equation 2 Δ (mP) = P_H-P_I Unlabeled (no FITC) peptide (I) is added to prevent F from binding to R So Y is its maximum value P_HFrom the plateau P that should satisfy equation 1._LTo To decrease. CB2^*CB2 from ACA-6701, according to 3B10^*This about -F substitution These titrations are shown in FIGS. 26 and 27, respectively. This tie tray An equation was obtained to explain It is: Where P_LIs the same as in Equation 1, where I is the concentration of unlabeled peptide competitor and Then K_I'Is the apparent dissociation constant of the peptide. Change these parameter values to c FP titration data obtained by fitting to the above equation Was.   The true dissociation constant of I is obtained from Equation 4.                       Equation 4 K_I= K_I'(1.0 + R / K_D) Where R and K_DIs defined by equation 1. R / K_DThe ratio is P_H'(From equation 3) And P_HAnd P_LBy using the value of (from equation 1) and equation 5, hand can get.                       Equation 5 R / K_D= (P_H'-P_L) / (P_H-P_H') Generally, the titration once defined in Equation 1 is performed as a “standard curve” If so, Equation 5 can be used to determine aPL antibody concentration. Therefore, K_I In addition to providing a method for determining Standardize all aPL antibody stock solution concentrations using only 5-10 μg of antibody And means for analyzing their binding activity / stability over time I do.dbFP Dissociation constant determined by cFP and Peptide antibody sequence KD ^a or K _I       CB2^*-F 6501 FITC-GPCILLARDRCG 482nM^a       CB2^*-F 6701 FITC-GPCILLARDRCG 512nM^a        CB2^*      6701 GPCILLARDRCG 35nM3B10 6701 AGPCLLLAPDRCPG 313nM ^a K determined from Equation 1 without considering the bivalent nature of the antibody_DK to modify the value_D The value was multiplied by two. These results indicate that CB2^*-F has two very different aPL antibodies (ACA-6501 and ACA-67 01) and demonstrate that they bind with high affinity equal to both. FITC based Removal of CB2 to ACA-6701^*Was improved 14 times. Binding of related peptide 3B10 Is CB2^*Of the binding to ACA-6701. This result Can be attributed to additional backbone residues on 3B10, but this result also indicates that CB2^*8 in Arginine at proline substitution. 5A12, a peptide similar to 3B10 Earlier NMR structural studies showed that this alternative proline provided structural rigidity. Was shown. CB2 is a much more flexible peptide and this position With arginine. More flexible peptides such as CB2 are more cross-reactive so is there. Because it is more adaptable to its shape to fit a given antibody binding site This is because it can be easily adjusted. Drug rigidification for binding affinity on) suggests Koehler et al., p. 251, GUIDEBOOK ON MOLECULER MODELING IN DRUG DES. It is discussed in IGN (Academic Press, edited by N. Cohen, 1996). Example 31:Peptide conjugate tolerant activity   Two different conjugates containing the same peptide induce antigen-specific tolerance in vivo The ability to conduct was tested. Briefly, mice are labeled with peptide-specific Immunogenic carrier keyhole limpet hemocyani to produce B cells (KLH). Three weeks later, five mice per group Groups of mice are treated with different doses of the test conjugate and one group Mice were not treated and served as controls. 5 days later, including control group All mice were boosted with KLH-conjugated peptide and 7 days later, all mice were Blood and collect their sera using a modified Farr assay Assays were made for antibodies. Antigen binding capacity (ABC) was determined by G.M. Iverson, "Assay f or in vivo adoptive immue response, '' Volume 2, Chapter 67, HANDBOOK OF EXPERIMEN TAL IMMUNOLOGY (Blackwell Scientific Publications, edited by Weir et al., 4th edition, Oxfor d, 1986), calculated for each individual serum sample. Next Use these values to calculate the mean and standard deviation for all individuals in the group. The difference was determined.   