APL immunoreactive peptides, conjugates thereof and methods of treatment for APL antibody-mediated pathologies

TECHNICAL FIELD
This invention is in the field of immunology and relates to compositions and methods for treating and diagnosing antiphospholipid (aPL) antibody-mediated pathologies. More specifically, the invention relates to diagnostic assays for quantitating the level of antiphospholipid antibodies in a biological sample. In addition, the invention relates to conjugates of chemically-defined nonimmunogenic valency platform molecules and immunospecific analogs of aPL-binding epitopes, said analogs lacking T cell epitopes. The invention also relates to a method of utilizing random peptide libraries to identify immunospecific analogs of aPL-binding epitopes.
BACKGROUND OF THE INVENTION
Antiphospholipid antibodies occur in autoimmune diseases such as systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome (APS) as well as in association with infections and drug therapy. APS is characterized by one or more clinical features such as arterial or venous thrombosis, thrombocytopenia and fetal loss. APS may be primary or it may be associated with other conditions, primarily SLE. (PHOSPHOLIPID-BINDING ANTIBODIES (Harris et al., eds., CRC Press, Boca Raton, Fla., 1991) ; McNeil et al. ADVANCES IN IMMUNOLOGY, Vol. 49, pp. 193-281 (Austen et al., eds., Academic Press, San Diego, Calif., 1991)). Approximately 30-40% of patients with SLE have aPL, however, 50% of patients with aPL antibodies do not have SLE. This 50% may have other autoimmune rheumatic diseases, miscellaneous conditions or they may have been subjected to drug therapy, particularly chlorpromazine. In one study of 70 patients, 26 males and 44 females, with primary APS (PAPS) but no evidence of SLE, the following features were observed: deep venous thrombosis (DVT) in 31; arterial occlusion in 31, particularly stroke or transient ischemia; myocardial infarctions in 15; recurrent fetal loss in 24; thrombocytopenia (TCP) in 32; 10 had a positive Coombs' test; Evans' syndrome in 7; anti-nuclear antibody (ANA) in 32, but less than 1:160 in 29; and antimitochondrial antibody (AMA) in approximately 24. (McNeil et al., supra.). Estimates vary but in about 5% of all stroke patients, aPL antibodies are thought to be an important contributing factor.
Transient aPL antibodies, such as those detected in a VDRL test, occur during many infections. Approximately 30% of patients possessing persistent aPL antibodies have suffered thrombic event. The presence of aPL antibodies defines a group of patients within SLE who display a syndrome of clinical features consisting of one or more of thrombosis, TCP, and fetal loss. The risk of this syndrome in SLE overall is around 25%; this risk increases to 40% in the presence of aPL antibodies and decreases to 15% in their absence. Because aPL antibodies appear to be directed at phospholipids in plasma membranes, it has been postulated that they may exert direct pathogenic effects in vivo by interfering with hemostatic processes that take place on the phospholipid membranes of cells such as platelets or endothelium. In patients with PAPS, the fact that aPL antibodies appear to be the only risk factor present is further evidence that these antibodies have a direct pathogenic role. Induction of PAPS by immunizing mice with human anticardiolipin antibodies is the best evidence yet that aPL antibodies are directly pathogenic (Bakimer et al. 1992 J. Clin. Invest. 89:1558-1563; Blank et al. 1991 Proc. Natl. Acad. Sci. 88:3069-3073).
It is now generally accepted that aPL antibodies recognize an antigenic complex comprised of .beta..sub.2 -glycoprotein I (.beta..sub.2 -GPI) and negatively-charged phospholipid, e.g., cardiolipin (McNeil et al. (1990) Proc. Natl. Acad. Sci. 87:4120-4124; Galli et al. (1990) Lancet i:1544-1547). .beta..sub.2 -GPI is a minor plasma glycoprotein found free and in association with lipoprotein lipids where it is also known as apolipoprotein H (apo H). It consists of five independently folding domains referred to as Sushi or short consensus repeat domains that resemble similar domains in other proteins. .beta..sub.2 -GPI has been reported to undergo antigenic and conformational changes upon binding phospholipid (Wagenkneckt et al. (1993) Thromb. Haemostas. 69:361-365; Jones et al. (1992) Proc. 5th Intl. Symp. Antiphospholipid Antibodies (Abstract S5)). The fifth domain of .beta..sub.2 -GPI is the site of lipid binding as well as the site recognized by aPL antibodies (Hunt J. and S. Krilis, (1994) J. Immunol. 152:653-659; Lauer et al. (1993) Immunol. 80:22-28). The pathological mechanism for aPL is unknown (McNeil et al., supra). Most explanations invoke endothelial cell function or platelet involvement (Haselaar et al. (1990) Thromb. Haemostas. 63:169-173). These explanations suggest that following blood vessel endothelial cell injury or platelet activation, the exposure or transbilayer migration of anionic phospholipid to the plasma-exposed surface may lead to .beta..sub.2 -GPI-binding and trigger aPL antibody formation.
aPL antibodies may be directly prothrombotic by reducing prostacyclin formation (Vermylen, J. and J. Arnout (1992) J. Clin. Lab. Med. 120:10-12); by direct interference with the action of coagulation proteins; or by blocking the ability of .beta..sub.2 -GPI to inhibit the intrinsic blood coagulation pathway, platelet prothrombinase activity, and ADP-mediated platelet aggregation (Arvieux et al. (1993) Thromb. Haemostas. 60:336-341).
A major new tool in medicinal chemistry in the search for lead compounds has been the advent of combinatorial libraries providing vast molecular diversity. Molecular diversity may arise from chemical synthesis or from biological systems (Scott., J. K. RATIONAL DRUG DESIGN (CRC Press, Weiner, D. B. and W. V. Williams, eds., Boca, Raton, Fla., 1994); Moos et al. (1993) Ann. Reports Med. Chem. 28:315-324). By displaying random peptides on the surface of filamentous phage, epitope libraries containing hundreds of millions of clones for probing by clinically significant antibodies have been created (Scott, J. K. and G. P. Smith (1990) Science 249:286-390; Cesareni, G. (1992) FEBS Lett. 307:66-70). Such phage libraries are prepared by incorporating randomized oligonucleotide sequences into the phage genome, usually the pIII gene, which encode unique peptide sequences on the surface of each phage. Following sequential rounds of affinity purification and amplification, those phage that bind antibody are propagated in E. coli and the binding peptides identified by sequencing the corresponding coding region of viral DNA. In most cases, subsequent study will involve corresponding synthetic peptides after establishing their ability to bind antibody. Phage-based libraries have been used to mimic discontinuous epitopes (Luzzago et al. (1993) Gene 128:51-57; BaLass et al. (1993) Proc. Natl. Acad. Sci. 90:10638-10642). The potential plasma instability of peptide-based drugs has been successfully overcome by N-terminal blocking or by the judicious use of amino acid analogs (Powell, M. F. (1993) Ann. Reports Med. Chem. 28:285-293).
At present there is no selective, immunospecific therapy for patients showing high titers of aPL antibodies. In many cases use of drugs such as aspirin, steroids, and warfarin has proven to be largely inadequate (PHOSPHOLIPID-BINDING ANTIBODIES (Harris et al., eds., CRC Press, Boca Raton, Fla., 1991); McNeil et al., supra)). Synthetic mimotope peptides, characterized by (i) the inability to activate T cells while (ii) retaining the ability to bind immune B cells, are used to tolerize in an antigen-specific manner. This technology is disclosed in co-owned, co-pending U.S. patent application, Ser. No. 08/118,055, filed Sep. 8, 1993, and U.S. Pat. No. 5,268,454, which are incorporated by reference herein in their entirety. As disclosed in the application and patent cited above, B cell tolerance entails administering such peptides conjugated to multivalent, stable, non-immunogenic valency platforms in order to abrogate antibody production via B cell anergy or clonal deletion after cross-linking surface immunoglobulin.
Although the exact molecular nature of the target epitopes recognized by aPL antibodies is unknown, the use of peptides derived from epitope libraries will allow for the construction of successful tolerogens. B cell tolerogens for the treatment of human systemic lupus erythematosus-related nephritis have also been disclosed in co-owned U.S. Pat. Nos. 5,276,013 and 5,162,515 which are incorporated by reference herein in their entirety.
DISCLOSURE OF THE INVENTION
This invention resides in the discovery of a method for identifying analogs of key epitopes recognized by aPL antibodies in patients suffering from PAPS, APS and other aPL antibody-mediated diseases such as recurrent stroke using random peptide phage libraries.
Accordingly, one aspect of the invention is an improved method for screening random peptide phage libraries in order to identify the peptide sequences which best mimic the epitopes recognized by aPL antibodies. This method comprises the steps of: (a) biopanning the library using methods modified from those known in the art; (b) eliminating very weakly-binding phage by micropanning the phage screened from step (a) by (i) incubating the phage in microplate wells coated with aPL antibody bound to Protein G, (ii) washing the microplate wells to remove unbound phage, (iii) eluting the bound phage, and (iv) infecting a microorganism such as E. coli with the eluted phage and counting the number of infected microorganisms by plating on agar; (c) determining the strongest-binding clones recovered in (b) by evaluation via phage-capture ELISA by (i) coating the wells of a microplate with aPL antibody, (ii) incubating the strongest-binding clones identified by micropanning in (b) in the coated wells and washing away unbound phage, (iii) quantitating the number of phage bound to the antibody using a conjugated goat anti-phage antibody in a colorimetric ELISA assay and, if several equivalent strongly-binding clones are identified, an additional round of (d) phage-ELISA on the strongest-binding phage-capture-ELISA clone. The phage-ELISA assay consists of (i) coating a uniform amount of different clones on wells of a microtitration plate followed by (ii) identifying the peptide inserts which most strongly bind aPL antibody by adding antibody to the wells and developing the reaction with an enzyme-labeled anti-human IgG conjugate. The random peptides displayed by the phage which have a high binding affinity to aPL antibody as measured by phage-ELISA, colony blot or phage-capture-ELISA represent the analogs of the aPL-specific epitope. These peptides are then synthesized and ranked for strength of binding using competition assays.
Another aspect of the invention is an aPL antibody-binding analog that (a) binds specifically to B cells to which the aPL epitope binds and (b) lacks T cell epitope(s).
Yet another aspect of the invention is a composition for inducing specific B cell tolerance to an APS or PAPS immunogen comprising a conjugate of a nonimmunogenic valency platform molecule and an aPL antibody-binding analog that (a) binds specifically to B cells to which the aPL immunogen binds and (b) lacks T cell epitope(s).

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows that the substitution of fish gelatin for adult bovine serum abolished all anticardiolipin (ACA) activity in an ELISA assay of a commercial aPL antibody standard. This result supported the findings of McNeil et al., supra, and Galli et al., supra, concerning the importance of .beta..sub.2 -glycoprotein I (.beta..sub.2 -GPI) in defining the target epitope(s) of ACA.
FIG. 2 shows that resin-bound analog 5A12 immunospecifically binds to affinity-purified IgG designated ACA-6501.
FIG. 3 illustrates that the aPL antibody-binding analogs derived from screening with ACA-6626 bound aPL antisera but did not bind normal sera. FIG. 4 also illustrates that the ACA-6501-5A12 analog immunospecifically binds ACA-6501 antiserum and is crossreactive with ACA-6626.
FIGS. 4 and 5 illustrate that while the aPL antibody-binding analogs derived from screening with methods within the instant invention bind preferentially with the screening antibody, a significant degree of crossreactivity was detected.
FIGS. 6A and B illustrate the GPL value for ACA-6501.
FIG. 7 shows the activity of affinity isolated ACA-6501 compared to GPL standard sera.
FIG. 8 illustrates the dramatic drop in sequence diversity by the fourth round of biopanning.
FIG. 9 shows three clones (3A12, 3B3 and 3A5) that exhibited a very strong immunospecific signal in the phage-capture ELISA.
FIG. 10 shows the strong signal exhibited by seven clones in a phage-ELISA using ACA-6501.
FIG. 11 shows the results of a competitive-binding ELISA obtained with peptides 5A12, CB2 and 3B10 using ACA-6501.
FIG. 12 illustrates the 50% inhibition values for peptides 139, (amino acid residues 3-14 of SEQ ID NO. 54) 142 (amino acid residues 2-14 of SEQ ID NO. 54) and 143 (SEQ ID NO. 54) with ACA-6501.
