WO2001002001A9 - PEPTIDE LIGANDS THAT BIND IgM ANTIBODIES AND BLOCK INTERACTION WITH ANTIGEN - Google Patents

Abstract

This invention relates to peptides which have the unique capacity to bind selectively to immunoglobulin molecules of class M of all or substantially all mammalian species without selectively binding to immunoglobulins of other classes. More specifically, such peptides bind in a manner that inhibits the specific binding of IgM antibodies to their antigens.

Description

TITLE: PEPTIDE LIGANDS THAT BIND IgM ANTIBODIES AND BLOCK

INTERACTION WITH ANTIGEN

INVENTORS: Pati M. Glee, Seth H. Pincus, James B. Burritt and Jim E. Cutler

RELATED APPLICATIONS

This application incorporates by reference the specification, claims and drawings of the following U.S. provisional applications: Application No. 60/142,048 filed July 2, 1999, Application No. 60/142,389 filed July 6, 1999, and Application No. 60/142,524 filed July 7, 1999.

FIELD OF THE INVENTION

This invention relates to peptides which have the unique capacity to bind selectively to immunoglobulin molecules of class M of all or substantially all mammalian species without selectively binding to immunoglobulins of other classes. More specifically, such peptides bind in a manner that inhibits the specific binding of IgM antibodies to their antigens.

FEDERAL FUNDING

The disclosed invention was supported by grants RO1AI42184, RO1AI24912, RO1 AI41502, and PO1 AI31769 from the National Institutes of Health, and grant 9704584S from the American Heart Association. The United States government has certain rights in the invention.

BACKGROUND Immunoglobulin molecules of class M (IgM) are antibodies that form the principal component of the primary immune response to antigens in mammals and they are particularly associated with an initial immune reaction to bacterial infections. The basic structure of an IgM molecule is similar to IgG, one of the members of the immunoglobulin family of molecules which is also secreted into serum. However, the heavy chain of an IgM molecule, designated μ, has an additional 20 amino acids at the C terminal end that form a "tail" on the molecule, compared to an IgG molecule. In its monomeric form IgM serves as an antigen receptor on the surface of virgin B cells and the longer C terminal "tail" forms a transmembrane domain.

In addition to extra length on the heavy chain, as it exists in serum IgM has a pentameric structure. Thus, IgM molecules are the macromolecules of the immunoglobulin family of compounds as they exist in serum. Serum IgM has a molecular weight of approximately

900,000. In the pentameric form IgM exhibits high antigen avidity and once antigen-complexed IgM fixes complement with high efficiency, resulting in high hemolytic efficiency. A red blood cell can be lysed by a single IgM complex (see IMMUNOLOGY, IMMUNOPATHOLOGY & IMMUNITY, Stewart Sell editor, 1996, Appleton & Lange, Stamford, CT, pp. 101-103; 118-120; 197-198; and 328-330).

1. Purification of IgM

A purified preparation of IgM would be of great benefit as a reagent in conducting basic research on the molecular structure of IgM and the interaction of IgM with various antigens and other components of the immune response, such as complement. The development of molecules effective for isolation of pure IgM molecules from serum has been difficult and there is a need for a purification technique that would be effective. U.S. Patent 5,077,391, issued Dec. 31, 1991 to Raison et al., is generally directed to the use of a protein known in the art as Clq bound to an insoluble matrix for chromatographic contact with serum or other biological samples to enrich for IgM molecules. A similar IgM binding method is generally discussed in U.S. Patent 5,112,952, in which a mannan binding protein is the operative IgM binding molecule. Pierce

Chemical Co., the indicated assignee of U.S. Patent 5,112,952, advertises a product identified as ImmunoPure® Immobilized Mannan-Binding Protein in their on-line catalog and indicates that the "recommended protocol should result in mouse IgM that is at least 90% pure by HPLC," see PIERCE Product Description No. 22212. However, mannan binding protein does not bind IgM exclusively and while functional with IgM from mouse it is not efficient at binding IgM found in the serum of other species, including human IgM molecules.

A few IgM binding peptides have also been described, for example in WO 98/26794 and EP 0752425 A2, which peptides interfere with IgM/receptor binding. These peptides are not IgM specific and they do not block IgM/ antigen binding. Technogen has offered a commercial product, KAPTIN-M™ that is advertised as a chromatographic matrix for purifying IgM molecules from any source and characterizes the ligand as "a low molecular weight synthetic peptidornimetic, capable of binding to the constant portion of IgM and not interfering with the antigen binding site," TECHNOGEN product pamphlet description.

2. Uses of Purified IgM B cells play a crucial role in both cellular immunity and antibody production. The ability to label and purify B cells in which IgM is membrane bound and functioning as an antigen receptor to enable further research involving these complex cells would be particularly beneficial. This is certainly true with respect to fetal and neonatal immune responses, in which IgM production is dominant. A source of such B cells with surface expressed IgM will allow researchers to conduct various analyses including determining antigen specificity, elucidating mechanisms for B cell ingestion, B cell activation cascades, and the specific cellular events associated with major histocompatibility complex display (for a general discussion of surface immunoglobulins on B cells see EVIMUNOLOGY THE SCIENCE OF SELF-NONSELF DISCRIMINATION, Jan Kleig editor, 1982, John Wiley & Sons, New York, NY, pp. 234-240). The peptides disclosed herein are useful for both labeling and purifying B cells in which IgM is surface bound.

Additional immunodiagnostic methods which will benefit from the ability to use the unique peptides disclosed herein include imaging interactions, antibody class identification, and identification of the specific characteristics of antibody/antigen complexes that are specific to or largely involve IgM immunoglobulins. Also, peptides which interact specifically with IgM in a cross-species manner facilitate any number of diagnostic methods for analyzing IgM specific antigen interactions that are observable across mammalian species and those which are limited to a particular species. Experimentation with IgM molecules in both the pentameric form found in serum and as a membrane-bound B cell receptor will be enhanced by the various methods and products disclosed herein.

Immunotherapy with serum preparations that have been enriched for IgM are generally discussed in U.S. Patent 5,612,033. Therapies involving peptides which interact with complex binding to the Fc portion of immunoglobulins is generally described in U. S. Patent 4,628,045. Such therapies also will be enhanced by the various methods and products disclosed herein.

SUMMARY OF THE INVENTION The present invention relates to peptides, or proteins comprising such peptides, that selectively bind to IgM antibodies, preferably substantially all IgM antibodies, but does not selectively bind to antibodies of other classes. In addition, the invention relates to peptides that cause release of specifically bound antigen from an IgM-antigen complex or that inhibit the specific binding of an antigen to IgM. Preferably, the peptides are capable of selectively binding to the IgM molecules of a plurality of mammalian species, including both the pentameric and monomeric forms of IgM, particularly human or murine IgM. Such peptides are preferably capable of purifying any and all IgM molecules from any and all mammalian species.

In one embodiment of the present invention, peptides are provided consisting of or comprising the sequence W-I-X_rX₂-X₃-X₄-W, wherein W = trytophan, I = isoleucine, X] = proline or serine, X₂ = glutamine, alanine, glycine or serine, X₃ = glutamic acid, alanine, serine, arginine, glycine, valine, lysine, asparagine or tyrosine, and X₄ = serine, aspartic acid or alanine.

In another embodiment of the invention, peptides are provided consisting of or comprising the sequence Xι-X₂-X₃-X₄-X₅-D-W wherein X_t = alanine, glycine or valine, X₂ = isoleucine or valine, X₃ = lysine, tyrosine, threonine or tryptophan, X₄ = serine, glycine or glutamine, X₅ = glycine, lysine or valine, D = aspartic acid and W = trytophan.

Another embodiment, peptides are provided consisting of or comprising the sequence I- W-Xj-X₂-D-W, wherein I = isoleucine, W = trytophan, X, = glutamine or serine, X₂ = arginine, lysine or asparagine, and D = aspartic acid. Preferred peptides are selected from the group consisting of SWISSRDWT,

SWISSKDWT and YDWTPSSAW or the group consisting of DWTDQMYDW and QKWISSAWD.

Multimeric forms of these peptides are contemplated as well.

The present invention also relates to nucleic acid molecules encoding the foregoing peptides, to recombinant vectors comprising such nucleic acid molecules and to cells transformed with such vectors.

In another aspect, the invention relates to antibodies elicited by the foregoing peptides.

Yet another aspect of the invention relates to a method of detecting the presence of IgM in a test sample comprising the steps of immobilizing an inventive peptide on a substrate; contacting the substrate with the test sample; and detecting IgM bound to the peptide. Related methods provide a technique for purifying IgM from a sample comprising the steps of immobilizing an inventive peptide on a substrate; contacting the IgM containing sample with the substrate; and removing the bound IgM from the immobilized peptide to obtain purified IgM, preferably by washing the substrate with a solution having a pH effective to cause the removal of bound IgM. In a preferred embodiment, the removed IgM is at least about 95% pure relative to other classes of immunoglobulin.

The invention also provides a method to isolate an antigen specific IgM population comprising the steps of immobilizing an antigen on a substrate; contacting the substrate with a sample containing IgM; and further contacting the substrate with an inventive peptide to remove IgM specifically bound to the antigen. In a preferred embodiment, the specifically bound IgM is at least about 95% antigen specific relative to other IgM molecules.

Thus, the present invention also relates to IgM populations, or subpopulations, produced by the foregoing methods. Such methods are applicable to samples such as ascites fluid, serum and hybridoma cell culture supernatant, by way of example.

In another of its aspects, the present invention relates to methods to isolate an antigen bound by a specific IgM population, comprising the steps of immobilizing the antigen specific IgM on a substrate; contacting the substrate with a sample containing the antigen; and further contacting the substrate with an inventive peptide to remove antigen specifically bound to the IgM.