One of the conjugates induced tolerance, while the other conjugate Did not inhibit the anti-peptide antibody over a period of time. Most likely about this difference One explanation is that the latter conjugate has a short in vivo half-life. this Inducing tolerance in vitro to address the problem, thereby considering half-life Was used, and then the cells were transferred to the irradiated recipient. Easily Collecting spleen cells from mice primed with the peptide bound to KLH, Then, in complete RPMI-1640 medium at 37 ° C for 2 hours with various doses of test conjugate. Incubated. One group of cells is injected without toleragen Incubated and served as a positive control. Wash cells and irradiate Shi Transferred to Pients and boosted with peptide bound to KLH. 7 days later, everything Blood from all mice and assay their sera for anti-protein antibodies. I did. Did not induce any detectable tolerance when tested in an in vivo model The conjugate induced tolerance when tested in this in vitro model. As a result Support the speculation that differences between conjugates are due to the short half-life of the conjugate. This temporary The conjugate is implanted with an osmotic pump to directly test the It was administered continuously during the period. These results clearly demonstrate this binding when injected with sustained release. The body induces tolerance and when injected in a bolus, as shown in Figure 32. Indicates that there was no.Study of (LJP685) ₄ / MTU-AHAB-TEG for tolerant activity in an in vivo model   Mice were treated with alum + pertussis LJP-685-KLH as adjuvant The first antigen was stimulated. Three weeks later, mice are dosed with a range of doses (LJP685)_Four/ MTU-AHAB Treated with -TEG conjugate. One group is untreated and controls Group. 5 days later, 10 μg of all mice including the control group Were boosted with LJP685-KLH and mice were bled 7 days later. Those sera Was analyzed for anti-LJP685 antibody by the modified Farr assay described above. Figure The results shown in 28 span the dose range of 1-50 nmole (LJP685)_Four/ MTU-AHAB-TEG coupling Indicates that treatment with the body had no detectable effect on the anti-LJP685 response .Testing of (LJP685) ₄ / MTU-DABA-TEG conjugate for tolerance induction in an in vivo model   Mice were primed with alum + pertussis LJP685-KLH. Three weeks later, Mice were treated with 5 nmol, 10 nmol, or 50 nmol of (LJP685)_Four/ MTU-DABA-TEG conjugate Was placed. One group was untreated and served as a control group. 5th After that, all mice including the control group were additionally exempted with 10 μg of LJP685-KLH. Mice were bled and seven days later, mice were bled. These sera were used to modify the modified Farr Analysis for anti-LJP685 antibody by Say. The results shown in FIG. 29 are (LJP685)_Four/ MTU-DABA-TEG conjugate is 5 nmol ED₅₀Induces tolerance in an in vivo model using Indicate thatTrial of (LJP685) ₄ / MTU-AHAB-TEG conjugate for tolerance induction in in vitro model Trial   Three weeks ago, spleen cells from mice primed with LJP685-KLH were harvested and 4 μM, 20 μM, or 100 μM (LJP) in whole RPMI-1640 medium at 37 ° C. for 2 hours. 685)_FourIncubation was performed using the / MTU-AHAB-TEG conjugate. One group of cells Were incubated without tolerogens and served as a positive control group. Wash cells, transfer to irradiated recipients and supplement with 10 μg LJP685-K-LH Quarreled. Seven days later, mice were bled and their sera were subjected to a modified Farr assay. Was analyzed for anti-LJP685 antibody. The results shown in FIG. 30 clearly show (LJP68 Five)_Four/ MTU-AHAB-TEG conjugate has an IC of less than 4 μM when tested in an in vitro model₅₀ Show that tolerance can be induced to achieveTrial of (LJP685) ₄ / MTU-DABA-TEG conjugate for tolerance induction in in vitro model Trial   Three weeks ago, spleen cells from mice primed with LJP685-KLH were harvested and In total RPMI-1640 medium, 0.4 μM, 1.3 μM, or 4 μM (LJ P685)_FourIncubation was performed using the / MTU-DABA-TEG conjugate. One glue of cells Were incubated without tolerance and served as a positive control group . Wash cells, transfer to irradiated recipients, and use 10 μg LJP685-KLH Boosted. Seven days later, mice were bled and their sera were modified with modified Fa Analyzed for anti-LJP685 antibody by rr assay. The results shown in FIG. 31 are (LJP68 Five)_Four/ MTU-DABA-TEG conjugate has an IC of less than 4 μM when tested in an in vitro model₅₀ Show that tolerance can be induced to achieveFor in vivo tolerance induction using a continuous delivery pump (LJP685) ₄ / MTU- Testing AHAB-TEG conjugate   Mice were primed with alum + pertussis LJP-685-KLH. 3 weeks later , Mice were divided into 5 groups, 5 mice per group. Day 1, 1 One One group was treated with a bolus of saline and another group was treated with 50 nmol (LJP685)._Four Treated with a bolus of the / MTU-AHAB-TEG conjugate. Osmotic pressures for the remaining three groups Implanted. In one group, the pump is filled with saline and Delivered at μL / hr for 3 days. The other two groups are (LJP685)_Four/ MTU-AHAB-TE A pump filled with G conjugate (50 nmol) was installed. 1 μL / hr for one group Pump for 3 days between delivery, and on the other hand 0.5 μL / hour for 7 days Reaching pump was installed. On day 5, surgically remove pump for 3 days Was. On day 7, all mice, including the control group, were treated with 10 μg of LJP685-K Boosted with LH. On day 10, the pump delivering for 7 days was surgically removed. 14 On the day, all mice were bled. These sera were used in a modified Farr assay. Thus, the anti-LJP685 antibody was analyzed. The results are shown in FIG.(LJP-peptide) ₄ / MTU-BMP-TEG conjugate for tolerance induction in an in vivo model Exam   Mice were primed with alum plus pertussis peptide-KLH. 3 weeks later Mouse (LJP-peptide)_FourTreated with / MTU-BMP-TEG conjugate. One group is No treatment and a control group. 5 days later, control group Were boosted with 10 μg of peptide-KLH, and 7 days later, Blood was collected from the mouse. The sera were subjected to anti-peptide anti-peptide by a modified Farr assay. The body was analyzed. The results show that (LJP-peptide) 4 / MTU-BMP-TEG conjugate Tolerance in Bomodel, (LJP685)_Four/ MTU-AHAB-TEG conjugate To indicate that

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 38/00 A61K 39/00 H 39/00 C07K 16/44 C07K 16/44 C12Q 1/70 C12Q 1 / 70 G01N 33/53 S G01N 33/53 33/531 A 33/531 A61K 37/02 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE) , LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG, B , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM , TR, TT, UA, UG, US, UZ, VN, YU (72) Inventor Johns, David S. United States of America 92127, San Diego, Florindo Road 11265 (72) Inventor You, Lin United States of America 92122, San Diego , Lombard Place 8933, Apartment 228

Claims (1)

[Claims] 1. An aPL analog that specifically binds to B cells to which the aPL epitope binds. 2. 2. The analog of claim 1, wherein said analog lacks a T cell epitope. 3. The analog of claim 1, wherein said analog is a peptide. 4. The peptide is CLILAPDRC, CLILTPDRC, CLLLAPDRC, CTILTLDRC, CLVLALDR C, CTILTPDRC, CILLAHDRC, CGNAADARC, CTNWADPRC, CGNIADPRC, CTNLTDSRC, CGN PTDVRC, GILLNEFA, GILTIDNL GILNALDYV, LSDPGYVRNIFH, or LTDPRYTRDISNFT 4. The analog of claim 3, comprising the sequence of D. 5. The peptide is AGPCLGVLGKLCPG, GPCLGVLGKLCPG, PCLGVLGKLCPG, CLGVLGK Sequence of LCPG, AGPCLGVLGKLCG, CLGVLGKLC, GPCILLARDRCG, or AGPILLARDRCPG The analog of claim 3, comprising: 6. The peptide comprises at least one proline, and further at least 4. An analogue according to claim 3, wherein one of said prolines has been replaced by α-methylproline. . 7. 4. The method of claim 3, wherein at least one L amino acid has been replaced with a D amino acid. analog. 8. 4. The method according to claim 3, wherein the peptide is cyclized by a disulfide bond. Nalog. 9. 9. The method according to claim 8, wherein the disulfide bond is replaced with a thioether bond. analog. 10. The peptide comprises at least one leucine, and at least 4. An analogue according to claim 3, wherein also one of said leucines has been replaced by isoleucine. 11. Conjugates of non-immunogenic valence platform molecules and (a) aPL immunogen Specifically binds to matching B cells and (b) deletes the T cell epitope of the immunogen Induces specific B cell tolerance to aPL immunogens, including aPL antibody-binding analogs Composition for 12. The aPL antibody-binding analog is CLILAPDRC, CLILTPDRC, CLLLAPDRC, CTILTL DRC, CLVLALDRC, CTILTPDRC, CILLAHDRC, CGNAADARC, CTNWADPRC, CGNIADPRC, C TNLTDSRC, CGNPTDVRC, GILLNEFA, GILTIDNL, GILNALDYV, LSDPGYVRNIFH, or The composition according to claim 11, which is a peptide comprising the sequence of LTDPRYTRDISNFTD. 