FIG. 13 (SEQ ID NO:1 through SEQ ID NO:4) illustrates the effects of substituting .alpha.-Me-Pro at positions 3, 9 and both 3 and 9.
FIG. 14 shows the percentage reduction in antibody response in mice resulting from the administration of the indicated dosages of MG-peptide-PEG.

MODES FOR CARRYING OUT THE INVENTION
Measurement of aPL antibodies in the clinical environment is still an imperfect art. A commercially available set of standard antisera (APL Diagnostics, Inc., Louisville, Ky.) allow generation of a standard curve for comparison of assays performed in various laboratories. A great deal of inconsistency exists, however, between the results obtained at these laboratories regarding the exact GPL and MPL, the unit of measurement for IgG and IgM antiphospholipid antibodies, respectively, ratings for given sera and the levels of GPL and MPL that are categorized as high, medium or low titer. The available commercial kits vary greatly in the values assigned to the commercially available standards (Reber et al. (1995) Thrombosis and Haemostat. 73:444-452). In spite of these limitations, there is general agreement that the epitopes recognized by antibodies in APS, PAPS and other aPL antibody-mediated diseases including recurrent stroke and recurrent fetal loss are located in the 5th domain of .beta..sub.2 -GPI and are exposed to the antibody following binding of .beta..sub.2 -GPI to cardiolipin.
A. Definitions
As used herein the term "B cell anergy" intends unresponsiveness of those B cells requiring T cell help to produce and secrete antibody and includes, without limitation, clonal deletion of immature and/or mature B cells and/or the inability of B cells to produce antibody.
"Unresponsiveness" means a therapeutically effective reduction in the humoral response to an immunogen. Quantitatively the reduction (as measured by reduction in antibody production) is at least 50%, preferably at least 75%, and most preferably 100%.
"Antibody" means those antibodies which are T cell dependent.
As used herein the term "immunogen" means an entity that elicits a humoral immune response comprising aPL antibodies. Immunogens have both B cell epitopes and T cell epitopes.
The term "analog" of an immunogen intends a molecule that (a) binds specifically to an antibody to which the immunogen binds specifically and (b) lacks T cell epitopes. Although the analog will normally be a fragment or derivative of the immunogen and thus be of the same chemical class as the immunogen (e.g., the immunogen is a polypeptide and the analog is a polypeptide), chemical similarity is not essential. Accordingly, the analog may be of a different chemical class than the immunogen (e.g., the immunogen is a carbohydrate and the analog is a polypeptide) as long as it has the functional characteristics (a) and (b) above. The analog may be a peptide, carbohydrate, lipid, lipopolysaccharide, nucleic acid or other biochemical entity. Further, the chemical structure of neither the immunogen nor the analog need be defined for the purposes of this invention.
An analog of an immunogen may also comprise a mimotope. The term "mimotope" intends a synthetic molecule which competitively inhibits the antibody from binding the immunogen. Because it specifically binds the antibody, the mimotope is considered to mimic the antigenic determinants of the immunogen. Like an analog of an immunogen, a mimotope (a) binds specifically to an antibody to which the immunogen binds specifically and (b) lacks T cell epitopes.
As used herein "valency platform molecule" means a nonimmunogenic molecule containing sites which facilitate the attachment of a discreet number of analogs of immunogens.
"Nonimmunogenic" is used to describe the valency platform molecule and means that the valency platform molecule elicits substantially no immune response when it is administered by itself to an individual.
As used herein "individual" denotes a member of the mammalian species and includes humans, primates, mice and domestic animals such as cattle and sheep, sports animals such as horses, and pets such as dogs and cats.
aPL immunogens that are involved in aPL antibody- mediated pathologies may be external (foreign to the individual) immunogens such as drugs, including native biological substances foreign to the individual such as therapeutic proteins, peptides and antibodies, and the like or self-immunogens (autoimmunogens) such as those associated with thyroiditis, antibody-mediated hypercoagulability (stroke), male infertility, myasthenia gravis and rheumatic fever.
B. Identification of aPL Antibody-binding Analogs
aPL antibody-binding analogs may be identified by screening candidate molecules to determine whether or not they (a) bind specifically to aPL antibodies to the immunogen and (b) lack T cell epitopes. Specific binding to aPL antibodies may be determined using conventional immunoassays such as the ELISA assay described in the examples below and the presence or absence of T cell epitopes may be determined by conventional T cell activation assays. In this regard, an analog which "binds specifically" to serum antibodies to the immunogen exhibits a reasonable affinity thereto. The presence or absence of T cell epitopes may be determined using a tritiated thymidine incorporation assay disclosed in Ser. No. 08/118,055. The presence of T cell epitopes can also be determined by measuring secretion of T cell-derived lymphokines by methods well known in the art. Analogs that fail to induce statistically significant incorporation of thymidine above background are deemed to lack T cell epitopes. It will be appreciated that the quantitative amount of thymidine incorporation may vary with the immunogen. Typically a stimulation index below about 2-3, more usually about 1-2, is indicative of a lack of T cell epitopes.
Mimotopes identified by methods within the present invention are synthesized by conventional methods and are screened in the same manner as other analogs of immunogens.
C. Preparation of Conjugates
The aPL antibody-binding analogs are coupled to a nonimmunogenic valency platform molecule to prepare the conjugates of the invention. Preferred valency platform molecules are biologically stabilized, i.e., they exhibit an in vivo excretion half-life often of hours to days to months to confer therapeutic efficacy, and are preferably composed of a synthetic single chain of defined composition. They will normally have a molecular weight in the range of about 200 to about 200,000, usually about 200 to about 20,000. Examples of valency platform molecules within the present invention are polymers such as PEG, poly-D-lysine, polyvinyl alcohol and polyvinylpyrollidone. Preferred polymers are based on polyethylene glycols (PEGs) having a molecular weight of about 200 to about 8,000.
Other valency platform molecules suitable for use within the present invention are the chemically-defined, non-polymeric valency platform molecules disclosed in co-owned, co-pending U.S. Ser. No. 08/152,506, filed Nov. 15, 1993, which is incorporated by reference herein in its entirety. Particularly preferred homogeneous chemically-defined valency platform molecules suitable for use within the present invention are derivatized 2,2'-ethylenedioxydiethylamine (EDDA) and triethylene glycol (TEG).
Conjugation of the aPL antibody-binding analog to the valency platform molecule may be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the analog and valency platform molecule.
Polypeptide analogs will contain amino acid sidechain moieties containing functional groups such as amino, carboxyl, or sulfhydryl groups that will serve as sites for coupling the analog to the carrier. Residues that have such functional groups may be added to the analog if the analog does not already contain same. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. In the case of carbohydrate or lipid analogs, functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry. For instance, primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent. In a similar fashion, the valency platform molecule may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.
The conjugates will normally be formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, they will typically be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. The conjugate will normally constitute about 0.01% to 10% by weight of the formulation. The conjugate is administered to an individual in a "therapeutically effective amount", i.e., an amount sufficient to produce B cell anergy to the involved immunogen and effect prophylaxis, improvement or elimination of the antibody-mediated condition being addressed. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Normally, a dose of about 1 .mu.g to about 10 mg conjugate/kg body weight, preferably about 10 .mu.g to about 1 mg/kg body weight, will be given, daily for three consecutive days. Other appropriate dosing schedules would be 3 doses per week, or one dose per week, or one dose every two to four weeks, or one dose on a monthly or less frequent schedule depending on the individual or the disease state. Repetitive administrations, normally timed according to B cell turnover rates, may be required to achieve and/or maintain a state of humoral anergy. Such repetitive administrations will typically involve treatments of about 1 .mu.g to about 10 mg/kg body weight or higher every 30 to 60 days, or sooner, if an increase in antibody titer is detected. Alternatively, sustained continuous release formulations of the conjugates may be indicated for some pathologies. Various formulations and devices for achieving sustained release are known in the art.
Anti-helper T cell treatments may be administered together with the conjugates. Such treatments usually employ agents that suppress T cells such as steroids or cyclosporin.
D. Nature of the antigen for aPL antibodies
Our initial studies on aPL antibodies coincided with the publication of reports questioning the nature of the antigenic site recognized by these antibodies. Initially these antibodies were thought to recognize the cardiolipin molecule much like those antibodies detected in a VDRL test. As shown in FIG. 1, substitution of fish gelatin for adult bovine serum in the anticardiolipin antibody (ACA) solid phase ELISA essentially abolished all ACA activity of an antibody preparation obtained commercially as an ACA standard. This finding indicated the ACA antibodies recognized a determinant on a serum protein as proposed by McNeil et al., supra and Galli et al., supra, rather than cardiolipin itself. This protein was shown by these authors to be .beta..sub.2 -GPI and, thus, the term ACA is really a misnomer but is still used today to refer to these antibodies.
The majority of autoimmune, IgG aPL antibodies recognize determinants on the .beta..sub.2 -GPI molecule. These epitopes may be formed or exposed on .beta..sub.2 -GPI only upon its binding to cardiolipin (a neo epitope). Alternatively, the epitope on .beta..sub.2 -GPI may exist in a single copy per .beta..sub.2 -GPI molecule and may have low affinity for aPL antibodies. Sufficient avidity to maintain an antibody-antigen interaction might be reached only with the alignment of two of these sites on adjacent .beta..sub.2 -GPI molecules by their binding to cardiolipin.
E. ACA ELISA
The necessity of large-scale testing in connection with GPL scoring of clinical samples and chromatographic purifications of research antibodies and .beta..sub.2 -GPI led to the development of an ELISA assay for aPL antibodies with performance in close agreement with a commercial kit and similar to the design found to have the best reproducibility (Reber et al., supra).
F. Immunoaffinity Purification of aPL Antibodies
In order to isolate the aPL antibodies, multilamellar, cardiolipin-containing dispersions (liposomes; also containing cholesterol and dicetylphosphate) are incubated with aPL plasma (or serum). These liposomes are pelleted from the serum by centrifugation. After washing, the liposome mixture is disrupted by 2% octylglucoside detergent and applied to a protein A-agarose column. Following extensive washings to first remove lipids and then to remove non-IgG components, IgG aPL antibody is eluted from protein A with mild acid, neutralized, buffer-exchanged, and tested in the ACA ELISA. This procedure yields aPL antibody enriched up to 10,000-fold that is devoid of any contaminating .beta..sub.2 -GPI as shown by western blotting with rabbit IgG anti-human .beta..sub.2 -GPI antisera.
G. Construction of Filamentous Phage Random Peptide Libraries
Eleven different fUSE 5 filamentous phage random peptide libraries on the p-III protein (five copies of p-III with peptide per phage) are constructed. These libraries provide a vast array of shapes and structures for the discovery of mimetic epitopes. Four libraries, designated "x" libraries, have peptide inserts that are 8, 10, 12, and 15 residues in length, respectively, and are flanked by proline residues on both the amino and carboxyl ends. The purpose of these proline residues is to disrupt any contribution to secondary structure that might arise from the native p-III protein and to project the insert into the solvent. The "y'" libraries contain cysteine-bounded inserts that are 6, 7, 8, 9, 11, and 13 amino acids long. The "y" library is the same as the "y'" library except that it lacks the 6 and 8 amino acid inserts. These peptide inserts for both "y" and "y'" libraries are flanked by cysteine residues at both the amino and carboxyl ends to form cyclic, more rigid structures. Proline residues are incorporated outside these cysteine residues for reasons similar to those for the "x" libraries above. The "x, " "y'," and "y" libraries are located five residues from the amino terminus of the native p-III protein. The "z" library consists of random eight amino acid inserts located at the amino terminus of the p-III protein and do not contain any flanking proline or cysteine residues. A combination of the "x," "y'" and "z" libraries represents eleven different libraries each with approximately one hundred million different peptide inserts.
These libraries are constructed by incorporation of random oligonucleotide sequences of the length appropriate to give the desired length insert into the p-III gene of fUSE 5 using standard molecular biology techniques. Following restriction endonuclease digestion of the fUSE 5 DNA, an excess of kinased oligonucleotides provided as gapped duplexes is added and ligated. The DNA is then electroporated into E. coli and inserts are selected by culturing in tetracycline-containing media. The phage from this culture (which contain the peptide insert) are isolated from the supernatant, washed and resuspended in buffer. Typically libraries are shown to have 7.times.10.sup.8 independent clones at 8.times.10.sup.12 transducing units per mL.