Yet another aspect of the invention relates to a method of isolating B cells from a sample, where the B cells having at least one IgM molecule integrally present on the cellular membrane, comprising the steps of immobilizing an effective amount of an inventive peptide on a substrate; contacting the substrate with the sample for a time and under conditions effective to permit binding of the B cells to the immobilized peptide; and isolating peptide bound B cells. In a preferred embodiment, the B cell-containing sample is obtained from peripheral blood lymphocytes or heparinized blood, by way of example, and may contain human B cells or B cells from other animals including mouse, rat, goat, rabbit, cow, horse, dog, guinea pig and sheep.

A further aspect of the invention relates to methods of treating a human diseases that are associated with IgM antibodies, such as rheumatoid factor binding to IgG, isohemaglutinin binding to red blood cells, autoimmune hemolytic anemia, paraneoplastic syndromes, cancer, multiple myeloma or autoimmune diseases. One such method involves administering to a patient a peptide of the invention, optionally a multimeric form of the peptide, in an amount effective to disrupt the binding of antigen to IgM antibodies. The invention also provides for a composition comprising a peptide of the invention in association with a therapeutic agent, and a method of administering said composition such that the peptide therein selectively localizes a therapeutic agent to IgM-expressing cells. Examples include but are not limited to therapeutic agents conjugated to said peptide, for example following production as a recombinant fusion protein or following chemical linking of a peptide of the invention to a therapeutic agent. A peptide of the invention might provide a means of removing IgM from serum, in an extracorporeal procedure involving the perfusion of patient serum over a peptide immobilized on a substrate, followed by return of the IgM-depleted serum to the patient.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

Figure 1 shows the MAb B6 reactivity to pill-displayed peptides from various phage clones. Lane 1 was stained with amido black to show the proteins contained in the high molecular mass standards. Lanes 2 - 18 contain the following phage clones blotted onto ntirocellulose membrane (NCM): lane 2- esl09, displayed sequence RDVAHKSMY, lane 3 - eslO QQGKYWTSM; lane 4- es81 EWSAQPIWD; lane 5- es40 NTRGMDWWE; lane 6 - ed9 EABYSKDWL; lane 7 - ed23 AGIWQKDWL; lane 8 - ed36 SWISSRDWT; lane 9 - ed42 AGAIWQRDW; lane 10 - ndl GGIVARLTG; lane 11 - nd8 LHYVRSYN; lane 12 - nd9 SAKDPLLGA; lane 13 - ns2 AGQDEWWG; lane 14 - ns4 KLRRAMHWD; lane 15 - nsl4 WVSKASTVW; lane 16 - es29 YYSSVPPGA; lane 17 - edl YDWIPSSAW; lane 18 - M13KBst library parent vector. The NCM was then incubated with IgM monoclonal antibody B6. Binding of antibody B6 was detected with enzyme-conjugated anti-mouse IgM. Phage clones in lanes 2-17 were present in the final round selection pool of selection on mAb B6.1. Only those in lanes 4, 6, 7, 8, 9 and 17 bound IgM.

Figure 2 shows immunoblots of phage edl and irrelevant clone J508,22 probed with various antibodies. Phage were run on SDS-PAGE gels and blotted onto nitrocellulose. Blot strips were incubated with various mAbs or polyclonal antibodies and appropriate secondary antibodies to determine immunoglobulin binding to the pffl-displayed peptides from clone edl and irrelevant clone J508,22. Clone edl displays peptide YDWIPSSAW and clone J508,22 displays peptide AQPQVRPIG. Clone J508,22 was selected as an irrelevant control phage which represents the epitope of mAb 44.1, an IgG (9). In each panel, the migration point of pill is labeled. A. Phage edl blot strips in lanes 1 - 13 were incubated with the following IgM mAbs: lane 1 - B6.1; lane 2 - 2B3.1, lane 3 - H9, lane 4 - A4.1, lane 5 - C3.4, lane 6 - JD3, lane 7 - A6.1, lane 8 - C6, lane 9 - 10G, lane 10 - mt9A, lane 11 - mt5A, lane 12 - mt6F, and lane 13 - P3x63. Lane 14 was incubated with a mouse anti-Candida polyclonal ascites, and lane 15 with a rabbit anti-Candida antiserum. Lanes 1 - 14 and control (no primary antibody) lane 16 were incubated with affinity purified AP-conjugated goat anti-mouse μ-chain specific secondary antibody. Lane 15 and control (no primary antibody) lane 17 received affinity purified HRP- conjugated goat anti-rabbit polyvalent Ig secondary antibody. B and C. Western blot strips of clone edl (panel B) and clone J508,22 (panel C) were probed with the following primary antibodies: lane 1 - IgM mAb S10, lane 2 - IgM mAb T17, lane 3 - IgG mAb 44.1, lane 4 - IgG mAb 6C5-H4, and lanes 5 and 6 - blocking buffer only. Secondary antibodies were 1 : 1000 dilutions of affinity purified AP conjugates: lanes 1, 2, and 5 - goat anti-mouse μ-chain specific antibody, and lanes 3, 4, and 6 - goat anti-mouse g-chain specific antibody. D. Western blot strips of clone edl. Lanes 1 and 3 were incubated with 1:500 human serum while control strips

2 and 4 were incubated in block alone. Secondary antibodies were 1 : 1000 dilutions of affinity purified AP conjugates: lanes 1 and 2 - goat-human g-chain specific antibody, and lanes 3 and 4 - goat anti-human μ-chain specific antibody.

Figure 3 shows binding of phage to IgM and IgG antibodies. Microtiter wells were coated with the indicated mAbs. Phage (109/well) were incubated with the mAbs. Unbound phage were washed out, and the remaining phage, bound to the antibody, were detected with anti-M13 antibody, enzyme-conjugated secondary antibody and colorimetric substrate. The results are shown as A405 (mean and SEM). S9 is an IgM antibody binding to a carbohydrate antigen, S10 and T17 are IgM anti-protein antibodies. Antibodies 924 and 41.1 are IgG. Figure 4 shows that phage and peptide conjugates bind to anti-GBS mAb S9. A. Binding of phage to S9. ELISA plates were coated with mAb S9. Varying amounts of phage were added to the wells, incubated and then washed. Phage binding was detected with rabbit anti-M13 antiserum followed by alkaline phosphatase-conjugated anti-rabbit Ig. The results are reported as A405. B. Binding of peptide to S9. ELISA plates were coated with mAb S9. Varying concentrations of edl -KLH conjugate, pep2 (WENWMMGNA)-KLH conjugate, or KLH were added to the wells, incubated and then washed. Binding was detected with rabbit anti-KLH antiserum followed by alkaline phosphatase-conjugated anti-rabbit Ig. The results are reported as A405.

Figure 5 shows binding of phage to cell-surface IgM. Murine B-cell lymphomas 5F5 (surface IgM+) and 4F10 (surface IgM-) were incubated with 3 X 109 phage, washed, and incubated with rabbit anti-M13 antiserum followed by FIT C-conjugated anti-rabbit Ig (two left sets of panels), or were incubated only with an FITC-conjugated anti-mouse μ-chain (right panel). Labeled cells were analyzed by flow cytometry. Cell number is on the vertical axis, fluorescence intensity on the horizontal.

Figure 6 shows that phage inhibit the binding of IgM but not IgG antibodies to GBS. Anti GBS antibodies were incubated with the indicated phage and then plated onto ELISA plates coated with GBS. The binding of antibodies to GBS was determined with enzyme-conjugated secondary antibodies and substrate. The results are A405 (mean and SEM, where no error bars are seen they are obscured by the symbols). Antibodies S9 and S10 are IgM, 2A6 and IB 1 are IgG. Antibodies S9, 1B1, and 2A6 all recognize the same antigen, the type III capsular polysaccharide, while S 10 identifies the Ibc protein antigen.

Figure 7 shows that phage and peptide conjugates inhibit antigen binding. A. Inhibition of S9 binding to GBS by phage. ELISA plates were coated with intact GBS. S9 antibody (O.lμg/ml) was premixed with varying numbers of different phage and then added to the ELISA plate. Following an incubation, the wells were washed and S9 binding detected with alkaline phosphatase anti mouse IgM. The results are reported as A405. B. Inhibition of S9 binding to

GBS by peptide-KLH conjugates. ELISA plates were coated with intact GBS. S9 antibody (O.lμg/ml) was premixed with varying concentrations of peptide-KLH conjugate and then added to the ELISA plate. Following an incubation, the wells were washed and S9 binding detected with alkaline phosphatase anti-mouse IgM. The results are reported as A405. Figure 8 shows inhibition of mAb binding to C. albicans carbohydrate antigens by intact phage edl. Microtiter wells were coated with C. albicans cell wall extract or buffer alone. Wells received mAb B6.1 (panel A) or mAb B6 (panel B); samples were uninhibited (mAb only), premixed and incubated with phage (mAb + edl or mAb + M13KBst), or added to wells with subsequent spiking of phage (mAb + later edl or mAb + later M13KBst) as described in the methods section. Absorbance values are the average of triplicate wells shown with standard deviations. Figure 9 shows inhibition of rheumatoid factor binding by phage edl. A human serum containing high titer RF was mixed with 1.8 X 1010 phage, or an equal volume of buffer, and with latex beads coated with aggregated rabbit IgG. The samples were placed on flat black cardboard slides and rotated gently. Fifteen mmutes later, agglutination was detected in samples with no phage and with the parental M13, but not in the sample with edl phage.