13. The aPL antibody-binding analog is AGPCLGVLGKLCPG, GPCLGVLGKLCPG, PCLGVLGK LCPG, CLGVLGKLCPG, AGPCLGVLGKLCG, CLGVLGKLC, GPCILLARDRCG, or AGPILLA The composition according to claim 11, which is a peptide comprising the sequence of RDRCPG. 14． 7. The aPL antibody binding analog is the analog of claim 6. 12. The composition according to 11. 15. 9. The aPL antibody binding analog is the analog of claim 7. 12. The composition according to 11. 16. 9. The aPL antibody binding analog is the analog of claim 8. 12. The composition according to 11. 17． 10. The aPL antibody binding analog is the analog of claim 9. 12. The composition according to 11. 18. The aPL antibody binding analog is the analog of claim 10. Item 12. The composition according to Item 11. 19. The non-immunogenic valency platform molecule comprises triethylene glycol. The composition according to claim 11. 20. 20. The set of claim 19, wherein said valency platform molecule comprises AHAB-TEG. Adult. 21. The valence platform molecule comprises compound 46, A-DABA-ATEG. 20. The composition according to 19. 22. Wherein the valence platform molecule comprises Compound 51, A-PABA-DT-TEG. Item 20. The composition according to Item 19, 23. 20. The method of claim 19, wherein the valence platform molecule comprises Compound 55, MP-TEG. A composition as described. 24. Wherein the valence platform molecule comprises Compound 60, A-PIZ-IDA-TEG. Item 20. The composition according to Item 19, 25. Wherein the valence platform molecule comprises Compound 68, A-PIZ-IDA-HB-TEG; 20. The composition according to claim 19. 26. The valency platform molecule comprises Compound 72, A-PIZ-MP-TEG. 20. The composition according to 19. 27. The non-immunogenic valency platform molecule comprises polyethylene glycol. The composition according to claim 11. 28. 29. The set of claim 28, wherein said valency platform molecule comprises DABA-PEG. Adult. 29. The non-immunogenic valence platform molecule comprises tetraaminobenzene The composition of claim 11. 30. The non-immunogenic valency platform molecule is heptaamino beta cyclodextrin 12. The composition of claim 11, comprising Trin. 31. The non-immunogenic valency platform molecule is tetraaminopentaerythritol The composition of claim 11, comprising toll. 32. The non-immunogenic valency platform molecule is 1,4,8,11-tetraazacyclo The composition of claim 11, comprising tetradecane (Cyclam). 33. The non-immunogenic valence platform molecule is 1,4,7,10-tetraazacyclo 12. The composition of claim 11, comprising dodecane (Cyclen). 34. The non-immunogenic valence platform molecule is Compound 63, Tetrakis-A-PIZ The composition of claim 11, comprising -PMA. 35. The non-immunogenic valency platform molecule comprises Compound 55, MP-TEG. 12. The composition according to claim 11. 36. The composition of claim 11, wherein the conjugate is derived from Tetrakis-BMB. 37. Non-immunogenic valency platform molecules including AHAB-TEG. 38. Non-immunogenic valency platform molecules including Compound 46, IA-DABA-ATEG. 39. Non-immunogenic valency platform molecules including Compound 51, BA-PABA-DT-TEG. 40. Compound 55, a non-immunogenic valency platform molecule including BMP-TEG. 41. Non-immunogenic valency platform molecules including Compound 60, BA-PIZ-IDA-TEG. 42. Compound 68, non-immunogenic valence platform containing BA-PIZ-IDA-HB-TEG Child. 43. Non-immunogenic valency platform molecules including Compound 72, BA-PIZ-HIP-TEG. 44. Non-immunogenic valence platform containing compound 63, tetrakis-BA-PIZ-PMA Molecule. 45. Administering an effective amount of the composition of claim 11 to an individual in need thereof. A method for treating an individual suffering from an aPL antibody-mediated disease, comprising: 46. 46. The method of claim 45, wherein said aPL antibody-mediated disease is seizure. 47. 46. The method of claim 45, wherein said aPL antibody-mediated disease is fetal death. 