H. Phage-screening methodology
The essence of screening phage display peptide libraries lies in the ability to collapse billions of potential candidate phage to a relative few with outstanding properties. The original screening protocols recommended by Scott, J. K. and G. P. Smith, (1990) Science 249:315-324 are significantly modified to facilitate the selection of the best epitopes for various aPL antibodies. These procedures are designed to apply greater stringency of selection as the screen progressed until a point is reached where a useful number of clones representing the best sequences can be thoroughly investigated. With some antibodies, the library does not appear to have sequences which bind very tightly and if a method with a high degree of stringency is applied to the screen, no clones survive that are specific. On the other hand, the library frequently yields many clones that represent good analogs of the antigen and it is necessary to employ a method with a high degree of stringency to identify the best epitopes. For that reason, assays were developed with varying degrees of stringency in order to identify the best epitopes from an epitope library screen. The assays are listed here in order of increasing stringency: Biopanning<Micropanning<Phage-Capture ELISA<Phage ELISA=Colony Blot=Peptide ELISA.
(i) Biopanning
"Biopanning" describes the technique wherein affinity-purified aPL antibody and phage bearing random peptide inserts are allowed to mix, following which antibody-specific recovery captures the bound phage. The phage confer tetracycline resistance to E. coli that are propagated in a tetracycline-containing medium and then isolated. Multiple rounds of biopanning enrich the number of immunospecific phage in a sample. Phage are always recovered at the end of three to five rounds of selection but may represent only sequences that are nonspecifically bound at low affinities for the selecting antibodies. A method for further evaluating these phage (micropanning) is required.
(ii) Micropanning
An estimation of the relative strength of binding of the phage to the aPL antibody can be determined by "micropanning. " Micropanning is carried out following three or more rounds of biopanning and uses the same antibody as employed in the biopanning method. The method consists of dilution of the phage from the last round of biopanning and analyzing fifty or more of these clones by micropanning. Micropanning is accomplished by growing each clone to a similar density and then incubating dilute phage at an optimal single concentration in microtitration wells previously coated with a constant amount of antibody. The optimal single concentration of phage is that concentration most likely to reveal the widest range of micropanning scores (from 0 to 4+) and, thus, permit the greatest discrimination among the clones being tested. It is based on the micropanning behavior of six randomly selected clones where the score is determined at each of several concentrations of phage obtained by serial dilution. Following the incubation with antibody, the unbound phage are washed away and the amount of bound phage is used as an indication of the affinity of the phage insert for the antibody. The amount of bound phage is determined by elution with mild acid followed by neutralization and infection of E. coli. The number of infected E. coli are then quantitated by plating the microorganisms on agar plates containing tetracycline and determining then colony densities achieved by each clone.
(iii) Phage-Capture ELISA
The phage-capture ELISA test was developed to provide an intermediate level assay to bridge the gap between the relatively low stringency of the micropanning assay and the high stringency of the phage- or peptide-ELISA assays. Preliminary studies show that some antibody preparations give too many positive clones by micropanning but none by phage-ELISA or peptide-ELISA. The limitation of the phage-ELISA described below is that only five copies of p-III are located on each phage and even with a large number of phage coated on a well, few copies of the insert are represented and detection requires that the antibody have a very high affinity for the insert. With the phage-capture ELISA, the signal is amplified many times which facilitates the detection of lower affinity, stable interactions between the antibody and the insert.
The phage-capture ELISA consists of the following steps. Microtitration wells are coated with aPL antibody and phage clones are added as in the micropanning assay. Unbound phage are washed away and the amount of bound phage is quantitated using an enzyme-conjugated goat antiserum which binds the major coat protein of the phage. Phage screened using phage-capture ELISA react with many aPL antibodies and provide a strong signal in subsequent ELISA assays. This intermediate level of sensitivity allows for greater efficiency in the peptide synthesis effort since few micropanning-positive phage are phage-capture ELISA positive. As a result, peptides synthesized from positive phage-capture ELISA phage are generally immunoreactive.
(iv) Phage-ELISA
This method of selecting phage requires very tight binding of the insert to the screening antibody. Phage are directly coated onto wells of a microtitration plate and incubated with the screening antibody. Following washes to remove unbound antibody, an anti-human IgG alkaline phosphatase conjugate is added to bind any aPL antibodies bound to the phage. APL antibodies are then detected by adding a calorimetric substrate to the well which will react with alkaline phosphatase according to methods well known in the art.
(v) Colony Blot
This assay allows large-scale colony screening of E. coli infected by biopanned phage. This procedure is an alternative to phage-ELISA for identifying immunoreactive clones and exhibits a comparable level of sensitivity without requiring culturing of individual phage clones prior to testing. In this assay, E. coli infected with phage from a round of biopanning are spread on a large diameter nitrocellulose (NC) membrane and cultured overnight on the surface of an agar plate containing tetracycline (Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982). Each colony results from infection by phage containing identical sequences. Several replicate transfer blots on NC are made using this NC "master" and are allowed to grow on the surface of an agar plate. Following the chemical and enzymatic disruption of phage-infected colonies on the blots, the phage may be probed by the techniques commonly used in Western blotting, i.e., staining or immunoblotting. Blots that have been blocked may be incubated with the screening aPL antibody. Following washes to remove unbound antibody, an anti-human IgG horseradish peroxidase conjugate is added to bind to any aPL-antibody that is bound to phage. The addition of a colorimetric substrate allows one to localize the discrete colonies in the master plate which represent immunospecific phage that may be cloned for further study.
(vi) Peptide-ELISA
Following DNA sequencing to determine the peptide insert sequences of the best-reacting phage in the assays described above, the corresponding peptides are made using standard Fmoc peptide chemistry as is well known in the art. For the peptide-ELISA assay, the peptides are made as branched tetravalent molecules, i.e., each molecule has four copies of the insert. Such a peptide can coat the well of a microtitration plate and still have epitopes exposed to the solution to allow binding by an antibody. The tetravalent peptides are synthesized by incorporating lysines as branch points at the first two couplings analogous to the methods used for Multiple Antigenic Peptides (MAPS) (Posnett et al. (1988) J. Biol. Chem. 263:1719-1725). A spacer consisting of glycine-serine-glycine-serine (SEQ ID NO:5) is added on each arm after the lysines and then the insert, including the framework amino acids found in the phage, proline-glycine at the carboxyl terminus and alanine-glycine-proline at the amino terminus. All amino acids in this synthesis are added one at a time using standard Fmoc methods.
These peptides are then assayed by ELISA which is carried out by coating the peptides on microtitration wells and then assaying their reactivity with aPL antibody in a standard ELISA format. In practice, the peptides usually bind very strongly to the original screening antibody and show some cross-reactivity with other aPL antibodies. Controls of non-aPL antibodies are included to eliminate nonspecific binding peptides.
(vi) Competitive Binding Peptide-ELISA
Once ELISA-positive peptides are identified, it is necessary to quantitate their relative binding affinity to the aPL antibodies and to determine whether or not two peptides bind the same population of antibodies in a given patient serum via a peptide-competition ELISA assay. In this assay, various monomeric peptides compete with tetravalent peptides coated on a microtitration plate well. To perform the assay, the peptides to be evaluated are synthesized as monomers, i.e., without the lysine branches employed in the synthesis of the tetravalent peptides, using standard Fmoc chemistry. The monomeric peptides are then purified and dissolved at known concentrations. Wells of a microtitration plate are coated with a tetravalent peptide known to bind to the aPL antibody. Serial dilutions of the monomeric peptides are incubated with a constant dilution of the aPL antibody. The dilution of the aPL antibody was previously determined by titering the antibody against the tetravalent peptide and selecting a dilution on the downslope of the titration curve. After incubating the antibody and monomeric peptides for one hour, the antibody/peptide solutions are added to the microtitration wells and a standard colorimetric ELISA is performed. The concentration of each monomeric peptide that decreases binding of the aPL antibody with the tetravalent peptide is determined by plotting the colorimetric readings obtained for each well. The 50% inhibition point is used as the measure of the relative strength of binding for the monomeric peptides.
(vii) Evaluation of amino acid contributions to binding by substitution and deletion synthesis
The desired epitope for tolerance induction should have as strong an interaction with as many of the aPL antibodies as possible but not contain any unnecessary residues. In order to deduce the minimum constitution of an epitope, analogs of each peptide are made (i) that lack given residues, for example, the framework residues at the carboxyl and/or amino termini are deleted, or (ii) in which amino acid substitutions have been made which differ from sequences found in the epitope library screen. These amino acid substitutions may be either natural, e.g., isoleucine for leucine, or unnatural, e.g., alpha methyl proline for proline. The effect of these deletions and/or substitutions are then measured via peptide-competition ELISA.
(viii) Grouping of aPL Sera Specificities by Mutagenesis of the 5th domain of .beta..sub.2 -GPI
For a tolerogen to be generally effective, it must bind a major portion of the aPL antibodies in the majority of patients. It is important to determine if several antibodies from different patients bind identical residues within the eighty-four amino acid 5th domain of .beta..sub.2 -GPI. If several antibodies bind identical residues, a single mimotope derived from the structural data of the peptides can be constructed which will react with all the antibodies. On the other hand, if the antibodies bind to different residues, a unique tolerogen would be required for each antibody. Site-directed mutagenesis was performed to identify the key residues involved in aPL antibody binding to the 5th domain of .beta..sub.2 -GPI. The resulting mutant .beta..sub.2 -GPIs were assayed for reactivity with several aPL antibodies. A fusion protein comprising the 5th domain of .beta..sub.2 -GPI and glutathionine S transferase (GST) was obtained from A. Steinkasserer and expressed in E. coli (Steinkasserer et al. (1992) FEBS Lett. 313:193-197). This fusion protein was successfully substituted for native .beta..sub.2 -GPI in the ACA ELISA. Amino acid substitutions are engineered using standard site-directed mutagenesis.
K. Isolation of Synthetic aPL Epitopes
Antibody ACA-6501, from a patient with a GPL score of 151 (high titer) and a history of recurrent stroke, fetal loss and no lupus, was immunoaffinity purified on cardiolipin liposomes. The antibody was used in four separate phage library screens, using the xy, the xyz, the xy'z, and a special pro/cys-bounded 7-mer library where, based on the previous screens, Arg was fixed at the seventh position. As shown in Table 1, 36 sequences were obtained in phage that micropanned (out of 140 tested). These appear quite homologous and the conserved DR residues at positions 6 and 7 are notably striking. The consensus sequence is CLLLAPDRC (SEQ ID NO:6). Despite this homology, only seven phage (see Table 2) were positive after phage-ELISA calorimetric testing. Screening the xy'z phage with affinity purified ACA-6626 (from a patient with a high but lower titer than ACA-6501) yielded five unique sequences that were phage-ELISA tested. None were color positive but two were positive when the ELISA immunoconjugate was developed with a chemiluminescent substrate. The sequence motif associated with ACA-6626 appears related but is different from that seen with ACA-6501 (see Table 3). Both antibodies preferred the cys-bounded (probably cyclized and constrained) epitopes over the open pro-bounded sequences.