GENERAL DESCRIPTION

The invention disclosed herein is directed to the surprising discovery of peptides which possess the unique and novel properties of specifically binding substantially to IgM class immunoglobulin molecules, in a non-antigen specific manner, while not specifically binding significantly to immunoglobulins of the other classes, such as IgG, IgE, and IgA. Even more surprising is that the IgM-binding properties of these peptides are not limited to any one mammalian species, but have shown binding affinity for the IgM immunoglobulins of many different species, including human, mouse, rat, rabbit, and goat. An additional unique feature of the peptides disclosed herein is their capacity to bind to the IgM molecules in a manner which disrupts or prevents the binding of an antigen to which the IgM antibodies are specifically directed, in both the pentameric and monomeric, B -cell-attached forms of the IgM antibody. This universal or ubiquitous interference with the antigen/antibody interactions in a non-antigen specific manner has not been reported in the prior art. In addition to the peptides themselves, this invention further provides efficient means of producing the peptides; antibodies which bind to the peptides; methods of purifying IgM and populations of B-cells comprising surface-attached IgM; various immunodiagnostic and immunotherapeutic methods related to the peptides and IgM-containing products; and, kits designed to implement the various methods. The present invention relates to a set of peptides with IgM-binding activity. These peptides were discovered through the use of a peptide-display phage library to select for peptides that mimic the antigens recognized by IgM antibodies. These peptides bound to IgM antibodies from mice, rabbits, and humans, but not to IgG antibodies from those species. Binding activity was seen when the peptides were displayed as chimeras on the termini of the pill protein on intact phage or when a synthetic peptide was conjugated to a carrier protein. IgM antibodies specific for both carbohydrate and for protein antigens were bound. Unconjugated peptides were not tested because of insolubility in water at physiologic pH. Peptides bound to multimeric IgM and to the monomeric cell-surface form of IgM. Most surprisingly, the peptides inhibited the binding of antigen to IgM antibodies, inhibition of antigen binding was shown for five distinct antibodies recognizing microbial carbohydrate and protein antigens and an aggregated protein autoantigen. The site of peptide binding on the IgM antibodies has not been defined. Because the peptides bind to all IgM's tested, but to no antibodies of other classes, it is likely that the site is located within the heavy chain constant regions. Since the peptides bind to IgM antibodies of multiple species, presumably this site is well-conserved. Because the peptides bind to monomeric IgM, the J-chain is an unlikely site of binding. Because of its effect on antigen binding, it is possible that the peptide binds the Fab. To determine whether these peptides represent a portion of a known IgM ligand, we performed a BLAST search utilizing the National Center for Biotechnology Information web site; no homologous protein sequences were identified. We have experimentally demonstrated that peptide binding does not affect complement activation, suggesting that the peptide binding site is not physically close to the C 1 q-binding site.

Although there, is variability in the sequence of the different IgM-binding peptides, there are several well conserved elements as well. A tryptophan is always found in the C-terminal or penultimate position, with an aspartate residue located on the N-terminal side of the tryptophan in the large majority of sequences. Interestingly when there is not an aspartate adjacent to the C- terminal tryptophan, elsewhere in the sequence there is an aspartate-tryptophan set, suggesting that this may be a minimum requirement for IgM binding. Many of the residues are hydrophobic. Clearly the avidity of binding is influenced by the sequence of the displayed peptide (figure 4), with the sequence YDWTPSSAW (edl) having the highest binding activity for murine IgM. Although data show that some of these peptides bind to IgM antibodies of other species, we have not determined whether the sequence requirements for binding are the same for species other than mouse.

The mechanism whereby the peptides inhibit the binding of antibody to antigen is intriguing, because the peptides do not appear to bind within the antibody variable region. Two potential mechanisms are steric hindrance or the induction of a conformational change in the IgM molecule that hinders the binding to antigen. Although steric hindrance has not been absolutely ruled out, for several reasons this is not believed to be the mechanism. The peptides bind to constant-region determinants and it is well established that anti-isotypic antibodies do not block antigen binding. While the phage displaying the peptides are quite large and could conceivably cause steric inhibition over a large area, the peptides conjugated to KLH are smaller and less likely to sterically hinder. Without being bound by this mechanism, it appears from the stoichiometry of the interaction that the mechanism is more than simple blocking of the antigen combining site on the IgM, since virtually complete inhibition of antigen binding was seen at approximately 1:1 molar ratio of displayed peptide to pentamer IgM (figure 7A).

However, to definitively prove that the mechanism of inhibition is not steric, one would construct a small soluble peptide capable of binding to IgM and then test that molecule for inhibition of antigen binding. If steric hindrance does not account for the inhibition of antigen binding, then it is possible that the peptide either induces or prevents a conformational change in the IgM molecule. There is clear evidence that IgM molecules undergo a conformational shift upon antigen binding (23,24), and it is presumed that this shift, a lifting of the Fab arms from the planar structure of the molecule, allows for greater accessibility of the antigen for the combining sites present on the large IgM molecule. Perhaps the IgM-binding peptides interfere with this process. Alternatively, the peptides might insert between unique CH1-CL1 interactions of the IgM and disrupt the antigen-binding site.

The IgM-binding peptides may represent potential artifacts when peptide-display phage libraries are used to epitope map IgM antibodies, since such peptides appear to bind to an IgM antibody in a manner analogous to antigen. It is only when the specificity of binding and antigen inhibition are tested on irrelevant IgM antibodies that the true nature of the peptide binding is revealed.

I. Definitions The term "analog" means peptide molecules that contain single or multiple substitutions, deletions and/or additions of any component(s) naturally or artificially associated with the IgM- binding peptides disclosed herein such as carbohydrate, lipid and/or other proteinaceous moieties. This term is not intended to encompass peptides which bind with specificity to IgM antibodies in the manner of an antigen per se. The term "antibody" as used herein, unless indicated otherwise, is used broadly to refer to antibody molecules. The term "antibody" shall have the ordinary meaning given to the term by a person of ordinary skill in the art of immunology.

Antibodies of the invention may be isolated from a hybridoma cell, the serum of a vertebrate, recombinant eukaryotic or prokaryotic cells transfected with a nucleic acid encoding the antibody, which may include plant cells, ascites fluid, or the milk of transgenic animals. Antibodies described herein also may contain alterations of the amino acid sequence compared to a naturally occurring antibody. In other words, the antibodies of the invention need not necessarily consist of the precise amino acid sequence of their native variable region or constant region framework, but contain various substitutions that improve the binding properties of the antibody to its cognate antigen or change the binding of the antibody to effector molecules such as complement or the Fc receptor. In another format, a minimal number of substitutions are made to the framework region in order to ensure reduced, and preferably, minimal immunogenicity of the antibody in humans. In preferred embodiments of recombinant antibodies of the invention, any non-human framework regions used may be altered with a minimal number of substitutions to the framework region in order to avoid large-scale introductions of non-human framework residues.

The term "antigen" means a molecule that is specifically recognized and bound by an antibody. The specific portion of the antigen that is bound by the antibody is termed the "epitope".

The term "humanized antibody" refers to an antibody which is substantially human in structure; that is, it derives at least substantially all of its constant regions from a human antibody even though all or apart of its variable regions are derived from some other species. "Human antibody" refers to an antibody which is encoded by a nucleotide of human origin and such nucleotides may be modified by the skilled artisan by known nucleotide manipulation techniques.

The term "inhibit" or "inhibition" as used herein refers to a decreased level of activity, binding, interaction or reaction, e.g., as between an IgM antibody and its specific antigen.

As used herein, "modulate" means an significant increase or decrease, or change in the qualitative nature of an endpoint. For example an increase or decrease in the severity of an immune response, or an alteration in the course of the immune response. The term "isolated" or "substantially pure" as used herein refers to an antibody or, for example, a fragment thereof, or a nucleic acid, or a peptide according to the present invention, which is substantially free of other antibodies, nucleotides, nucleic acids, proteins, peptides, lipids, carbohydrates or other materials with which it is naturally associated, or associated as a consequence of its manufacture. One skilled in the art would be able to isolate or to substantially purify, e.g., the peptides, nucleic acids and antibodies described herein using conventional methods for antibody or protein purification.

As used herein, "nucleic acid" is defined as RNA or DNA that encodes a peptide as defined above, or is complementary to nucleic acid sequence encoding such peptides, or hybridizes to such nucleic acid and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least 75% sequence identity, preferably at least 80°/o, and more preferably at least 85°Λ>, with the peptide sequences. Specifically contemplated are genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbone or including alternative bases whether derived from natural sources or synthesized. Such hybridizing or complementary nucleic acids, however, are contemplated as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a peptide according to the present invention.

The term "peptide" means an amino acid containing molecule which when designated by sequence reads left to right from the amino to the carboxyl end and includes modified amino acids as well as unmodified amino acids. While small peptides are exemplified the intention is to include any proteinaceous material which contains the described peptides in a manner in which they retain the IgM interactions characterized herein.

The terms "selective binding" and "selectively bind" mean that, in a mixture of antibodies of multiple classes, the peptide preferentially binds to antibodies of the IgM class wherein at least about 50% of the peptide binds IgM antibodies, more preferably about 70%, more preferably about 80 to 85%, even more preferably about 90% and most preferably at about 95% or more of the peptide binds IgM antibodies relative to its binding to antibodies of other classes. "Stringent conditions" are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium titrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 °C. Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2 x SSC and 0.1% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. The terms "selective binding" and "selectively bind" mean that, in a mixture of antibodies of multiple classes as well as other serum or cellular proteins and products, the peptide preferentially binds to antibodies of the IgM class wherein at least about 50% of the peptide binds IgM antibodies, more preferably about 70%, more preferably about 80 to 85°/o, even more preferably about 90% and most preferably at about 95% or more of the peptide binds IgM antibodies relative to its binding to antibodies of other classes. The term "therapeutically effective" generally mean that the, e.g., peptide is effective to decrease the number of IgM-antigen complexes otherwise found in the circulation of a host or a localized site. In addition, the phrase "therapeutically effective" means that the peptides, nucleic acids or antibodies or pharmaceutical compositions thereof according to the present invention are able to interact with such IgM-antigen complexes associated with diseases that are mediated in part by the presence of IgM-antigen complexes. Preferably, the treatment methods of the present invention are effective to reduce by at least about 20%, more preferably 40%, even more preferably 60% and most preferably 90% or such disease associated IgM-antigen complexes in an infected mammalian host in a therapeutic course of treatment.