48. The method according to claim 45, wherein the aPL antibody-mediated disease is anti-phospholipid antibody syndrome (APS). The described method. 49. Wherein the aPL antibody-mediated disease is primary anti-phospholipid antibody syndrome (PAPS), Item 46. The method according to Item 45. 50. 46. The method of claim 45, wherein said aPL antibody-mediated disease is thrombosis. 51. An antibody that specifically binds to an aPL antibody isolated from a human suffering from an aPL antibody-mediated disease A method for identifying pitope analogs, comprising:   (a) preparing a phage random peptide library;   (b) screening the library with aPL antibodies to identify aPL mimetic epitopes Wherein the screening step includes the following steps: Steps to do:     (i) screening the library by biological selection;     (Ii) Phage isolated by biological selection in (i) for microselection Thus further screening; and     (iii) phage containing the aPL antibody high affinity binding peptide recovered in (ii) Identifying by an assay, How to include 52. Biological selection of phage random peptide library and binding to aPL antibody A method for identifying and isolating a peptide comprising:   (A) affinity-purified aPL antibody with random peptide insert Reacting with phage;   (b) recovering phage having a random peptide insert that binds to the aPL antibody Performing the step;   (c) infecting the microorganism with the phage recovered in (b); and   (d) culturing the infected microorganism in a medium containing an antibiotic to isolate the phage Process, A method comprising: 53. Micro-selection of phage random peptide library, high aPL antibody A method for identifying and isolating a peptide having a high binding affinity, comprising:   (a) Isolate phage with random peptide insert by biological selection Process;   (b) coating the phage recovered in step (a) with an aPL antibody bound to protein G; Incubating in the prepared microplate wells;   (c) washing the microplate wells to remove unbound phage;   (d) eluting the bound phage; and   (e) infecting the microorganism with the phage recovered in (d); and   (f) culturing the infected microorganism in a medium containing an antibiotic and isolating the phage , A method comprising: 54. 52. The method of claim 51, wherein the immunoassay is a phage capture ELISA. The law:   (a) In microplate wells coated with aPL antibody, by microselection Incubate phage with isolated random peptide insert Process;   (b) washing away unbound phage;   (c) incubating a labeled anti-phage antibody against the well;   (d) washing away unbound labeled anti-phage antibody;   (e) adding a labeled substrate; and   (f) measuring the signal generation of the substrate to identify high affinity binding phage, A method comprising: 55. 55. The method of claim 54, wherein said label is an enzyme. 56. 55. The method of claim 54, wherein said substrate is colorimetric. 57. Further performing a phage capture ELISA assay for high affinity binding phage 55. The method of claim 54, further comprising:   (a) coating a uniform amount of phage on the microplate well;   (b) incubating the aPL antibody in the well,   (c) washing away unbound antibody,   (e) incubating a labeled anti-aPL antibody with the conjugated aPL antibody;   (f) washing away unbound labeled anti-aPL antibody;   (g) adding a substrate to the well; and   (h) measuring the signal generation of the substrate and measuring the relative binding affinity of the phage Process, A method comprising: 58. 58. The method of claim 57, wherein said label is an enzyme. 59. 58. The method of claim 57, wherein said substrate is colorimetric. 60. 52. The immunoassay is a colony blot immunoassay. The method described in:   (a) chilling microorganisms infected with phage having random peptide inserts Culturing on a membrane on a heaven-containing culture medium;   (b) applying the microorganism cultured in (a) to a membrane on an agar-containing culture medium; Replication transcription by blotting on;   (c) incubating the transcribed microorganism;   (d) lysing the microorganism;   (e) digesting the microorganism;   (f) blocking the membrane;   (g) incubating the membrane with an aPL antibody;   (h) washing away unbound aPL antibody;   (i) incubating a labeled anti-aPL antibody with the membrane;   (j) washing away unbound labeled anti-aPL antibody;   (k) adding a substrate; and   (l) measuring the signal generation of the substrate, and identifying a high affinity binding phage, A method comprising: 61. 