TABLE 1______________________________________ACA-6501 Phage Library SequencesCloneSequence______________________________________xy Library2101(SEQ ID NO:7)CNILVLDRCxyz Library5A12(SEQ ID NO:8)CLILAPDRC2D7(SEQ ID NO:9)CLLLAPDRC3B6(SEQ ID NO:10)CLVLALDRC3E4(SEQ ID NO:11)CIFVALDRC3E7(SEQ ID NO:12)CILLAHDRC2H1(SEQ ID NO:13)CIILAPGRC3C10(SEQ ID NO:14)CILLAKNRC3C5(SEQ ID NO:15)CIVLVPDRC2F4(SEQ ID NO:16)CLVIALDRC5B1(SEQ ID NO:17)CWFRSQSSC3E11(SEQ ID NO:18)CSPILRGNC3E8(SEQ ID NO:19)CHKFFWLTCxy'z Library2A10(SEQ ID NO:20)CTILAPDRC2G12(SEQ ID NO:21)CLLITPDRC2G11(SEQ ID NO:22)CLLITHDRC2F10(SEQ ID NO:23)CNILVLDRC2E3(SEQ ID NO:24)CPLITHDRC2D12(SEQ ID NO:25)CLVLAADRC3B10(SEQ ID NO:26)CLLLAPDRC3F2(SEQ ID NO:27)CFFHFDHSC2D3(SEQ ID NO:28)CPLHTHHTCCustom (X) R LibraryG11(SEQ ID NO:29)CTILTPDRC2H5(SEQ ID NO:30)CTILTPDRC2H2(SEQ ID NO:31)CTILTLDRC2H10(SEQ ID NO:32)CTLLTPDRC2E10(SEQ ID NO:33)CIQLTPDRC1B7(SEQ ID NO:34)CHLLTPDRC2H1(SEQ ID NO:35)CLILTPDRC2H12(SEQ ID NO:36)CSILAPDRC1A4(SEQ ID NO:37)CNLLALDRC2H6(SEQ ID NO:38)CNLLAIDRC1C3(SEQ ID NO:39)CLLLAIDRC1D10(SEQ ID NO:40)CTILTQDRC2H4(SEQ ID NO:41)CNIITRDRC2G12(SEQ ID NO:42)CILHAAHRC1A9(SEQ ID NO:43)CSSKSYWRC______________________________________
TABLE 2______________________________________ACA-6501 Colorimetric ELISA-positive PhageCloneSequence______________________________________5A12(SEQ ID NO:8)CLILAPDRC2H1(SEQ ID NO:35)CLILTPDRC3B10(SEQ ID NO:26)CLLLAPDRC2H2(SEQ ID NO:31)CTILTLDRC3B6(SEQ ID NO:10)CLVLALDRCG11(SEQ ID NO:29)CTILTPDRC3E7(SEQ ID NO:12)CILLAHDRC______________________________________*Corresponds to consensus or average sequence
TABLE 3______________________________________ACA-6626 xy'z Phage Library SequencesCloneSequence______________________________________4B11(SEQ ID NO:44)CGNAADARC4D3(SEQ ID NO:45)CTNWADPRC4D3(SEQ ID NO:46)CGNIADPRC4G7(SEQ ID NO:47)CTNLTDSRC4A2(SEQ ID NO:48)CGNPTDVRC______________________________________
ACA-6644 is another high-titered aPL antibody that was used to screen the pooled p-III phage libraries according to methods described herein. The following sequences were discovered: ##STR1##
These sequences all were derived from the component epi.sup.z library that lacks phage framework residues at the N-terminus. When synthesized as peptides the sequences were immunoreactive with several ACA sera including ACA-6644 and ACA-6501. Analysis revealed unsuspected homologies with the sequences previously obtained with ACA-6501 as illustrated in Table 4.
TABLE 4______________________________________ACA-6644/CBdGILTIDNL(SEQ ID NO:50)ACA-6501/2H2CTILTLDRC(SEQ ID NO:31)ACA-6644/CBfGILALDYV(SEQ ID NO:51)ACA-6501/2F10CNILVLDRC(SEQ ID NO:7)ACA-6644/CBcGILLNEFA(SEQ ID NO:49)ACA-6501/1D10CTIITQDRC(SEQ ID NO:40)______________________________________
The convergent sequence homology from two very dissimilar source libraries screened by these two aPL antibodies suggests that the sequences may mimic a major, perhaps immunodominant, region in the native target antigen.
Screening of the p-III libraries with ACA-6701 yielded two unique sequences with a high degree of internal homology but unlike others previously obtained with other aPL antibody. The sequences are as shown:
ACA-6701/3B1 L S D P G Y V R N I F H (SEQ ID NO:52)
ACA-6701/3E1 L T D P R Y T R D I S N F T D (SEQ ID NO:53)
As resin-bound peptides, the sequences were strongly immunoreactive with the parent serum (ACA-6701) but were minimally cross-reactive with other aPL antibodies. Thus, this may be a new, probably rare mimotope.
L. Immunoreactivity of aPL-related Peptides with Affinity Purified IgG aPL Antibody
Phage sequences obtained with affinity-purified ACA-6501 and found to be phage-ELISA-positive were synthesized on a solid support recently developed for combinatorial synthetic peptide libraries. This support, a Rapp resin, has a high peptide density and uses a hydrophilic polyethyleneglycol spacer before the first amino acid is coupled. The synthesis resulted in resin-bound peptide that was ideally suited for antibody binding studies. As shown in FIG. 2, peptide 5A12 (sequence CLILAPDRC) (SEQ ID NO:8) dramatically outperformed an unrelated control peptide while not significantly binding normal IgG. Similar results were obtained with the other phage-ELISA-positive peptides tested. In the experiment shown, resin peptide-bound affinity-purified ACA-6501 aPL antibody was detected by an immunoconjugate color reaction.
M. aPL Serum Antibody Reactivity with Synthetic Peptides
The discovery that resin-bound peptides could bind aPL immunospecifically using serum significantly enhanced the ability to test aPL antibodies. As shown in FIG. 3, the peptide derived from screening with ACA-6626 bound aPL antiserum but did not demonstrate significant binding of normal serum. FIG. 3 also illustrates the immunospecific-binding behavior of ACA6501-5Al2 peptide towards aPL serum. Binding of normal serum to peptide 5Al2 was nil in data not shown.
N. Crossreactivity of Synthetic Peptides Towards aPL Antiserum That Was Not the Source of the Library Screening Antibody
Two peptides were selected for aPL sera testing, one (5A12) representing the ACA-6501 screen and another one (4D3) representing the ACA-6626 screen. As shown in FIGS. 4 and 5, each of the peptides reacted preferentially with the parent serum of the screening antibody. However, a significant degree of crossreactivity was detectable especially between ACA-6501 and ACA-6626. A survey of 19 patient sera with low or no GPL score and of 13 pathologic sera with moderate to high GPL carried out with the 5A12 resin-bound peptide showed 8 of the 13 pathologic samples with detectable peptide binding while only 2 out of 19 control samples showed low peptide binding. These results evidence that synthetic peptides are useful for diagnostic or prognostic assays in stroke patient care.
O. New Synthetic Peptide Methodologies
The identification of new candidate sequences by aPL library screening requires testing of the new synthetic peptides for antibody binding. With Rapp resin of known peptide substitution, it is possible to carry out quantitative binding studies such as saturation binding analysis and equilibrium measurements using radiolabeled aPL IgG. Peptide synthesis allows the molecular dissection of the mimotope by selective synthesis. This includes the modification of each amino acid along the chain with the goal of enhancing antibody binding. Selective synthesis reveals the relative importance of each amino acid in the sequence. If necessary, selective substitution at particular residue locations can be designed to maintain B cell reactivity while abolishing any T cell reactivity discovered during T cell assays.
P. Use of .alpha.-methyl Proline Substitution for Proline to Enhance Peptide Immunoreactivity and Confer Resistance to Protease Attack
Proline residues have a special significance due to their influence on the chain conformation of polypeptides. They often occur in reverse turns on the surface of globular proteins. In the phage epitope libraries of the present invention, all random peptide inserts are flanked by boundary prolines. In addition, most of the mimotopes discovered with ACA-6501 have a third proline which, based on computer-based predictions, likely exists as part of a .beta.-turn. .beta.-turn mimetics can be used to enhance the stability of reverse turn conformations in small peptides. Such a mimetic is (S)-.alpha.-methyl proline (.alpha.-MePro), a proline analog that, in addition to stabilizing turn conformations, confers resistance to protease degradation. Protease resistance is a desirable property for a potential drug designed to act in the plasma. Peptide ACA-6501/3B10 AGPCLLLAPDRCPG (SEQ ID NO:54) (insert highlighted) is a consensus peptide. It has a sequence featuring the most prevalent residue at each position based on a comparison with 35 other homologous sequences. Due to its representative character, the sequence was subjected to a number of systematic modifications and deletions and its activity subsequently evaluated by aPL antibody binding. Among the most important findings was the discovery that the prolines at the 3 and 9 positions are important for activity. Proline-3 is derived from the phage framework and is not part of the random insert. The most dramatic effect was obtained by the substitution of .alpha.-MePro for proline at the 9-position. This substitution led to a six-fold enhancement in immunoreactivity.
All articles, patents and patent applications cited herein are incorporated by reference herein in their entirety.
The following examples are intended to further illustrate the invention and its uniqueness. These examples are not intended to limit the scope of the invention in any manner.
EXAMPLES
Example 1
Measurement of Anticardiolipin Antibodies (ACA) in serum 6501
Even numbered wells of an Immulon I microtitration plate (Dynatech Laboratories, Inc., Chantilly, Va.) were coated with 50 .mu.g cardiolipin (Sigma Chemical, St. Louis, Mo.) in 30 .mu.L ethanol per well. The plate was dried overnight at 4.degree. C. and blocked with 200 .mu.L of 10% adult bovine serum (Irvine Scientific Co., Santa Ana, Calif.) in phosphate-buffered saline (ABS/PBS) for 2 hours at room temperature (RT). The plates were washed 5 times with Tris-buffered saline (TBS) prior to the addition of 10 .mu.L of aPL standards and the test serum, ACA-6501. The aPL standards (APL Diagnostics, Inc., Louisville, Ky.) were reconstituted according to the manufacturer's instructions and diluted 1:50 with 10% ABS-PBS. The test serum, ACA-6501, was diluted 1:50 to 1:2,000 in serial dilutions with 10% ABS-PBS was added to selected duplicate wells and incubated for 2 hours at RT. The plate was washed five times with TBS and 100 .mu.L of 1:1,000 goat-anti-human-IgG/alkaline phosphatase conjugate (Zymed, South San Francisco, Calif., Cat. No. 62,8422) in 10% ABS-PBS was added and incubated for 1 hour at RT. Again, the plate was washed five times with TBS and the assay was developed by adding 100 .mu.L phenolphthalein monophosphate (PPMP) substrate solution (Sigma, Cat. No. P-5758) diluted 1:26 with deionized water from the prepared stock solution of 0.13M PPMP and 7.8M 2-amino-2-methyl-1-propanol adjusted to pH 10.15 with HCl. After approximately 30 minutes, the reaction was stopped with 50 .mu.L of 0.2M dibasic sodium phosphate (Mallinckrodt, Analytical Reagent) added per well. The optical density was read at 550 nm in a microplate autoreader (Bio-Tek Instruments, Winooski, Vt., Model EL311). The optical density of the odd-numbered control wells (blank, without Cardiolipin (Cl)) was subtracted from the optical density of the even-numbered wells. The absorbance readings of the aPL standards were plotted using Graph Pad Prizm (Graph Pad Software, Inc., San Diego, Calif.) to generate the GPL (IgG phospholipid) standard curve. The diluted 6501 test serum absorbance readings were used to calculate GPL scores based on the GPL standard curve.