The terms "variable region" and "constant region" used in reference to antibody and immunoglobulin molecules have the ordinary meaning given to the term by a person of ordinary skill in the art of immunology. Both antibody heavy chains and antibody light chains may be divided into a "variable region" and a "constant region." The point of division between a variable region and a contrast region may be determined by the person of ordinary skill in the art by reference to standard texts describing antibody structure. See, e.g., Kabat et al, "Sequences of Proteins of Immunological Interest: 5th Edition" U.S. Department of Health and Human Services, U.S. Government Printing Office (1991). The abbreviations used in this specification have the meanings that would be known to the skilled artisan from the context of their use. Some of these abbreviations include: A, absorbance; AP, alkaline phosphatase; DPBS, Dulbecco's PBS; GBS, group B streptococci; HRP, horse radish peroxidase; KLH, keyhole limpet hemocyanin; NCM, nitrocellulose membrane; PDPL, peptide-display phage library; RF, rheumatoid factor; TBS, tris buffered saline.

II. DETAILED DESCRIPTION A. Phage Display The present invention is based on the selection of a peptide-display phage library on IgM antibodies, from which the present inventors have identified a panel of phage expressing peptides that bind to IgM antibodies in general, but not to antibodies of other classes. A synthetic peptide corresponding to one of the displayed peptide sequences also binds to IgM antibodies. The peptides bind to both soluble pentameric antibodies and to monomeric cell surface IgM. The phage-displayed and synthetic peptides inhibit the binding of IgM antibodies to antigen. These peptides may create confounding artifacts when IgM antibodies are used for epitope mapping studies. Nonetheless, the peptides may have both experimental and therapeutic utility.

The use of bacteriophage display peptide libraries (PDPL), termed epitope or "mimotope" libraries, greatly facilitates identifying peptide ligands for proteins of interest (for reviews, see (1,2). Inspired by advances in peptide synthesis (3), Smith and colleagues pioneered PDPL construction in which vectors were designed for expression of random peptides fused to coat proteins, usually pill or pNffl, of filamentous phage (2). Thus, a multitude of different oligopeptides are surface-displayed by the viral particles and available for interaction and affinity selection with a ligate of choice. DΝA sequence information from the selected clones reveals the sequence of the displayed peptide. Most applications of PDPL technology have focused on protein-to-protein interactions. However, recent data from various labs have indicated the feasibility of finding peptide structures that mimic nonproteinaceous ligands such as biotin (4) or oligosaccharide epitopes (5-8). Phage-display technology was applied to search for structural equivalents of microbial polysaccharide epitopes. Carbohydrate epitopes had previously been identified from Candida albicans and group B streptococci (GBS) that elicit protective antibody responses, as well as other carbohydrate epitopes that elicit nonprotective antibodies. The antibodies used to select the phage were of the IgM class, which are typical of T-independent, anti-carbohydrate responses. While using these IgM antibodies to select for carbohydrate-mimetic phage (8), a population of phage that bind to all IgM antibodies regardless of antigenic specificity were identified, presumably recognizing determinants in the constant regions. These phage were characterized, and the resulting data demonstrate, surprisingly, that the phage and peptides derived from them can inhibit the interaction between antigen and IgM antibody.

B. Materials and Methods

J404 nonapeptide PDPL. The J404 PDPL utilized in the studies reported here was constructed by one of the inventors (JBB) and is described elsewhere (1,9,10). The library displays random 9-mer peptides from the N-terminal portion of the pffi capsid protein of kanamycin resistant, filamentous bacteriophage M13KBst. The J404 library contains an estimated 5 x 10⁸ different nonapeptides at high titer (1 x 10¹³ pfu/ml).

Antibodies. Anti-Candida IgM mAbs B6.1 and B6 were isolated as previously described (11) and produced in serum-free medium (BG 101 Liquid Kit, Irvine Scientific, Santa Ana, CA). The antibodies were concentrated by ammonium sulfate precipitation, and exhaustively dialyzed against Dulbecco's phosphate-buffered saline (DPBS, Sigma Chemical Co.). Anti-GBS IgM mAbs S7, S9, and S10 (12) were prepared as mouse ascites and the IgM fraction isolated by distilled water dialysis. S7 and S9 are directed against carbohydrate antigens, while S10 is specific for the GBS Ibc protein. Monoclonal anti-GBS mAbs 1B1 and 2A6 are specific for the GBS type III capsular polysaccharide (8), as is the IgM S9 (12). Additional mAbs and polyclonal antibodies were used to assess peptide binding and immunoglobulin class specificity. Murine IgM mAbs utilized included: H9 and C6 (13); mt9A, mt5 A, mt6F, P3x63(14); 2B3.1 ,

A4.1, C3.4, JD3 (M. Riesselman and J.E.C., unpublished), and T17 directed against the synthetic peptide (Y,E)-A~K (15). Murine IgG mAbs included 6C5-H4 (gift from Kevin C. Hazen, University of Virginia), 44.1 (9), and 924 (16). Polyclonal preparations included murine ascites, rabbit and human sera. Preparation of selection matrices. Antibody affinity matrices for interaction with the

J404 PDPL were mAb B6.1 adsorbed to polystyrene dishes, MAb B6.1 conjugated to Sepharose 4B (CL-4B-200, Sigma) or mAb S10 conjugated to Sepharose beads. Polystyrene dishes (Falcon 35 mm) were coated for 2 hours at room temperature with 1 mg mAb B6.1/ml DPBS and washed five times with cold DPBS. For mAb-conjugated Sepharose matrices, the Sepharose 4B was activated with CNBr as previously described (17) and coated with mAb B6.1 or S10 (3 mg per ml packed beads, 16 h at 4° C), washed, and blocked in 1% bovine serum albumin (BSA)

(ICN Biomedical.s, Inc., Aurora, OH) in DPBS prior to incubation with phage.

Interaction of the J404 library with IgM MAb matrices. All antibody-coated dishes and beads were pre-blocked for 1 hour in DPBS + 1% BSA prior to incubation with the library. Three independent selections (mAb B6.1-dish, MAb B6.1-Sepharose, and mAb SlO-Sepharose) were performed with aliquots from the J404 PDPL. Manipulation of the nonapeptide PDPL and appropriate Escherichia coli K91 host cells were essentially as described (9). Briefly, aliquots of the nonapeptide PDPL (approx. 7.5 x 1011 phage) were diluted in phage buffer (50 mM Tris- HC1, pH 7.5, 150 mM NaCl, 0.5% v/v Tween-20 and 0.1- 1.0% BSA) and incubated with the blocked matrices (16 hours with rotation, 4o C). After thorough washing with phage buffer, bound phage were eluted with 0.1 M glycine buffer pH 2.2 and immediately neutralized by addition of Tris base.

A few microliters of the eluted phage were removed for titering and the remaining phage were amplified in 'starved' E. coli K91 to a titer of 1012 to 1013 plaque forming units/ml. Subsequent selections were carried out against the same IgM mAb as the initial selection. Half of the amplified phage were diluted in phage buffer and incubated with a fresh mAb-coated selection matrix for a second round of affinity selection. The eluted phage were titered and amplified as above, and half were subjected to a third round of selection.

Analysis of the third round phage pools. The third round selection pools of phage were analyzed by sequencing of phage clones as follows. An appropriate dilution of phage pool was plated, single plaques were excised, and phage minipreps (9) were prepared in kanamycin- containing Luria broth and harvested to provide single-stranded template DNA for sequencing with Sequenase v.2.0 (USB/Amersham). The phage templates were primed with a gene III specific primer which anneals approximately 50 nucleotides from the 27-mer insert as described (9). Clones were tested for binding to IgM mAbs by plaque lifts, immunoblots, and ELISA. Preparation and analysis of immunoblots. SDS-PAGE, transfer of proteins and immunoblot preparations were essentially as described (18). Briefly, phage samples were electrophoresed in 12.5% polyacrylamide gels and proteins transferred to nitrocellulose membranes (NCM, BA83, Schleicher and Schuell, Keene, NH) with a semidry blotting system (MilliBlot-SDE, Millipore, Bedford, MA). NCM were briefly rinsed in transfer buffer and dried. For immunoblotting, membranes were rehydrated in water, washed for 10 minutes in DPBS and blocked (1 -2 hours, 23° C) in a fresh solution of Dulbecco's PBS (DPBS) containing

5% (wt/vol) nonfat milk and 0.1 - 0.2% Tween 20 (BLOTTO). BLOTTO was used for all antibody dilutions and wash steps. Blocked membranes were incubated overnight (4o C) with monoclonal (5 μg/ml or a 1:200 dilution of ascites preparations) or polyclonal antibodies (1:200 dilution of serum samples). Blots were washed 3 times (10 minutes each) and incubated with alkaline phosphatase (AP) or horseradish peroxidase (HRP)-conjugated secondary antibodies.

AP conjugate incubations were followed by washes (3 x 15 m) in 0.1 M Tris base pH 9.5, 0.1 M NaCl, 1 mM MgC12 and development with nitroblue tetrazolium and 5-bromo-4-chloro-3- indolyl phosphate-p-toluidine. Blots incubated with HRP conjugates were immersed in 3,3'- diaminobenzidine plus H₂O₂ in 50 mM Tris-HCl pH 7.4 for detection. Plaque lifts of phage clones were prepared by placing NCM circles on overlay plates for

1 - 2 hours and washing 3x with tris buffered saline (TBS). Lifts were blocked and processed as described above for western blots.