61. The method of claim 60, wherein said membrane is nitrocellulose. 62. 61. The method of claim 60, wherein said microorganism is digested with lysozyme. 63. 61. The method of claim 60, wherein said blocking solution is gelatin. 64. 61. The method of claim 60, wherein said label is an enzyme. 65. 61. The method of claim 60, wherein said substrate is colorimetric. 66. A parent that specifically binds to an aPL antibody isolated from a human suffering from an aPL antibody-mediated disease Methods for assaying and ranking compatible binding property epitopes are also included. Yes,   (a) Cover the wells of the microtitration plate with cardiolipin Process;   (b) non-specific binding to the cardiolipin and to the wells of the plate Mature bovine or human serum as a source of β2-GPI to prevent Step;   (c) Incubate a solution of the monomeric analog and the high titer aPL antibody for a predetermined time. Step;   (d) transferring the aPL antibody / analog mixture to the microtitration plate And incubating for a predetermined time;   (e) washing the wells to wash away unbound aPL antibodies;   (f) adding anti-human IgG conjugated to the label to the wells of the plate, and Incubating for a while;   (g) washing the wells to remove unbound anti-human IgG conjugate;   (h) adding a substrate for the labeled conjugate and performing a substrate / labeling reaction for a predetermined time;   (i) measuring the final product of the substrate / labeling reaction and determining the amount of aPL antibody bound to the well; Quantifying;   (j) calculating the percentage of inhibition of binding of the aPL antibody, if any, determining the affinity for the aPL antibody, A method comprising: 67. 67. The method of claim 66, wherein the conjugate is labeled with an enzyme. 68. 67. The method of claim 66, wherein said substrate is colorimetric. 69. a in a body fluid collected from a subject suspected of having a PL antibody-mediated disease A diagnostic immunoassay for determining the presence of a PL antibody,   (a) Contacting a sample of body fluid with an analog of the epitope that specifically binds to the aPL antibody The step of causing;   (b) detecting the aPL antibody bound to the analog, A diagnostic immunoassay, comprising: 70. An immunoassay according to claim 69,   (a) Bind wells of microtitration plate specifically to aPL antibody Coating with an analog of the epitope;   (b) washing the wells to wash away unbound analog;   (c) adding a test sample of body fluid to the well and incubating for a predetermined time; About;   (d) washing the well to remove unbound test sample;   (e) adding anti-human IgG conjugated to the label to the wells of the plate, and Incubating for a while;   (f) washing the wells to remove unbound anti-human IgG conjugate;   (g) adding the substrate of the labeled conjugate, and performing the substrate / labeling reaction for a predetermined time; Step;   (h) measuring the final product of the substrate / labeling reaction and detecting the anti-aPL antibody in the test sample. The process of determining existence, An immunoassay, comprising: 71. 71. The method of claim 70, wherein the label is an enzyme and the substrate is colorimetric. Immunoassay described. 72. Formulating a peptide or other bioactive molecule   R¹S (CH_TwoCH_TwoO)_nCH_TwoCH_TwoO (CH_Two)_mCO_TwoR^Two A hydrophilic linker for attaching to the valency platform molecule using n = 0 to 200, m = 0 to 10, R¹= Protecting groups such as H or trityl, R^Two= H Or alkyl or aryl such as 4-nitrophenyl ester. 73. 73. The linker of claim 72, wherein m = 0-2. 74. The aPL analog comprises a non-immunogenic valency platform molecule and a sulfhydride. 12. The conjugate of claim 11, wherein the conjugate is linked by a ril-containing moiety.