Example 2
Anticardiolipin Antibody (ACA) Purification from Serum 6501
In a 25 mL round-bottom flask (Kontes Scientific Co., Vineland, N.J.) a mixture of 1.2 mL cardiolipin (Sigma Chemical, St. Louis, Mo., #C-1649), 0.464 mL cholesterol (Sigma Diag., St. Louis, Mo., #965-25), 0.088 mL of 5 mg dicetylphosphate (Sigma Chemical, St. Louis, Mo., D-2631) per mL chloroform was dried for approximately 5 minutes in a Rotavap (Buchi, Switzerland). Following the removal of solvent, 2 mL of 0.96% (wt./vol.) NaCl (J. T. Baker, Inc., Phillipsburg, N.J.) Baker analyzed reagent) was added and mixed in a Vortex Genie Mixer (Scientific Industries, Inc., Bohemia, N.Y.) for 1 minute. The liposome suspension was incubated for 1 hour at 37.degree. C. Meanwhile, serum 6501 was spun at 600.times.g in a Sorvall RT 6000 centrifuge (Dupont Co. Wilmington, Del.) for 10 minutes at 8.degree. C. Four mL of the supernatant was placed in a 25 mL round-bottom flask with 1 mL of the prepared liposome suspension and the mixture was incubated with agitation at medium speed in an orbital shaker, Tektator V (Scientific Products, McGraw Park, Ill.) for 48 hours at 4.degree. C., and an additional 2 hours at 37.degree. C. Twenty mL of cold TBS was added and the mixture was transferred into a 50 mL polycarbonate centrifuge tube (Nalge Co., Rochester, N.Y.) and centrifuged at 27,000.times.g for 15 minutes at 4.degree. C. in an RC3 centrifuge in an SS-34 rotor (Sorvall-Dupont, Wilmington, Del.). The precipitate was washed 3 times with 25 mL of cold 0.96% NaCl using the RC3 centrifuge. The pellet was dissolved in 1 mL of 2% (wt/vol) solution of n-octyl-.beta.-D-glucopyranoside (Calbiochem, La Jolla, Calif.) in TBS and applied to a 0.6 mL protein A/cross-linked agarose (Repligen Corporation, Cambridge, Mass.) column which had been pre-washed with 15 times bed volume of 1M acetic acid and equilibrated with 15 times bed volumes of TBS. The antibody-protein A/agarose column was washed with 40 times bed volume of 2% octylglucopyranoside to remove lipids, followed by extensive washings with TBS until the optical density of the eluate at 280 nm approached the baseline. The bound antibody was eluted with 1M acetic acid. One mL fractions were collected, neutralized immediately with 0.34 mL 3M Tris (Bio-Rad, electrophoresis grade reagent) per fraction and kept in an ice bath. The optical density of each fraction was determined at 280 nm in a spectrophotometer (Hewlett-Packard, 8452A Diode Array Spectrophotometer, Palo Alto, Calif.). Fractions containing antibody were pooled, concentrated and washed 4 times with TBS in Centricon-30 concentrators (Amicon Division, W. R. Grace & Co., Beverly, Mass.) per manufacturer's protocol. The final yield of purified antibody from 4 mL of serum 6501 was determined by reading the optical density at 280 nm of an aliquot from the concentration, where 1 mg=1.34 OD.sub.280. The average yield obtained was 750 .mu.g antibody from 4 mL of serum 6501. The purified antibody was tested for ACA activity and checked for purity with Laemmli SDS-PAGE.
Example 3
Construction of a p-III Library Vector Preparation
fUSE 5 (Scott, J. K. and G. Smith, supra) was used as the vector for the construction of p-III libraries, a variation of the method of Holmes, D. S. and M. Quigley (1981), Anal. Biochem. 144:193) to generate the double-stranded replicative form (RF). Briefly, an 800 mL culture of E. coli K802, harboring fUSE 5, was grown in 2YT medium (Difco Labs, Ann Arbor, Mich.) containing 20 micrograms/mL tetracycline for 18 hours at 37 degrees with vigorous shaking. Cells were collected by centrifugation and resuspended in 75 mL STET. STET consists of 8% sucrose in 50 mM Tris/HCl pH 8.0, 50 mM EDTA and contains 0.5% Triton X-100. Lysozyme, 10 mg/mL in STET, was added to a final concentration or 1 mg/mL. After 5 minutes at RT, three equal aliquots were placed in a boiling water bath with occasional shaking for 3.5 minutes. The viscous slurry was centrifuged for 30 minutes at 18000.times.G and an equal volume of isopropanol was added to the supernatant. The solution was cooled to -20.degree. C. and the nucleic acids were collected by centrifugation. The RF was isolated from a CsCl gradient as described by Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2d ed., 1989).
Preparation of the random insert
The DNA for insertion was generated by the "gapped duplex" method described by Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382. In this method, an oligonucleotide containing a degenerate region in the middle is surrounded by short constant regions on each end of the oligonucleotide. Two shorter, complementary oligonucleotides are annealed to form a "gapped duplex" possessing overhangs that are complementary to the sticky ends produced by the restriction endonuclease used to digest the vector. In this case, the longer degenerate oligonucleotide has the sequence: 5' GGGCTGGACCC(NNK).sub.x CCGGGGGCTGCTG 3' (SEQ ID NO:55) where N=A or C or G or T, K=G or T, and x is the number of codons in the random regions. EpiGS2 was designed to base pair to the 5' end of the degenerate oligonucleotide and has the sequence 5' GGGTCCAGCCCCGT 3' (SEQ ID NO:56). Similarly EpiGS3 was designed to anneal to the 3' end of the degenerate oligonucleotide and has the sequence 5' CAGCCCCCGG 3' (SEQ ID NO:57). When correctly annealed, the three oligonucleotides form a "gapped duplex" which, when inserted into fUSE 5 digested with Sfi1, restores the reading frame of p-III with the random insert near the 5' end.
Oligonucleotides described above were prepared by excision from polyacrylamide gels. One nanomole of EpiGS2, EpiGS3, and 50 picomoles of the degenerate oligonucleotide were kinased separately in 66 microliter volumes. The three oligonucleotides were then pooled, NaCl was added to 50 mM and the mixture was heated to 65.degree. C. for 5 minutes followed by slow cooling to RT. The annealed oligonucleotides were then cooled on ice and used immediately in the ligation reaction with fUSE 5. The ligation reaction consisted of 10 micrograms of fUSE 5 DNA digested to completion with Sfi1, 30 .mu.L of the "gapped duplex" solution and 1000 U of T4 ligase in a total volume of 450 microliters. The ligation was incubated for 18 hours at 16.degree. C. The mixture was then phenol-chloroform extracted, precipitated with ethanol and the precipitate dissolved in 20 .mu.L of water.
Generating and Amplifying the Library
The ligated DNA was introduced into E. coli by electroporation (Dower et al. (1988) Nucleic Acid Res. 16:6127-6145). Frozen electrocompetent MC1061 cells (0.1 mL) were mixed with 4 .mu.L of ligated DNA in a cold 2 mm cuvette and subjected to 2.5 kV, 5.2 mS pulse by a BTX electroporation device (BTX Corp., San Diego, Calif.). Immediately after the pulse, 1 mL of SOC, a cell growth medium (see Dower et al., supra) was added. Five separate electroporations were carried out, pooled, and incubated at 37.degree. C. for 1 hour. At that time, samples were removed and diluted to determine the total number of clones generated. The balance of the mixture was diluted in 1L of 2YT (1.6% Peptone, 140, 1% Yeast Extract, and 0.5% NaCl, Difco Labs, Ann Arbor, Mich.) containing 20 micrograms per mL of tetracycline and grown for 18 hours at 37.degree. C. while shaken at 275 rpm. Phage were purified by 2 rounds of PEG/NaCl precipitation and resuspension in 1.2 mL TBS containing 0.02% sodium azide. Particle number was estimated by absorbance at 269 nm. The titer of the phage was determined by mixing 10 .mu.L of dilutions of phage with 10 microliters of starved E. coli.
Example 4
Screening a p-III Library with aPL Antibody
Affinity-purified ACA-6501 (affACA-6501, 10 .mu.g; 7 .mu.l of 1.44 mg/mL stock solution) was incubated for 2 hours at RT in siliconized 1.4 mL microfuge polypropylene tubes with pooled phage from the x, y and z libraries (epi.sup.xyz) �11 .mu.L+8.5 .mu.L+2 .mu.L, respectively; total volume=21.5 .mu.L, .about.10.sup.10 clones! in a final volume of 100 .mu.L Tris-buffered saline, pH 7.4 (TBS) with 0.5% bovine serum albumin (BSA). During this incubation, the final steps in preparing freshly starved E. coli, strain K-91, were carried out. A suspension of E. coli freshly grown in 2YT medium for about 5 hours with 250 rpm shaking at 37.degree. C. was spun at 1000.times.g for 10 minutes at RT in 50 mL polypropylene tubes. Twenty mL of 80 mM NaCl was added to the packed E. coli pellet and then incubated for 45 minutes at 37.degree. C. at 100 rpm. Following centrifugation as above, the starved E. coli pellet was suspended in 1 mL of 50 mM ammonium phosphate/80 mM NaCl and used later for phage amplification. Protein G-agarose beads were washed 2.times.in TBS/BSA and 2 times in TBS/0.5% Tween-20 and stored at 4.degree. C. as a 50% suspension in TBS/Tween.
Two hundred .mu.L of the protein G-agarose bead suspension was then added to the ACA-6501/phage mixture and incubation continued for an additional 1 hour at RT. At this point, the mixture was chilled and washed 3 times with cold TBS/Tween and the precipitate was collected in a microfuge in a cold room. The washed beads were transferred to new microfuge tubes prewashed with TBS/BSA and TBS/Tween to prevent non-specific adherence. After three additional washes with TBS/Tween, the beads with bound ACA-6501-bearing phage were eluted with 300 .mu.L 0.2N HCl/glycine, pH 2.1 by tumbling for 10 minutes at RT. Following centrifugation at 16,000.times.g, the acidic eluate supernatant was collected and an additional 100 .mu.L of elution solution was added to the bead pellet and the procedure repeated. After 10 minutes, the phage-containing eluates (representing the unamplified first round phage) were pooled (.about.400 .mu.L) and placed in a sterile 17.times.100 mm polypropylene cell culture tube to which was added 50 .mu.L 0.5M NaCl followed by pH neutralization with 2.5M Tris base (usually .about.25-35 .mu.L). An equal volume of the starved E. coli suspension was added immediately and then incubated for 10 minutes at 37.degree. C. at 100 rpm. The mixture was then transferred to a 250 mL sterile culture flask containing 25 mL 2YT with 20 .mu.g/mL tetracycline (Tet) and incubated overnight at 37.degree. C. at 250 rpm.
To isolate amplified phage from overnight cultures, the suspension was centrifuged at 12,000.times.g for 10 minutes in polycarbonate tubes and the pellet discarded. After heating the supernatant at 70.degree. C. for 30 minutes in polypropylene tubes, the material was spun again in polycarbonate tubes and the supernatant saved. To the supernatant, 1/4 volume of 20% (w/v) polyethylene glycol, molecular weight 8000 (PEG 8000) was added to precipitate phage. The solution was mixed by inversion 100 times and then incubated at 4.degree. C. for 2 hours. After centrifugation at 35,000.times.g for 30 minutes at 4.degree. C., the phage-containing pellet was resuspended in .about.0.5 mL of TBS/BSA and transferred to a 1.4 mL microfuge tube. After a 1 minute spin in a microfuge at 16,000.times.g, the supernatant was transferred to a clean tube and labeled first round amplified phage.
During second, third and fourth rounds of biopanning, 75 .mu.L of amplified phage from the preceding round was incubated with 7 .mu.L affACA-6501 in a final volume of 100 .mu.L. For fifth round phage, affACA-6501 was diluted first at 1:1000, then treated as described for the other rounds. All subsequent steps were carried out as described for the first round. Phage from five rounds of biopanning were spot-titered on 2YT/Tet plates to determine phage concentration. The spot titer of amplified phage requires an initial phage dilution of 1.times.10.sup.6 in TBS/BSA or 2YT media. For each round, 10 .mu.L of the dilute phage was incubated with 40 .mu.L of starved E. coli for 10 minutes at 37.degree. C. with no shaking. Following the addition of 950 .mu.L of 2YT/dilute Tet (0.2 .mu.g/mL), the mixture was incubated for 30-45 minutes at 37.degree. C. with 250 rpm shaking. Ten .mu.L aliquots of neat and diluted phage solutions, 1:10, 1:100, and 1:1000, were spotted in 2YT/dilute Tet using agar plates containing 20 .mu.g/mL Tet.
Micropanning
Immulon type 2 plates were coated with protein G. Protein G was prepared at 10 .mu.g/mL in 0.1M NaHCO.sub.3 and 100 .mu.L per well was added to the wells of microtitration plates and incubated overnight at 4.degree. C. After discarding excess protein G solution from plates, each well was blocked with 250-300 .mu.L 2YT for 1 hour at RT with agitation on an oscillating platform. Tris-buffered saline, pH 7.4/0.5% Tween 20 (TBS/Tween), was used with an automatic plate washer to wash the wells 4 times with 200 .mu.L. One hundred .mu.L affACA-6501 (or control normal IgG), diluted to 2.5 .mu.g/mL with 2YT, was added to washed wells. The plate was transferred to a cold room rotator near the end of a 1 hour incubation at RT on a rotating platform.
Phage to be tested by micropanning were obtained from the agar plates generated by biopanning. Each clone to be tested was transferred using sterile toothpicks to a separate well of a round-bottom 96-well microtitration plate (Corning, Corning, N.Y.) containing 250 .mu.L 2YT/Tet per well and cultured overnight at 37.degree. C. Clone designations are based on the screening antibody, the biopanning round of origin, and the location of the clone in the overnight culture plate, e.g., ACA-6501/3B10 refers to the clone isolated by ACA-6501 in the third round located in the well designated B10 on the microtitration plate. Following overnight incubation, phage cultures were centrifuged using a microtitration plate holder at 1300.times.g for 10 minutes at RT. Supernatants constituted the source of "neat" phage.