ELISA. ELISA was used to measure phage binding to IgM and inhibition of IgM antibody binding to antigen. ELISA to determine phage binding to antibody were performed as follows. Immulon II microtiter plates (Dynatech, Chantilly, VA.) were coated with 0.2 - 1.0 μg mAb per well and blocked with PBS/1% bovine serum albumin for 2 hours at room temperature. Varying concentrations of phage or KLH-conjugated peptides derived from the phage were added to wells in a 100 μl volume and incubated. Plates were washed with PBS 0.1% Tween-20 and rabbit anti-M13 antiserum (a gift from Al Jesaitis, Montana State University) or rabbit anti KLH antiserum was added and the plates were incubated. Plates were washed and then alkaline phosphatase-conjugated anti-rabbit Ig (Zymed, South San Francisco, CA) was added. We previously determined that this antiserum does not react with mouse Ig. Following an incubation, the plates were washed and the substrate p-nitro phenyl phosphate (Sigma, St. Louis, MO) was added. The absorbance at 405 nm (A405) was measured using an EL-320 Microplate reader (BIO-TEK, Winooski, VT). ELISA assays for phage inhibition of mAb interaction with Candida albicans antigens were carried out by coating microtiter wells with 0.5μg (based on dry weight) C. albicans cell wall extract (19), blocking 1 hour with 1% Ficoll 400 in TBS and then 2 hours in BLOTTO. The inhibition assay was performed by either preincubating the antibody with phage, or by adding the phage.to microtiter wells after the antibody solution was present. For the preincubation inhibition assays, mAbs were diluted (0.25 μg/ml) in BLOTTO alone or mixed with 1.8 x 1012 phage/ml phage and rotated for 1 hour prior to placing 100 μl of the mixture into the cell wall extract-coated wells (2 hours). For the later addition of phage, mAbs were incubated in wells for 1 hour, and then phage added to the microtiter wells and additionally incubated for 1 hour. Wells were washed and an HRP-conjugated goat anti-mouse polyvalent Ig was added and incubated 2-4 hours. Substrate containing o-phenylenediamine, H2O2, in 0.1 M sodium citrate (pH 5.0) was added and the color developed for 10-30 minutes. Reactions were stopped by the addition of 10% H2SO4 and A490 measured.

GBS were coated onto microtiter wells using poly-l-lysine and glutaraldehyde as described elsewhere (8). Plates were blocked with 1% BSA. IgM anti-GBS antibodies were mixed with phage or with KLH-conjugated peptides derived from the phage and incubated in microtiter wells at 40 for 16 hours. The plates were washed and incubated with alkaline phosphatase-conjugated anti-Ig for 6 hours, followed by washing and addition of p-nitrophenyl phosphate. A405 was then determined. Latex agglutination for measurement of rheumatoid factor(RF). Latex beads coated with aggregated IgG were obtained from REFSCAN Kit (Becton Dickinson, Cockeysville, MD). Serum containing high titered RF activity were either contained within the kit or were obtained from the Division of Rheumatology, University of Utah School of Medicine. Pretitered dilutions of serum were premixed with phage and then added to the latex beads. The mixture was stirred and allowed to incubate with slow shaking for 20 minutes prior to photography.

Flow cytometry for cell surface IgM. Murine B-lymphoma cells expressing cell surface IgM (designated 5F5) or differentiated to express IgA (4F10) (20,21) were incubated with phage in PBS BSA/ 0.1% sodium azide, washed, incubated with rabbit anti-M13 antiserum, washed, and then stained with FITC- conjugated anti-rabbit Ig (Cappell Laboratories, Durham, NC). Alternatively, cells were directly stained with FITC conjugated anti-mouse IgM (Cappell). Preparation of synthetic peptide - carrier protein conjugate. The nonapeptide displayed by phage clone edl was chosen for conjugation to keyhole limpet hemocyanin (KLH) with a heterobifunctional crosslinker, m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) (Pierce Chemical Co.). A synthetic peptide containing a 4 amino acid extension including cysteine for crosslinking (YDWIPSSAWGPPC) was made(Bio-Synthesis, Inc., Lewisville, TX).

Briefly, 10 mg KLH (Imject( KLH, Pierce) in degassed, nitrogen-sparged 0.05 M citrate- phosphate buffer pH 5.0 was stirred gently with 2 mg crosslinker for 1 hour at room temperature under a N² cap, then passed over a Sephadex G-25 column to separate the carrier protein-linker product from unreacted crosslinker. The MBS-KLH was placed into a fresh glass tube and stirred 6 hours (N² cap, room temperature) with 5 mg of the synthetic edl peptide which was dissolved in 50 μl dimethyl formamide. The sample was dialyzed against three liters of the citrate-phosphate buffer pH 5.0 to remove free peptide and then dialyzed against three changes of DPBS. Aliquots of the edl-KLH conjugate preparation were stored at -20° C prior to use. Independent IgM selections enrich for phage expressing similar peptide sequences.

Phage were selected in four distinct selections on three different monoclonal antibodies, immobilized either on Sepharose beads or on polystyrene dishes. In each case, the final selection pools contained phage clones that bound to multiple IgM antibodies in ELISA or immunoblot analysis (see below). Nonapeptide sequences from individual IgM-binding phage clones are shown in Table 1. Phage displaying some sequences, especially those designated edl, ed-4, and ps-1, were isolated considerably more frequently than the others. All sequences contain a tryptophan residue in the C-terminal or penultimate position, and the sequences shown in Table 1 are aligned to that residue. While there is variability in the sequences capable of binding to IgM, certain motifs are strongly represented, such as aspartate on the amino terminal side of the conserved tryptophan and W I S/P S/Q X D W in many clones. The absolute or minimal sequence requirements necessary for IgM binding activity have not been established. Table 1. Peptide Ligands of IgM Antibodies

PHAGE CLONE SEQUENCES

Binding of phage and peptide to IgM antibodies. Immunoblotting and ELISA were used to demonstrate binding to IgM by phage and peptides derived from the phage. Phage clones were initially identified as binding to IgM by plaque lift immunoblots (data not shown). Immunoblots of SDS-PAGE separated phage proteins were used to demonstrate that IgM binding by phage involves the chimeric plJJ-displayed peptide and also to show the specificity of binding. IgM-binding or non-binding irrelevant phage were subjected to SDS-PAGE, blotted onto nitrocellulose and were incubated with either one of two different IgM mAbs, or a murine IgG mAb. The results of the immunoblot with the IgM mAb B6 is shown in figure 1. Signal occurred at the appropriate mobility for the plll-nonapeptide chimeric protein in the lanes containing the IgM-binding phage (lane 4 - es81, lane 6 - ed9, lane 7 - ed23, lane 8 - ed36, lane 9 - ed42, and lane 17- edl), but not with any of the irrelevant phage clones (lanes 2,3,5,10-16) nor with the parental phage M13KBst (lane 18). Duplicate blots were prepared and tested against a second IgM mAb, B6.1, or an IgG mAb, 6C5-H4. The identical pattern was seen with the IgM, and no reactivity with the IgG (data not shown).

Phage clone edl was chosen for further analysis. A preparative blot of the phage proteins was evaluated for reactivity with additional IgM antibodies, including murine mAbs and polyclonal Abs from various species, as well as to IgG antibodies (figure 2). All IgM mAbs bound the edl pill-displayed nonapeptide (figure 2A, lanes 1 through 13). Because the mAbs in figure 2A were primarily directed against carbohydrate antigens, two mAbs against protein antigens, T17 and S10, were tested for binding to edl. Figures 2B and 2C, lanes 1 and 2, demonstrate that these IgMs also bind to edl, but not to an irrelevant phage. In lanes 3 and 4, we showed that two IgG mAbs are not bound by edl (figure 2B), but that one of the IgGs will bind to phage displaying the epitope recognized by that IgG(figure 2C). IgM antibodies in a murine polyclonal ascites (figure 2 A, lane 14) are bound by e l. IgM antibodies from human and rabbit serum samples also bound the pill band (figure 2A, lane 15, and figure 2D, lane 3). The edl pill did not bind IgG antibodies, neither mAbs as described above, nor IgGs found in the affinity purified secondary antibodies utilized for the control strips (figure 2A, lanes 16 and 17, figure 2B, lanes 5 and 6, and figure 2D, lanes 1, 2, and 4).

ELISA was performed to demonstrate the specificity and avidity of phage binding. Specificity of binding of the phage to IgM antibodies is shown in Table 2. Two different IgM binding phage (S 10-4 and S 10-8) were compared with three different antibody-specific phage

(8) in an ELISA in which microtiter wells were coated with different antibodies. SI 0-4 and S10- 8 bound to wells coated with each of the IgM, but not IgG, antibodies, while the antibody- specific phage only bound to the selecting antibody. Figure 3 shows that phage edl and SI 0-4 bind to IgMs displaying specificity for both carbohydrate (S9) and protein antigens (S10 and T17), but do not bind to IgG antibodies. The parental phage M13KBst and an irrelevant phage, J508,22 do not bind to the IgMs. Since J508,22 represents the epitope bound by mAb 44.1 (9), it binds well to the IgG mAb 44.1.

The relative avidity of phage binding to IgM is shown in figure 4A. Seven different IgM binding phage and the parental phage M13KBst were serially diluted and the IgM binding activity measured. The results demonstrate at least a tenfold difference in relative avidity between those phage with the highest avidity (edl, ed42) and those with the lowest (ed9). The ability of A peptide derived from the phage to bind to IgM antibodies was tested. A synthetic peptide corresponding to the edl displayed sequence was conjugated to KLH. The conjugate, but not the peptide, was water soluble. Thus the conjugate was used in these experiments. Microtiter wells were coated with IgM mAb S9 and incubated with either KLH, KLH conjugated to the edl peptide, or KLH conjugated to an irrelevant peptide (designated pep2). Following an incubation and washing, binding was detected with rabbit anti-KLH antiserum and alkaline phosphatase anti-rabbit Ig. The ELISA results are shown in figure 4B and demonstrate that binding occurred with the KLH-edl conjugate, but with neither KLH, nor KLH-pep2.