Initial micropanning was restricted to six clones which were tested at dilutions expanded by factors from 1:10 to 1:10.sup.6. The results from these pilot clones were used to suggest the appropriate single dilution that would yield gradable results for clones in the source plate for each round. Plates representing cultured, randomly chosen clones from the 3rd, 4th, and 5th rounds of biopanning were diluted to 1:100,000 from the "neat" solution using 2YT in microtitration plates with a final volume per well of 250 .mu.L. From the last plate representing the desired dilution, 100 .mu.L was added to the plate containing protein G-bound ACA-6501 and normal IgG prepared as described above. The incubation of dilute phage with aPL antibody or control IgG was carried out for 2 hours at 4.degree. C. on a flat rotator. After 9 washes with TBS/Tween in an automated plate washer, the IgG-bound phage was eluted with 20 .mu.L of 0.2N HCl-glycine/0.1% BSA, pH 2.2. The elution incubation continued for 10 minutes at RT, during which time a new Corning microtitration plate was prepared containing 20 .mu.L of freshly starved E. coli per well and kept chilled. One hundred forty .mu.L of 29 mM Tris was added to the plate containing the phage eluates in order to neutralize the pH, following which 20 .mu.L of phage suspension was transferred from each well to the corresponding well in the plate containing starved E. coli. After a 10 minute incubation at 37.degree. C., 200 .mu.L 2YT/dil Tet was added and incubation carried out an additional 30 minutes at 37.degree. C. Using multichannel pipettors, 10 .mu.L from each well was spotted on a large 2YT/Tet agar plate while retaining the original 8.times.12 well pattern and orientation from the last microtitration plate. After allowing the spots to dry for 30 minutes, the plate was incubated overnight at 37.degree. C. The following day, colonies were semiquantitatively scored from 0 to 4+, with 0 symbolizing<10 colonies; .+-., 10-20; 1+, 20-50; 2+, 50-70% confluent; 3+, 70-90% confluent; and 4+representing>90% confluent colonies. Of the 94 clones that were examined at a dilution of 1:10.sup.5 �representing 81 third, 6 fourth, and 7 fifth round clones!, six clones had micropanning scores of zero, three scored 1+, 14 scored 2+, 62 scored 3+, and 9 scored 4+. A survey of random clones from the plates representing the second through fifth rounds of biopanning was carried out by G-track DNA sequencing as described below. The results showed a dramatic drop in sequence diversity by the fourth round of biopanning (see FIG. 8), therefore, a second plate was micropanned using phage at a dilution of 1:3.times.10.sup.5 this time including clones from an earlier, second, round. A total of 94 clones were tested, including 29 which had scored high at 1.times.10.sup.5 dilution �26 from the 3rd round, 1 from the 4th found, and 2 from the 5th round! plus 65 clones from the 2nd round that had not been previously tested. Of the 94 clones tested at a dilution of 1:3.times.10.sup.5, 26 scored zero, 11 scored .+-., 10 scored 1+, 13 scored 2+, 34 scored 3+, and zero scored 4+.
G-track DNA Sequencing
Single-stranded viral DNA was isolated from cultures incubated overnight at 37.degree. C. To prepare cultures, 2YT/Tet, either as 2 mL in tubes or 250 .mu.L in microtitration plate round-bottom wells, was inoculated with individual phage from spread plates of previously grown cultures in microtitration plates. The purification of phage by 20% PEG/2.5M NaCl precipitation of culture supernatants as well as the isolation or release of virion DNA by phenol-chloroform extraction or by alkali denaturation was performed as described in Smith, G. P. and J. K. Scott, "Libraries of peptides and proteins displayed on filamentous phage" (1993) Meth. Enzymol. 217:228-257 for cultures in tubes and as described in Haas, S. J. and G. P. Smith G. P., "Rapid sequencing of viral DNA from filamentous phage" (1993) BioTechniques 15:422-431 for phage in microtitration plates. The dideoxy nucleotide chain termination DNA sequencing technique of Sanger et al. (Sanger et al., "DNA sequencing with chain terminating inhibitors" (1970) Proc. Natl. Acad. Sci. 74:5463-5467) was carried out using a commercial Sequenase kit (U.S. Biochemical/Amersham, Arlington Heights, Ill.) as described by Smith and Scott, supra, for tube culture phage DNA and as in Haas and Smith, supra, for phage DNA from microtitration plates. By using only the ddCTP termination mixture, G tracking or the sequence pattern suggested by a single base (G) was obtained following electrophoresis on 7% polyacrylamide/urea sequencing gels and exposure by autoradiography. Standard gel electrophoresis and autoradiography procedures were followed (Sambrook et al., supra)
G-tracking of 76 clones from the micropanning plate tested at a phage dilution of 1:1.times.10.sup.5 from the ACA-6501 library screen revealed 11 unique sequences, while 64 clones from the second micropanning plate tested at a phage dilution of 1:3.times.10.sup.5 showed 30 unique sequences. Conventional DNA sequencing using all four dideoxynucleotide triphosphates was applied to the phage clones with the highest micropanning scores and unique sequences and resulted in the 12 sequences shown in Table 1 for the xyz library.
Phage-Capture ELISA
Clones ACA-6635/3A12, 3B3, 3C8, 3A5, 3C9, and 3B7 were grown as 3 mL cultures. Affinity purified ACA-6635 was diluted to 2.5 .mu.g/mL in phosphate-buffered saline, pH 7.2, and 100 .mu.L added to Immulon-2 microtitration plate wells. After 2 hours, the plates were washed 3 times with TBS/Tween in an automated plate washer with no shaking. The plate was then blocked with 150 gL 0.1% BSA (globulin-free) in PBS per well. After 1 hour at 4.degree. C., the plate was washed 3 times as previously described. After centrifuging each phage culture 3 minutes at 17,000.times.g, each supernatant was diluted 1:10 in 0.% BSA/PBS and 100 .mu.L added to each of the wells coated with affinity purified ACA-6635 and then incubated for 2 hours at 4.degree. C. Plates were then washed with TBS/Tween as before. Horseradish peroxidase-conjugated sheep IgG anti-M13 phage antibody (Pharmacia, Inc., Piscataway, N.J.) was diluted 1:5,000 in 0.1% BSA/PBS and 100 .mu.L applied to each well. Following incubation for 1 hour at 4.degree. C., the plate was washed 4 times as before. One hundred .mu.L of substrate prepared according to the conjugate manufacturer's instructions was added to each well. After 19 minutes, the absorbance at 405 nm of each well was read in the automated microplate absorbance reader (Biotek, Winooski, Vt.) The seven clones tested from the ACA-6635 phage library screen were selected because of their high micropanning scores and negative phage-ELISA scores (see below). As shown in FIG. 9, three clones (3A12, 3B3, and 3A5) out of the seven tested exhibited a very strong immunospecific signal in the phage-capture ELISA.
Phage-ELISA
Three mL cultures were prepared from 35 clones previously isolated with affinity purified ACA-6501 in several phage library screens as well as one fUSE 2 phage clone lacking a peptide insert which was used as a control. After centrifugation for 3 minutes at 17,000.times.g, 100 .mu.L from each phage supernatant (adjusted to -2.times.10.sup.11 particles/mL based on absorbance) was added to microtitration plate wells (Falcon, Becton-Dickinson Labware, Lincoln Park, N.Y.) and allowed to incubate overnight at 4.degree. C. Following four washes with TBS 7.4, the plate was blocked with 125 .mu.L of 0.5% BSA/TBS for 1 hour at RT. After another four TBS washes, 100 .mu.L of affACA-6501 previously diluted to 2.5 .mu.g/mL in TBS/BSA was added to each well and allowed to incubate 1 hour at 37.degree. C. Following an additional four washes, 100 .mu.L anti-human IgG diluted 1:1000 in TBS/0.5% Tween was added to each well. After 1 hour at RT, enzyme substrate was added and the incubation allowed to proceed for 2 hours. Following the addition of 50 .mu.L 0.2M Na.sub.2 HPO.sub.4 to stop the reaction, absorbance was measured at 550 nm in the automated plate absorbance reader. As shown in FIG. 10, seven of the clones had significant signals in the phage-ELISA with ACA-6501: 5A12, 3B6, 3E7, 3B10 (same sequence as 2D7), 2G11, 2H1, and 2H2. The sequences for these clones are shown in Table 2.
Peptide-ELISA
In the standard protocol, stock solutions of tetrameric peptide in dimethylformamide were diluted 1000 times to 10 .mu.g/mL in pH 9.5 carbonate buffer. Each microtitration plate well was coated overnight at 4.degree. C. with 100 .mu.L of dilute peptide followed by blocking with buffered albumin. Peptide-coated microtitration plates were incubated 1-2 hours at room temperature with aPL sera at several dilutions starting at 1:50. Following washes, the presence of peptide-bound human IgG was determined with enzyme-conjugated anti-human IgG according to standard ELISA procedures.
Competitive Binding Peptide-ELISA
Each well of an Immulon II plate (Dynatech Laboratories, Inc., Chantilly Va.) was coated with 100 .mu.L of a solution containing 10 .mu.g tetravalent peptide ACA 6501/3B10 in 50 mM sodium carbonate, pH 9.5, containing 35 mM sodium bicarbonate (Fisher Scientific, Pittsburgh, Pa., reagent grade) for at least 1 hour at RT, except for three wells used as blank controls. The liquid was then removed from the wells and 200 .mu.L of 0.5% (wt/vol) BSA (Sigma Chemical, St. Louis, Mo., #A7638) in TBS was added per well including the blank wells for blocking and incubated for at least 1 hour at RT. Four 1.5 mL microfuge tubes were numbered 1 to 4. The following reagents were mixed in the first microfuge tube (Brinkman Instruments, Westbury, N.Y.): 30 .mu.L of 5% BSA; 284 .mu.L TBS; 8 .mu.L of a stock solution of approximately 400-500 .mu.g/mL of monomeric peptide (ACA-5A12 or -CB2 or -3B10 or scrambled -3B10 as negative control) in TBS; and 8.2 .mu.L of 1:10 diluted serum 6501 in 0.5% BSA-TBS.
The following reagents were mixed in the second microfuge tube: 30 .mu.L of 5% S BSA; 290 .mu.L TBS; 2 .mu.L of a stock solution of approximately 400-500 .mu.g/mL of monomeric peptide (ACA-5A12 or -CB2 or -3B10 or scrambled -3B10 as negative control) in TBS; and 8.2 .mu.L of 1:10 diluted serum 6501 in 0.5% BSA-TBS. The following reagents were mixed in the third microfuge tube: 30 .mu.L of 5% BSA, 287 .mu.L of a 1:10 dilution of approximately 400-500 .mu.g/mL of monomeric peptides (5A12, CB2, 3B10, or scrambled sequence 3B10 control), and 8.2 .mu.L of ACA-6501 serum previously diluted 1:10 in 0.5% BSA-TBS. Diluted peptides were obtained after a ten-fold dilution of peptide stocks at 400-500 .mu.g/mL in TBS using 0.5% BSA-TBS as diluent. The following reagents were mixed in the fourth Eppendorf microfuge tube: 60 .mu.L 5% BSA; 584 .mu.L TBS and 16.5 .mu.L of 1:10 diluted serum 6501 in 0.5% BSA-TBS. The blocked plate was washed 5 times with TBS. The solution in the first microfuge tube was added to triplicate wells, 100 .mu.L per well. Identical amounts of the solutions in the second, third and fourth microfuge tubes were also added to triplicate wells. An aliquot of 100 .mu.L of the solution in the fourth microfuge tube was added to each of the three blocked blank wells of the microtitration plate. The plate was incubated for 1 hour at RT with agitation at 40 rpm in an orbital shaker (American Dade, Miami, Fla., Rotator V) and then washed 5 times with TBS. An aliquot of 100 .mu.L of 1:1000 diluted goat-anti-human-IgG/alkaline phosphatase conjugate (Zymed, South San Francisco, Calif., Cat. no. 62-8422) in 0.5% BSA-TBS was added and incubated for 1 hour at RT. The plate was then washed 5 times with TBS and the assay was developed by adding 100 .mu.L PPMP diluted substrate solution as described in Example 1. After 20 minutes, the reaction was stopped by adding 50 .mu.L of 0.2M Na.sub.2 HPO.sub.4 (Mallinckrodt, St. Louis, Mo., reagent grade) per well. The optical density was read at 550 nm in a microplate reader (Bio-Tek Instruments, Winooski, Vt., Model EL 311). The optical density at 550 nm versus the amount of the peptide per well was plotted in Graph Pad Prizm (Graph Pad Software, Inc., San Diego, Calif.). The amount of peptide required for 50% inhibition of binding of serum 6501 to tetravalent 3B10 was calculated from the graph.