Table 2. Binding of phage to IgM, but not to IgG

Antibody Coating Wells ¹

1. Wells were coated with IgM (S7, S9, B6.1), IgG (924) at 5 μg/ml or no antibody.

2. The indicated phage (109) were added to the wells. Phage binding was detected with rabbit anti- M13 antiserum and alkaline phosphatase-conjugated anti-rabbit Ig.

3. A₄₀₅, mean of duplicate samples.

Phage bind to cell-surface IgM. Indirect immunofluorescence and flow cytometry were used to demonstrate that IgM-specific phage bind to the monomeric cell-surface form of IgM (figure 5). Two different B-cell lymphoma lines were used, one expressing cell-surface IgM (5F5), the other IgA (4F10). FITC-conjugated anti-mouse IgM antibodies were used to demonstrate that 5F5 does express IgM and 4F10 does not (right hand panels). Binding of phage to the cells was detected by first incubating the cells with phage, washing, and then with rabbit anti-M13 antiserum, followed by FITC-conjugated anti-rabbit Ig. As shown in the left hand panels of figure 5, SI 0-4 phage bind to the IgM expressing cells, but not to those with cell surface IgA. The parental phage, M13KBst, bind to neither (center panels).

Inhibition of antigen binding by phage and phage-derived peptides. The ability of the IgM-binding phage to inhibit antigen binding by IgM antibodies was tested. These experiments were performed in multiple different antigenic systems.

The ability of phage to block the binding to GBS of two anti-GBS mAbs directed against two different carbohydrate epitopes (12) is shown in Table 3. For each mAb, the inhibition caused by the IgM-binding phage SI 0-4 was compared to the inhibition seen with mAb-specific phage described in a previous publication (8). Phage S10-4 inhibited both mAbs, the parental M13KBst inhibited neither, and each mAb-specific phage inhibited its respective mAb, but not the other. The inhibition obtained with SI 0-4 was equal or greater than that obtained with the mAb-specific phage, which presumably mimics the antigenic structure and binds within the antibody combining site. Figure 6 shows that both edl and SI 0-4 inhibit thel-άnding of two IgM antibodies, one specific for carbohydrate antigen (S9) the other for a protein structure (S10), to GBS. However, they inhibit neither of two IgG antibodies (2A6 and 1B1) with the same antigenic specificity as S9. The parental phage, M13KBst does not inhibit antigen binding at all. Because the phage inhibited the binding of IgM antibody to antigen, we sought to determine whether the phage bound within the combining site by performing a competitive inhibition, attempting to block the binding of phage to antibody S9 with purified GBS capsular polysaccharide. No inhibition was seen (data not shown).

The comparative ability of different IgM-binding phage to inhibit binding to GBS by mAb S9 is shown in figure 7A. The results demonstrate that there are marked differences in the degree of inhibition by phage with different displayed sequences, and not surprisingly, the relative inhibition correlates with the avidity of phage binding to the IgM antibody (figure 4A). We next tested whether antibody binding to antigen could be inhibited with a peptide corresponding to the sequence displayed by the edl phage (figure 7B). The results showed that the KLH-edl conjugate inhibited binding of antibody S9 to GBS, but neither KLH alone nor KLH conjugated to an irrelevant peptide had that effect.

The ability of IgM-binding phage to inhibit the interaction between IgM anti-Candida albicans antibodies (19) to cell wall extract from C. albicans is shown in figure 8. As with the GBS, two different antibodies with different epitope specificity were tested. Inhibition of both antibodies, mAb B6.1 (figure 8A) and mAb B6 (figure 8B) was observed with phage edl, but not with M13KBst. Much greater inhibition was seen when the phage and antibody were premixed and then added to antigen, than when the phage were added after the initial antigen- antibody interaction had occurred.

Human rheumatoid factor is an anti-IgG autoantibody, primarily of the IgM class. Figure 9 shows that edl phage, but not M13KBst, inhibited the rheumatoid factor-induced agglutination oflgG-coated latex beads. IgM-binding phage do not inhibit complement activation. By use of ELISA we tested the ability of phage to inhibit complement activation (22). IgM mAb and antigen were allowed to interact, then phage and human complement-containing serum was added. The binding of Clq and C3b to the immune complexes was assessed with specific antisera. There was no difference in complement binding in the presence of IgM-binding phage, control phage, or no phage (data not shown).

Table 3. Inhibition of antibody building to GBS by phage.

Antibody

1. Binding of the indicated antibody (0.3/μg/ml) to ELISA wells coated with GBS was detected with alkaline phosphatase-conjugated anti-mouse IgM.

2. Antibodies were premixed with 10¹⁰ of the indicated phage prior to addition to ELISA plate.

3. A₄₀₅, mean of duplicate samples.

C. IgM Binding Peptides IgM-binding peptides of the present invention include peptides defined by the following three generic formulae:

(A) A peptide comprising the sequence W-I-X_rX₂-X₃-X₄-W, wherein W = trytophan, I = isoleucine, Xj = proline or serine, X₂ = glutamine, alanine, glycine or serine, X₃ = glutamic acid, alanine, serine, arginine, glycine, valine, lysine, asparagine or tyrosine, and X₄ = serine, aspartic acid or alanine. (B) A peptide comprising the sequence X_rX₂-X₃-X₄-X₅-D-W wherein X_! = alanine, glycine or valine, X₂ = isoleucine or valine, X₃ = lysine, tyrosine, threonine or tryptophan, X₄ = serine, glycine or glutamine, X₅ = glycine, lysine or valine, D = aspartic acid and W = trytophan. (C) A peptide comprising the sequence I-W-X X₂-D-W, wherein I = isoleucine,

W = trytophan, X_t = glutamine or serine, X₂ = arginine, lysine or asparagine, and D = aspartic acid.

Other peptides with binding characteristics may have the formulae: (D) D-W-I-D-Q-M-Y-D-W (E) Q-K-W-I-S-S-A-W-D

Various modifications to these peptides may be made by one skilled in the art that preserve the functional activity described in the specification. For example, residue X3 in Formula A, above, may be substituted, e.g., by nonpolar amino acids which are not explicitly recited. However, the relevant binding activity between the peptides and IgM may be assayed by techniques described in this specification. Similarly the ability of a peptide to inhibit the binding of an IgM molecule to its specific antigen also may be assayed by techniques described in this specification and otherwise known to the skilled artisan. Specific peptide embodiments which are particularly effective for IgM binding are SWISSRDWT, SWISSKDWT and YDWIPSSAW. The critical peptide sequence can be embedded in a longer polypeptide without losing the IgM binding facility.

D. Chemical Synthesis of Peptides

The coding sequence of peptides according to the present invention may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.

225-232, etc). Alternatively, the amino acid sequence of the peptides themselves may be produced using conventional chemical synthesis method. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer). The newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton T. (1983) Proteins, Structures and Molecular Principles, W. H. Freeman and Co., New York, N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton). Additionally, the amino acid sequence of the peptides of the present invention, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins (i.e. chimera or fusion proteins), such as proteinaceous therapeutic agents, or any part thereof, to produce a variant polypeptide. Chemical methods can also be applied to conjugate a peptide of the invention with a therapeutic agent that is not proteinaceous.

E. Nucleotides Encoding Peptides

The present invention further provides nucleic acid molecules that encode the peptides having formulae (A) - (E) and the related proteins herein described. Modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the peptide sequence during translation can be made without destroying the activity of the peptide. Such substitutions or other alterations result in peptides having an amino acid sequence encoded by a nucleic acid falling within the contemplated scope of the present invention. The present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989). In the preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.

The choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule. Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N. J.

Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form a rDNA molecules the contains a coding sequence. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al, J. Mol. Anal. Genet. 1:327-341, 1982.) Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co- transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.

F. Transformed Host Cells

The present invention further provides host cells transformed with a nucleic acid molecule that encodes a peptide of the present invention. The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line. Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NTH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines.

Any prokaryotic host can be used to express a rDNA molecule encoding a peptide of the invention. The preferred prokaryotic host is E. coli. Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al, Molecular Cloning. A Laboratory Mammal. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed, see, for example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc. Natl. Acad. Sci. USA 76:1373-76, 1979.

Successfully transformed cells, i.e., cells that contain a rDNA molecule of the present invention, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J Mol. Biol. 98:503, 1975, or Berent et al, Biotech. 3:208, 1985 or the proteins produced from the cell assayed via an immunological method.

G. Production of Recombinant Peptides

The present invention further provides methods for producing a peptide according to the present invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a peptide typically involves the following steps: First, a nucleic acid molecule is obtained that encodes a peptide of the invention, such as the a nucleic acid molecule that is known to encode peptides of the formulae (A) - (E). The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the peptide open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant peptide. Optionally the recombinant peptide is isolated from the medium or from the cells; recovery and purification of the peptide may not be necessary in some instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be synthesized. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the peptide and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.

Recombinant chimera or fusion proteins comprising a peptide of the invention, or fragments or variants thereof, can also be produced using the above methods. Such fusion proteins can contain one or more copies of the peptide of the invention, and one or more copies of one or more peptides that are not peptides of the invention. For example, a nucleic acid sequence encoding a peptide of the invention may be fused to a nucleic acid sequence encoding a cytokine or a fragment of a cytokine, which may or may not be modified to optimize the activity of the resultant fusion protein.

H. Production of Antibodies Antibodies are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for some applications, use of monoclonal preparations may be preferred in certain circumstances. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten. polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin. Antibodies prepared from any mammalian host are contemplated, such as human antibodies and murine antibodies, as well as cbimeric antibodies and humanized antibodies.

I. Therapeutic Uses of Peptides The peptides described in this specification may have both experimental and therapeutic utility. If the affinity of interaction between peptide and IgM molecule is sufficiently high, the peptides may be used to purify IgM antibodies on affinity matrices. Because the peptides bind to cell surface IgM, they may function as agents that activate B cells. The ability of the peptides to block the interaction between IgM antibodies and antigen may be used to prevent deleterious antigen-antibody interactions or disrupt or dissociate IgM-containing immune complexes, and as a consequence modulate an immune response. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, rats and mice, or in vitro. As used herein, a human patient can be any individual, as long as that individual is in need of modulation of a pathological or biological process associated with IgM antibodies.

In addition, the peptides have potential utility as carriers for therapeutic agents or immunotoxins, specifically targeting IgM-expressing cells, IgM-receptor bound lymphocytes, B cells and their derivatives, as well as tumor cells that develop from these cell types (lymhoma and plasmacytoma cells, for example), and other tumor cells including those that dedifferentiate and produce IgM antibodies, for example in paraneoplastic syndromes. In this application, the therapeutic agents associated with a peptide of the invention may be toxic to the cell, may kill the cell, or may otherwise alter cellular events, for example by regulating the production of IgM. Therapeutic agents can include but are not limited to plant or bacterial toxins modified to eliminate their native targeting domains, radionuclides, cytotoxic drugs, and immunoregulatory drugs and proteins (for example cyclosporin A or cytokines). The therapeutic agents may be in association with at least one peptide of the invention by any know means, including but not limited to via covalent bond, via conjugation, via commingling in a preparation or delivery vehicle, or via any carrier which provides sufficient proximity and presentation of the peptide and the therapeutic agent. Alternatively, IgM-depleted preparations can be generated by extracorporeal perfusion of a sample over subsfrate-immobilized peptides of the invention, and the IgM-depleted preparation returned to the patient. The sample may be blood, plasma or serum, for example the sample may be serum from a human patient suffering from a disease associated with IgM antibodies.

Such pathologic IgM antibody interactions can occur in hemolytic transfusion reactions, autoimmune diseases such as autoimmune hemolytic anemia and rheumatoid arthritis, in paraneoplastic syndromes associated with Waldenstrom's macroglobulinemia, in multiple myeloma and in other cancers. More specific examples of pathological IgM antibody interactions include, but are not limited to rheumatoid factor binding to IgG, and isohemagglutinin binding to red blood cells (for example, in a transfusion reaction), and paraneoplastic syndrome due to IgM antibody binding to neural tissue. In addition, IgM preparations purified via complexing to the peptides described herein can be used in the treatment of bacterial infections in children, particularly infants, or to boost the protective immunity by administering such purified IgM.

The peptides and compositions of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, a peptide or composition of the present invention can be administered before, after or in combination with other known drugs typically prescribed for these conditions according to generally accepted medical practice. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time. The peptides and compositions of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage amounts will be determined by established protocols for the administration of therapeutic peptides and will be dependent upon, for example, the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 μg kg body wt. The preferred dosages comprise 0.1 to 10 μg kg body wt. The most preferred dosages comprise 0.1 to 1 μg/kg body wt. In addition to the pharmacologically active agent, the peptides and compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipopbilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

It is to be understood that both the foregoing description and the following examples are exemplary and explanatory only are not restrictive of the invention, as claimed. All references and other documents identified herein are incorporated by reference in their entireties.

EXAMPLES

Example 1: IgM Purification from Sample Using Peptide

A peptide comprising a sequence in accordance with any one of formulae (A) - (E) is used to prepare an affinity matrix. The peptide may be synthetically or recombinantly produced or isolated from any native source that contains the peptide. As one of skill in the art will recognize, a large variety of complexing materials are available that result in insoluble or immobile protein substrate conjugates, including agarose, latex, magnetic or polyacrylamide beads, silica and polystyrene. Once immobilized, the peptide/substrate conjugate is loaded into an incubation vessel, such as a chromatography column made of glass, plastic, or metal. A large variety of such vessels are available commercially.

The conjugate is equilibrated with a suitable buffer, such as PBS, TBS, BBS, or a non- saline buffer. A liquid sample containing IgM is then loaded onto the column. In particular, the sample will be mammalian or animal serum, such as human serum, to obtain polyclonal IgM fractions, or mouse ascites containing a monoclonal IgM antibody of interest. The column is incubated at room temperature for an amount of time sufficient to allow binding of the IgM to the immobilized peptide, e.g., 15-60 minutes, then washed with one or more equilibration buffers, such as Tris or NaCl. After washing, the bound IgM molecules are eluted from the incubation vessel with a dissociation agent, which alters the pH, the salt concentration or the hydrophobic interactions involved in the peptide/IgM binding. In addition, bound IgM can be eluted from the column by detergents, such as sodium dodecylsulfate (SDS).

The eluted antibody fractions are then dialyzed against distilled water, or alternatively against PBS with 0.02% sodium azide if they are to be stored, or any other suitable dialyzing solution. Specific details for these steps are known in the art and taught by laboratory manuals, such as Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY. See for example the discussion in Chapter 8 of Harlow as well as the pertinent sections of the book referred to therein.

Example 2: Method of detecting IgM in a Sample Using Peptide A peptide comprising a sequence in accordance with any one of formulae (A) - (E) is used to prepare a detectable conjugate. The peptide may be synthetically or recombinantly produced or isolated from any native source that contains the peptide. As one of skill in the art will recognize, a large variety of materials are available that can be employed as detectable labels. Labels appropriate for peptide conjugation include, but are not limited to, enzymes, such as horseradish peroxidase, alkaline phosphotase, acid phosphotase and luciferase; fluorochromes, such as fluorescein; radioisotopes, such as ¹²⁵1, ³⁵S and ¹⁴C; and colloidal gold particles. The steps involved in conjugating these detectable labels to peptides, as well as appropriate storage procedures, are well known to those of skill in the art and are disclosed with specificity in laboratory manuals, such as Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. The labeled peptide is then diluted in an appropriate buffer to a concentration appropriate for the label, the detection means, and the required sensitivity for the IgM detection contemplated. An effective amount of the buffered labeled peptide is brought into contact with a sample suspected of containing IgM antibodies. The contact should be maintained for an amount of time effective to allow binding between the labeled antigen and any IgM molecules present in the sample, e.g., about 15-60 minutes or longer if necessary. After incubation, the materials are washed with buffer to remove any unbound material. The presence of the labeled antigen/IgM complex is detected by means suitable for detecting the label, e.g., chromogenic assay, fluorescence , radioactivity or electron density.

An alternative detection means is to immobilize the peptide in the manner described above in Example 1; however, the preferred substrates for this analysis are solid substrates, such as nitrocellulose and polyvinylchloride. A sample suspected of containing IgM is brought into contact with the immobilized peptide. After the material is washed, it is contacted with a labeled antibody capable of binding IgM. Such labeled IgM recognition antibodies are well known in the art. The specific details of this protocol is set forth in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, and other similar laboratory manuals.

Example 3: B-cell purification Using Bound Peptide

A peptide comprising a sequence in accordance with any one of formulae (A) - (E) is used to prepare an immobilized peptide in accordance with example 1, above. A sample containing B cells in which IgM is an integral receptor on the cell membrane, such as peripheral lymphocytes prepared from blood or heparinized blood or a bone marrow sample, preferably peripheral lymphocytes prepared from human (or other mammalian) blood are incubated with the immobilized peptide under conditions effective for binding the cells to the immobilized peptide and for a sufficient time to allow such binding to occur. After the material is washed to remove any unbound material, the captured B cells are eluted from the immobilized peptide via use of a dissociation agent, which alters the pH, the salt concentration or the hydrophobic interactions involved in the peptide/IgM binding. Protocols for cell specific isolation procedures are well known to those of skill in the art, such as those for cell plating. For general review of these protocols see CURRENT PROTOCOLS IN IMMUNOLOGY, Greene Publishing Associates and John Wiley & Sons, 1991. and other similar laboratory manuals.

Example 4: Antigen Purification Using Bound IgM and Peptide Elution In view of the capacity of the peptides described in formulae (A) - (E) to disrupt an

IgM/antigen conjugate and cause the IgM to release the antigen, these peptides can be used as elution reagents in the preparation of IgM or antigens that are specifically bound by IgM. Affinity chromatography matrices are prepared with either immobilized IgM or immobilized antigen, in accordance with the preceding examples and standard procedures, such as those taught by Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY, 1988, CURRENT PROTOCOLS IN IMMUNOLOGY, Greene Publishing Associates and John Wiley & Sons, 1991. and other similar laboratory manuals. The immobilized antigen or immobilized IgM is used in a chromatography vessel to capture the other molecule from a sample or from an admixture containing either the IgM or antigen, respectively.

Typically, the captured molecules would be eluted with a dissociation agent which affords a shift in pH or salt concentration, or eluted with a detergent composition. Dissociation agents are not desirable in those instances in which either or both of the antigen and IgM molecules are sensitive to the change in condition or are altered, e.g., by a detergent. Eluting the bound molecules using an effective amount of at least one of the peptides set forth in formulae (A) - (E) will avoid such negative effects and can successfully separate the non-immobilized antigen or IgM from the antigen/IgM complex.

Example 5: Use of a Kit to Detect IgM A test kit containing immobilized peptide according to formulae (A) - (E), a composition which can specifically bind to IgM and which comprises a moiety capable of detection, an appropriately formulated dissociation agent, and instructions for the performance of a method of detecting IgM in a sample is provided. The immobilized peptide is loaded into or onto a chromatographic means in accordance with the instructions contained in the kit. A sample expected to contain IgM molecules is added to the chromatographic means and incubated in accordance with the instructions contained in the kit. After any suggested washing steps, the IgM-specific labeled composition is added to the chromatographic means in accordance with the instructions contained in the kit. After any necessary washing step or additive step critical to detection of the label, a final step of detecting the label and thus the bound complex, in accordance with the instructions in the kit.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims.