Example 5
Truncation Experiments of Peptide 3B10 and the Resulting Peptide Competition ELISA Results
Microtitration plates (96-well, flat bottom polystyrene, Immulon-2, Dynatech Laboratories, Inc., Chantilly, Va.) were coated with 100 .mu.L/well for 1 hour at RT with tetrameric ACA-6501/3B10 peptide at 10 .mu.g/mL in carbonate buffer, pH 9.6 (15 mM Na.sub.2 CO.sub.3 /35 mM NaHCO.sub.3). After the liquid from the wells was removed, each well was blocked for 1 hour at RT with 200 .mu.L 0.5% (wt/vol) BSA (globulin-free, cat. no. A7638, Sigma Chemical Co., St. Louis, Mo.) in TBS. Three wells on the plate were left uncoated by tetravalent peptide to serve as blank control wells.
During the blocking step, each soluble, monomer peptide to be tested was set up in three test tubes (Eppendorf micro test tubes, Brinkmann Instruments, Westbury, N.Y.) each containing 30 .mu.L 5% BSA/TBS, 8.2 .mu.L ACA-6501 serum (at 1:10 dilution with 0.5% BSA/TBS), plus a variable volume of the peptide/TBS stock and the necessary volume of TBS buffer to yield a final volume of 330 .mu.L. The 330 .mu.L volume was sufficient to generate triplicate 100 .mu.L samples for each peptide concentration that was tested for its ability to block ACA-6501 binding to the tetravalent peptide-coated plate. For peptide 139, which was truncated at the amino terminus and lacks the framework ala-gly contribution normally tested, the concentration of the stock solution was approximately 340-400 .mu.g/mL TBS and aliquots of 19 .mu.L, 75 .mu.L, and 292 .mu.L were removed to prepare the three peptide concentration tubes. For peptide 142 (lacking only the N-terminal ala) and peptide 143 (not truncated), stock solution concentrations were 400-500 .mu.g/mL in TBS. Aliquots from each stock solution of 1 .mu.L, 4 .mu.L, and 16 .mu.L were removed to set up the three concentration tubes for each peptide. For the peptide monomer control tube (lacking peptide), a tube with a final volume of 660 .mu.L was prepared containing 60 .mu.L 5% BSA, 16.5 .mu.L of a 1:10 dilution of ACA-6501 and 583.5 .mu.L TBS, i.e., the same final concentrations (0.5% BSA and ACA-6501 serum at 1:400) as the 330 .mu.L tubes but with twice the volume and without peptide.
Following the blocking incubation, the plate coated with tetravalent peptide was washed 5 times with TBS. From each 330 .mu.L tube containing peptides 139, 142 and 143 at different concentrations, 100 .mu.L was added to coated triplicate wells. Three 100 .mu.L aliquots from the 660 .mu.L control tube without peptide were each added to coated wells and three additional 100 .mu.L aliquots were each added to uncoated blank wells. The plate was incubated at 40 rev/min on a rotary orbital shaker (Rotator V, American Dade, Miami, Fla.) for 1 hour at RT and then washed 5 times with TBS. One hundred .mu.L/well of goat antihuman IgG/alkaline phosphatase conjugate (Cat. no. 62-8422, Zymed, South San Francisco, Calif.) diluted 1:1000 in BSA/TBS was added to the microplate. After incubation for 1 hour at RT, the plate again was washed 5 times with TBS. Color development followed the addition of 100 .mu.L/well of freshly prepared dilute PPMP substrate solution. A dilute solution of PPMP (phenolphthalein monophosphate, cat. no. P-5758, Sigma Chemical Co., St. Louis, Mo.) was prepared by making a 1:26 dilution with water of the PPMP stock solution (0.13M PPMP, 7.8M amino-2-methyl-1-propanol adjusted to pH 10.15 with HCl). After 30 minutes, the reaction was stopped by adding 50 .mu.L/well of 0.2M Na.sub.2 HPO.sub.3 (reagent grade, Mallinckrodt, St. Louis, Mo.). Absorbance measurements at 550 nm were carried out on a microplate reader (Bio-Tek Instruments, Winooski, Vt.) and the A.sub.550nm vs. peptide added per well results plotted using Graph Pad Prizm (Graph Pad Software, Inc., San Diego, Calif.). As shown on FIG. 11, a horizontal line has been drawn corresponding to half-maximal the binding reaction obtained in the absence of monomer peptide competitive inhibition. The amount of peptide necessary for 50% inhibition can be read at the intersection of this line with the plot for each peptide tested. The 50% inhibition values are shown on FIG. 12. The results indicate that while the loss of the N-terminal Ala had no negative consequences, the additional loss of Gly increased by about 8-fold the concentration necessary to achieve 50% inhibition.
Example 6
Substitution of alpha methyl proline into 3B10 and the resulting ELISAs
Testing of peptides ACA-6501/3B10 and analogs in which prolines at the 3 and 9 position were replaced by .alpha.-Me-Pro was carried out using the methodology described in Example 5. Peptides 3B10, 726 (.alpha.Me-Pro substituted at the 3 position), 727 (.alpha.Me-Pro substituted at the 9 position), and 728 (.alpha.Me-Pro substituted at both the 3 and 9 positions) were tested as soluble monomer peptides in a competitive-binding ELISA using tetramer peptide 3B10-coated microtitration plates and ACA-6501 serum.
Microtitration plates were coated with tetramer 3B10 peptide as described for Example 5. For each of the four peptides tested, three peptide concentrations were prepared in tubes. As in Example 5, these twelve tubes had final concentrations of 0.5% BSA/TBS and ACA-6501 serum at a final dilution of 1:400 in a final volume of 330 .mu.L. All peptide stock solutions were at 400-500 .mu.g/mL TBS. To tubes containing 30 .mu.L of 5% BSA/TBS and 8.2 .mu.L of ACA-6501 serum (diluted 1:10 with 0.5% BSA/TBS), aliquots of 1 .mu.L, 4 .mu.L, or 16 .mu.L of each of the four peptide stock solutions were added in addition to an appropriate volume of TBS to achieve a final volume of 330 .mu.L. A control tube with no competitor peptide present was prepared with a final volume of 660 .mu.L as described in Example 5.
Following the blocking incubation of the tetravalent peptide-coated plate, three 100 .mu.L aliquots from each of the peptide concentration tubes for each of the four peptides as well as the control tube containing no peptide and blank controls were tested as described in Example 5. The microtitration plate ELISA procedures as well as the data handling were performed as described in Example 5. As shown in FIG. 13, peptide 727, where .alpha.-Me-Pro was substituted at the 9 position, was significantly more active than unmodified peptide 3B10 or the analog with both prolines changed (peptide 728). Peptide 726, which was substituted at position 3, lost activity as a result of the substitution.
Example 7
Conjugation of MG (myasthenia gravis) peptide to di-DABA PEG-3350
The peptide, Cys-Glu-Lys-Ile-Trp-Arg-Pro-Asp-Leu-Val-Leu-Gly(SEQ ID NO:58), corresponding to residues 83 to 92 of the human acetylcholine receptor (AC.sub.H R) alpha subunit was synthesized using standard Fmoc chemistry. 10 mg of the peptide was dissolved in 1 mL of 0.2M TRIS, pH 8.3. Five mg of N,N'-bis�3.5-bis(iodoacetamido)benzoyl! derivative of .alpha.,.omega.-bis�N-(2-aminoethyl)carbamoyl!poly-(ethylene glycol) (di-DABA PEG 3350) as described in U.S. patent application, Ser. No. 08/152,506, filed Nov. 15, 1993, which is incorporated by reference herein in its entirety, was added and the reaction was monitored by reverse-phase HPLC and found to be nearly complete after 20 minutes. The conjugate was purified from the unreacted peptide on a 2.2.times.25 cm Vydac C-18 column using reverse phase HPLC with an acetonitrile gradient from 32 to 40% over 10 column volumes. The fractions containing conjugate were pooled and lyophilized prior to use in tolerization.
Example 8
T cell assay for activation
Tritiated thymidine uptake by peptide-stimulated T cells was monitored in 96-well round bottom plates. A single-cell suspension of draining lymph node cells (mice) or isolated peripheral blood lymphocytes (human), 5.times.10.sup.5 were mixed with between 1 and 30 .mu.g of peptide in a final volume of 150 .mu.L per well and incubated for 5 days at 37.degree. C. in 5% CO.sub.2. At that point, 1 micro curie of labeled thymidine was added and incubated for an additional 15-24 hours. The harvested cells were collected on filters and counted by liquid scintillation spectrometry.
Example 9
Tolerization with the MG peptide in the Immunized Mouse Model (IMM)
Mice primed with alum and pertussis plus 10 .mu.g human MG.sup.83-92 peptide (see Example 7) conjugated to keyhole limpet hemocyanin (KLH) were treated after 3 weeks with various intravenous doses (shown on FIG. 14) of the same peptide conjugated to DABA PEG as described in Example 7 above. Five days later, mice were boosted with the MG-peptide KLH conjugate. After an additional 5 days, blood was taken from the mice and analyzed for antibodies against the peptide in an ELISA assay using MG-peptide-PEG coated on the wells of a microtitration plate. As shown in FIG. 14, administration of the conjugate had no effect on the antibody level against KLH but essentially abrogated the response to the MG peptide at the higher levels of tolerogen administration. These results confirm that a peptide tolerogen made according to the methodology of Example 8 is an effective tolerogen at relatively low levels.
Example 10
Abbreviated Description of Screen with 6626 Antibody and the Corresponding Sequences
Affinity purified ACA-6626 (AffACA-6626) was isolated by affinity purification from 8 mL of ACA-6626 plasma as previously described. AffACA-6626 (10 .mu.g) was incubated with the epitope xy'z phage library consisting of a pool of all p-III component libraries in a final volume of 100 .mu.L as previously described for ACA-6501 biopanning. Following three rounds of biopanning, randomly selected phage from the second and third rounds were tested by micropanning. Only a few clones were weakly immunopositive at a 1:1000 dilution. An additional 4th round of biopanning was carried out. Micropanning of 94 fourth round clones revealed 43 immunopositives, some at phage dilutions as high as 1:100,000. G-Tracking DNA sequencing of the 43 immunopositive clones carried out as previously described for ACA-6501 revealed 5 unique sequences. After conventional four base DNA sequencing, the translated amino acid sequences of Table 3 were obtained.
Example 11
Identification of Sequences Specific for the ACA from Patient 6644
The epi.sup.xy'z phage display library was screened using methods similar to those in Example 4 with ACA affinity purified antibody from patient number 6644. A colony blot assay as described previously was employed as the final identification step prior to peptide synthesis. Approximately 150 colonies were plated on the original nitrocellulose membrane and assayed. Antibody from patient 6644 was used at a concentration of 1 .mu.g/mL. Of the 150 colonies plated on the nitrocellular membrane and assayed, only 4 were strongly positive and 2 weakly positive in this screen. Sequencing of the inserts of the six positive phage selected by this screen revealed that the inserts were all derived from the 8-mer library with a free amino-terminus (epi.sup.z):
Gly-Ile-Leu-Ala-Leu-Asp-Tyr-Val-Gly-Gly(SEQ ID NO:59) (3 inserts)
Gly-Ile Leu-Thr-lle-Asp-Asn-Leu-Gly-Gly(SEQ ID NO:60) (1 insert)
Gly-Ile-Leu-Leu-Asn-Glu-Phe-Ala-Gly-Gly(SEQ ID NO:61) (2 inserts)
Example 12
Summary of Phage Library Screen with ACA-6641.
AffACA-6641 was isolated from 4 mL of plasma taken from patient number 6641. AffACA-6641 (10 .mu.g) was incubated with the pooled p-III phage libraries in a final volume of 100 .mu.L as described previously. Following four rounds of biopanning, 45 clones from the 3rd and 4th rounds were tested by micropanning. Of the 45, 23 scored negative. The 3rd round phage yielded two clones that scored 4+, two that scored 3+and two that scored 2+. From the 4th round, one clone scored 4+, one scored 3+and three scored 2+. G-tracking DNA sequencing revealed six unique sequences. Only one, clone 3G3, was strongly positive in the phage-capture ELISA. Four base DNA sequencing gave the following translated peptide sequence:
C L G V L G K L C(SEQ ID NO:62).
Example 13
Peptide conjugation to non-immunogenic, multivalent carriers
Several tetravalent platforms for the development of B cell tolerogens have been developed as described in co-owned and co-pending U.S. patent applications, Ser. Nos., 08/118, 055, filed Sep. 8, 1993, 08/152,506, filed Nov. 15, 1993, and U.S. Pat. Nos. 5,268,454, 5,276,013 and 5,162,515 which are incorporated by reference herein in their entirety. Candidate peptides selected by aPL antibody screens of epitope libraries are conjugated and tested for modifications of immunochemical behavior such as antibody binding. ##STR2## Compound 2
A solution of 8.0 g (5.7 mmol) of 1 in 50 mL of absolute EtOH and 35 mL of cyclohexene is placed under nitrogen, and 500 mg of 10% Pd on carbon is added. The mixture is refluxed with stirring for two hours. When cool, the mixture is filtered through Celite and concentrated to give 5.0 g of 2 as an oil. .sup.1 H NMR (50/50 CDCl.sub.3 /CD.sub.3 OD) d 1.21 (m, 8H), 1.49 (m, 8H) 1.62 (m, 8H), 2.19 (t, J=7.4 Hz, 8H), 2.67 (t, J=7.4 Hz, 8H), 3.36 (bd s, 16H), 3.67 (s, 4H), 3.71 (m, 4H), 4.21 (m, 4H).
Protected peptide with free carboxyl (PHN-peptide-CO.sub.2 H
A peptide is synthesized with standard solid phase methods using FMOC chemistry on a Wang (p-alkoxybenzyl) resin, using trifluoroacetic acid (TFA) stable protecting groups (benzyl ester or cyclohexyl ester on carboxyl groups and CBZ on amino groups). Amino acid residues are added sequentially to the amino terminus. The peptide is removed from the resin with TFA to provide a peptide with one free carboxyl group at the carboxy terminus and all the other carboxyls and amines blocked. The protected peptide is purified by reverse phase HPLC.
Peptide--Platform Conjugate, 4
The protected peptide (0.3 mmol) is dissolved in 1 mL of dimethylformamide (DMF), and to the solution is added 0.3 mmol of diisopropylcarbodiimide and 0.3 mmol of HOBT. The solution is added to a solution of 0.025 mmol tetraamino platform, 2 in 1 mL of DMF. When complete, the DMF is removed under vacuum to yield a crude fully protected conjugate 3. The conjugate, 3, is treated with HF in the presence of anisole for 1 hour at 0.degree. to give conjugate 4. Purification is accomplished by preparative reverse phase HPLC. ##STR3##
Compound 5--Platform with Four Carboxylic Acid Groups
Succinic anhydride (1.0 g, 10 mmol) is added to a solution of 861 mg (1.0 mmol) of 2 and 252 mg (3.0 mmol) of NaHCO.sub.3 in 20 mL of 1/1 dioxane/H.sub.2 O, and the mixture is stirred for 16 hours at RT. The mixture is acidified with 1N HCl and concentrated. The concentrate is purified by silica gel chromatography to provide 5.
Protected peptide with free amine (H.sub.2 N-peptide-CONH.sub.2)
A peptide is synthesized with standard solid phase methods on an amide resin, which results in a carboxy terminal amide after cleavage from the resin, using TFA stable protecting groups (benzyl ester or cyclohexyl ester on carboxyl groups and CBZ on amino groups). Amino acid residues are added sequentially to the amino terminus using standard FMOC chemistry. The peptide is removed from the resin with trifluoroacetic acid to provide a protected peptide with a free amine linker. The protected peptide is purified by reverse phase HPLC.
Peptide--Platform Conjugate, 7
A solution of 0.05 mmol of protected peptide with free amine, (H.sub.2 N-peptide-CONH.sub.2), 0.1 mmol of diisopropylethyl amine, and 0.01 mmol of 5 in 1 mL of DMF is prepared. BOP reagent (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate) (0.1 mmol) is added, and the mixture is stirred until the reaction is complete as evidenced by analytical HPLC. The peptide protecting groups are removed by treatment with HF in the presence of anisole at 0.degree. to give conjugate with protecting groups removed, 7. Compound 7 is purified by preparative reverse phase HPLC. ##STR4##
Compound 9
Concentrated sulfuric acid (100 uL) is added to a 60.degree. C. solution of 4.48 g (17.2 mmol) of triphenyl methanol and 1.62 g (15.3 mmol, 1.3 mL) of 3-mercaptopropionic acid in 35 mL of EtOAc. The mixture is stirred at 60.degree. C. for 10 minutes, allowed to cool to RT, and placed on ice for 1 hour. The resulting white solid is collected by filtration to give 4.52 g (75%) of 9.
Compound 10
Dicyclohexyl carbodiimide (2.41 g, 11.7 mmol) is added to a 0.degree. C. solution of 2.72 g (7.8 mmol) of 9 and 1.08 g (7.8 mmol) of p-nitrophenol in 41 mL of CH.sub.2 Cl.sub.2. The mixture is stirred for 16 hours allowing it to come to RT. The mixture is filtered to remove DCU, and the filtrate is concentrated. The residue is crystallized from hexane/CH.sub.2 Cl.sub.2 to give 3.17 g (86%) of 10 as pale yellow crystals.
Compound 11--cyclic thioether peptide with mercaptopropionyl linker attached
A solution of a cyclic thioether peptide (an analogue of a disulfide cyclized peptide in which one sulfur is replaced with a CH.sub.2) and sodium bicarbonate in water and dioxane is treated with p-nitrophenyl ester 10. If the peptide contains lysine, it must be appropriately blocked. The resulting modified peptide is treated with trifluoroacetic acid to provide thiol linker modified peptide 11.
Compound 13--conjugate of cyclic thioether Peptide and bromoacetylated platform
To a He sparged solution of 0.10 mmol of thiol modified peptide 11 in 100 mM sodium borate pH 9, is added 0.025 of bromoacetylated platform 12 as a 40 mg/mL solution in 9/1 MeOH/H.sub.2 O). The solution is allowed to stir under N.sub.2 atmosphere until conjugation is complete as evidenced by HPLC. The conjugate is purified by reverse phase HPLC.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 62(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 3(D) OTHER INFORMATION: /product="OTHER"/label= MeP/note= "alpha-methyl proline"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AlaGlyXaaCysLeuLeuLeuAlaProAspArgCysProGly1510(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 9(D) OTHER INFORMATION: /product="OTHER"/label= MeP/note= "alpha-methyl proline"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:AlaGlyProCysLeuLeuLeuAlaXaaAspArgCysProGly1510(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: group(3, 9)(D) OTHER INFORMATION: /product="OTHER"/label= MeP/note= "alpha-methyl proline"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AlaGlyXaaCysLeuLeuLeuAlaXaaAspArgCysProGly1510(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AlaGlyProCysLeuLeuLeuAlaProAspArgCysProGly1510(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GlySerGlySer(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:CysLeuLeuLeuAlaProAspArgCys15(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy (details pg. 16)(B) CLONE: 2101(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CysAsnIleLeuValLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz(details pg. 16)(B) CLONE: 5A12(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CysLeuIleLeuAlaProAspArgCys15(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 2D7(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:CysLeuLeuLeuAlaProAspArgCys15(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3B6(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:CysLeuValLeuAlaLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3E4(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:CysLeuPheValAlaLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3E7(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CysIleLeuLeuAlaHisAspArgCys15(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 2H1(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CysIleIleLeuAlaProGlyArgCys15(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3C10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:CysIleLeuLeuAlaLysAsnArgCys15(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3C5(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:CysIleValLeuValProAspArgCys15(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 2F4(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:CysLeuValIleAlaLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 5B1(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:CysTrpPheArgSerGlnSerSerCys15(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3E11(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:CysSerProIleLeuArgGlyAsnCys15(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xyz (details pg. 16)(B) CLONE: 3E8(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:CysHisLysPhePheTrpLeuThrCys15(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2A10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:CysThrIleLeuAlaProAspArgCys15(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2G12(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:CysLeuLeuIleThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2G11(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:CysLeuLeuIleThrHisAspArgCys15(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2F10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:CysAsnIleLeuValLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2E3(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:CysProLeuIleThrHisAspArgCys15(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2D12(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:CysLeuValLeuAlaAlaAspArgCys15(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 3B10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:CysLeuLeuLeuAlaProAspArgCys15(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 3F2(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:CysPhePheHisPheAspHisSerCys15(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 2D3(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:CysProLeuHisThrHisHisThrCys15(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: G11(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:CysThrIleLeuThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H5(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:CysThrIleLeuThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H2(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:CysThrIleLeuThrLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:CysThrLeuLeuThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2E10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:CysIleGlnLeuThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 1B7(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:CysHisLeuLeuThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H1(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:CysLeuIleLeuThrProAspArgCys15(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H12(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:CysSerIleLeuAlaProAspArgCys15(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 1A4(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:CysAsnLeuLeuAlaLeuAspArgCys15(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H6(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:CysAsnLeuLeuAlaIleAspArgCys15(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 1C3(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:CysLeuLeuLeuAlaIleAspArgCys15(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 1D10(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:CysThrIleIleThrGlnAspArgCys15(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2H4(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:CysAsnIleIleThrArgAspArgCys15(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 2G12(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:CysIleLeuHisAlaAlaHisArgCys15(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: Custom (X)6(B) CLONE: 1A9(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:CysSerSerLysSerTyrTrpArgCys15(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 4B11(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:CysGlyAsnAlaAlaAspAlaArgCys15(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 4D3(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:CysThrAsnTrpAlaAspProArgCys15(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 4C7(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:CysGlyAsnIleAlaAspProArgCys15(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 4G7(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:CysThrAsnLeuThrAspSerArgCys15(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(vii) IMMEDIATE SOURCE:(A) LIBRARY: xy'z (details pg. 16)(B) CLONE: 4A2(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:CysGlyAsnProThrAspValArgCys15(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:GlyIleLeuLeuAsnGluPheAla15(2) INFORMATION FOR SEQ ID NO:50:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:GlyIleLeuThrIleAspAsnLeu15(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:GlyIleLeuAlaLeuAspTyrVal15(2) INFORMATION FOR SEQ ID NO:52:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:LeuSerAspProGlyTyrValArgAsnIlePheHis1510(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:LeuThrAspProArgTyrThrArgAspIleSerAsnPheThrAsp151015(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:AlaGlyProCysLeuLeuLeuAlaProAspArgCysProGly1510(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:GGGCTGGACCCNNKCCGGGGGCTGCTG27(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:GGGTCCAGCCCCGT14(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:CAGCCCCCGG10(2) INFORMATION FOR SEQ ID NO:58:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:CysGluLysIleTrpArgProAspLeuValLeuGly1510(2) INFORMATION FOR SEQ ID NO:59:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:GlyIleLeuAlaLeuAspTyrValGlyGly1510(2) INFORMATION FOR SEQ ID NO:60:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:GlyIleLeuThrIleAspAsnLeuGlyGly1510(2) INFORMATION FOR SEQ ID NO:61:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:GlyIleLeuLeuAsnGluPheAlaGlyGly1510(2) INFORMATION FOR SEQ ID NO:62:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:CysLeuGlyValLeuGlyLysLeuCys15__________________________________________________________________________

Number	Name	Date
5162515	Conrad et al.	Nov 1992
5268454	Barstad et al.	Dec 1993
5276013	Conrad et al.	Jan 1994
5344758	Krilis et al.	Sep 1994
5472883	Matsuura et al.	Dec 1995
5498538	Kay et al.	Mar 1996
5506110	Matsuura et al.	Apr 1996
5552391	Coutts et al.	Sep 1996

APL immunoreactive peptides, conjugates thereof and methods of treatment for APL antibody-mediated pathologies

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