REFERENCES

All of the preceding publications and patent applications as well as those in the following list are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

1. Burritt, J. B., C. W. Bond, K. W. Doss, and A. J. Jesaitis. 1996. Filamentous phage display of oligopeptide libraries. Analyt. Biochem. 338:1.

2. Smith, G. P., and J. K. Scott. 1992. Peptide-display phage libraries. Methods in Enzymology. 217:228.

3. Geysen, H. M., S. J. Rodda, T. J. Mason, G. Tribbick, and P. G. Schoofs. 1987. Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods. 102:259.

4. Devlin, J. J., L. C. Panganiban, and P. E. Devlin. 1990. Random peptide libraries: a source of specific protein binding molecules. Science. 249:404. 5. Oldenberg, K. R., D. Loganathan, I. J. Goldstein, and P. G. Schultz. 1992. Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc. Nat. Acad. Sci.

USA. 89:5393.

6. Hoess, R., U. Brinkmann, T. Handel, and I. Pastan. 1993. Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3. Gene. 128:43. 7. Valadon, P., G. Nussbaum, L. F. Boyd, D. H. Margulies, and M. D. Scharff. 1996.

Peptide libraries define the fine specificity of anti-polysaccharide antibodies to Cryptococcus neoformans. J. Mol. Biol. 261:11.

8. Pincus, S. H., M. J. Smith, J. B. Burritt, and P. M. Glee. 1998. Peptides that mimic the protective antigen of group B streptococcal type III capsular polysaccharide. J. Immunol. 160:293.

9. Burritt, J. B., M. T. Quinn, M. A. Jutila, C. W. Bond, and A. J. Jesaitis. 1995. Topological mapping of neutrophil cytochrome B epitopes with phage-display libraries. J. Biol. Chem. 270:16974.

10. DeLeo, F. R., L. Yu, J. B. Burritt, L. R. Loetterle, C. W. Bond, A. J. Jesaitis, and M. T. Quinn. 1995. Mapping sites of interaction of p47-phox and flavocytochrome b with random- sequence peptide phage display libraries. Proc. Nat. Acad. Sci. USA. 92:7110. 11. Han, Y., and J. E. Cutler. 1995. Antibody response that protects against disseminated candidiasis. Infect. Immun. 63:2714.

12. Pincus, S. H., A. O. Shigeoka, A. A. Moe, L. P. Ewing, and H. R. Hill. 1988. Protective efficacy of IgM monoclonal antibodies in experimental group B streptococcal infection is a function of antibody avidity. J. Immunol. 140:2779.

13. Brawner, D. L., and J. E. Cutler. 1986. Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans. Infect. Immun. 51:327.

14. Li, R.-K., and J. E. Cutler. 1991. A cell surface/plasma membrane antigen of Candida albicans. J. Gen. Microbiol. 137:455.

15. Pincus, S. H., C. J. Stocks, Jr., and L. P. Ewing. 1982. Monoclonal anti-(T,G) A--L antibodies: characterization of fine specificity and idiotype expression. Mol. Immunol. 19:1551.

16. Pincus, S. H., R. L. Cole, E. M. Hersh, D. Lake, Y. Masuho, P. J. Durda, and J. McClure. 1991. In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein. J. Immunol. 146:4315.

17. Poor, A. H., and J. E. Cutler. 1979. Partially purified antibodies used in a solid phase radioimmunoassay for detecting candidal antigenemia. J. Clin. Microbiol. 9:362.

18. Glee, P. M., P. Sundstrom, and K. C. Hazen. 1995. Expression of hydrophobic proteins by Candida albicans in vivo. Infec. Immun. 63:1373.

19. Han, Y., T. Kanbe, R. Cherniak, and J. E. Cutler. 1997. Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies. Infect. Immun. 65:4100.

20. Pascual, D. W., J. Xu-Amano, H. Kiyono, J. McGhee, and K. L. Bost. 1991. Substance P acts directly upon cloned B lymphoma cells to enhance IgA and IgM production. J.

Immunol. 146:2130.

21. Arnold, L. W., T. A. Gardina, A. C. Whitmore, and G. Haughton. 1988. Ig isotype switching in B lymphocytes. Isolation and characterization of clonal variants of the murine Ly 1+ B cell lymphoma, CH12, expressing isotypes other than IgM. J. Immunol. 140:4355. 22. Horgan, C, K. Brown, and S. H. Pincus. 1992. Effect of H chain V region on complement activation by immobilized immune complexes. J. Immunol. 149:127 . 23. Burton, D. R., and J. Woof. 1992. Human antibody effector function. Adv. in Immunol. 51:1.

24. Feinstein, A., N. Richardson, and M. J. Taussig. 1986. Immunoglobulin flexibility in complement activation. Immunol. Today. 7:169. 25. Shindo T, Ueda H, Makishima F, Suzuki E, Nishimura H. 1992 Efficient selection of mu m-mutants from mu m-expressing myeloma cells by treatment with ricin A-conjugated anti-mu antibody. Somat Cell Mol Genet. 18:553-8.

26. Bregni M, Lappi DA, Siena S, Formosa A, Villa S, Soria M, Bonadonna G, Gianni AM. 1988 Activity of a monoclonal antibody-saporin-6 conjugate against B-lymphoma cells. J Natl Cancer hist. 80:511-7.

27. Glennie MJ, McBride HM, Stiipe F, Thorpe PE, Worth AT, Stevenson GT. 1987 Emergence of immunoglobulin variants following treatment of aB cell leukemia with an immunotoxin composed of antiidiotypic antibody and saporin. J Exp Med. 166:43-62.

28. Terman, D.S. Young J.B., Shearer, W.T., Ayus, C, Lehane, D., Mattioli, C, Espada, R., Howell, J.F., Yamamoto, T., Zaleski, H.I., Miller, L., Frommer, P., Feldman, L., Henry, J.F.,

Tillquist, R., Cook, G., and Daskal, Y. 1981. Preliminary observations of the effects on breast adenocarcinoma of plasma perfused over immobilized protein A. New England Journal of Medicine, 305(20): 1195-2000.

29. Gurland, H.J., Lysaght, M.J., Samtleben, W., Schmidt, B., and Stoffher, D. 1984. Apheresis without substitution fluid: devices, directions and limitations. Life Support Syst,

2(l):39-45.

30. McLeod, B.C., Price, T.H., and Drew, MJ. 1997. Apheresis principles and practice of apheresis. publ. AABB Press, Bethesda, MD.

Claims

CLAIMS We claim:

1. A peptide that selectively binds to all IgM antibodies but does not selectively bind to antibodies of other classes.

2. A peptide that causes release of specifically bound antigen from an IgM-antigen complex.

3. A peptide that inhibits the specific binding of an antigen to IgM.

4. The peptide of any of claims 1 to 3, wherein the peptide is further capable of selectively binding to the IgM molecules of a plurality of mammalian species.

5. The peptide of any of claims 1 to 3, wherein the peptide is further capable of selectively binding to both the pentameric and monomeric forms of IgM.

6. The peptide of any of claims 1 to 3, wherein the peptide is further capable of purifying any and all IgM molecules from any and all mammalian species.

7. A peptide comprising the sequence W-I-X_rX₂-X₃-X₄-W, wherein W = trytophan, I = isoleucine, X_! = proline or serine, X₂ = glutamine, alanine, glycine or serine, X₃ = glutamic acid, alanine, serine, arginine, glycine, valine, lysine, asparagine or tyrosine, and X₄ = serine, aspartic acid or alanine.

8. A peptide comprising the sequence X_rX₂-X₃-X₄-X₅-D-W wherein X_l = alanine, glycine or valine, X₂ = isoleucine or valine, X₃ = lysine, tyrosine, threonine or tryptophan, X₄ = serine, glycine or glutamine, X₅ = glycine, lysine or valine, D = aspartic acid and W = trytophan.

9. A peptide comprising the sequence I-W-X_rX₂-D-W, wherein I = isoleucine, W ÷ trytophan, X_! = glutamine or serine, X₂ = arginine, lysine or asparagine, and D = aspartic acid.

10. A peptide selected from the group consisting of SWISSRDWT, SWISSKDWT and YDWIPSSAW.

11. A peptide selected from the group consisting of D-W-I-D-Q-M-Y-D-W and Q-K- W-I-S-S-A-W-D.

12. A peptide consisting of the sequence W-I-Xι-X₂-X₃-X₄-W, wherein W = trytophan, I = isoleucine, X_x = proline or serine, X₂ = glutamine, alanine, glycine or serine, X₃ = glutamic acid, alanine, serine, arginine, glycine, valine, lysine, asparagine or tyrosine, and X₄ = serine, aspartic acid or alanine.

13. A peptide consisting of the sequence X_rX₂-X₃-X₄-X₅-D-W wherein X_! = alanine, glycine or valine, X₂ = isoleucine or valine, X₃ = lysine, tyrosine, threonine or tryptophan, X₄ = serine, glycine or glutamine, X₅ = glycine, lysine or valine, D = aspartic acid and W = trytophan.

14. A peptide consisting of the sequence I-W-X^X^D-W, wherein I = isoleucine, W = trytophan, Xj = glutamine or serine, X₂ = arginine, lysine or asparagine, and D = aspartic acid.

15. A nucleic acid molecule encoding the peptides of any of claims 1-14.

16. A recombinant vector comprising the nucleic acid of claim 15.

17. A transformed cell comprising the vector of claim 16.

18. An antibody elicited by a peptide according to any of claims 1-14.

19. A method of detecting the presence of IgM in a sample to be tested comprising the steps of: a) immobilizing a peptide according to any of claims 1-14 on a substrate; b) contacting the immobilized peptide with a test sample; and c) detecting IgM bound to the peptide.

20. A method of purifying IgM from a sample comprising the steps of: a) immobilizing a peptide according to any of claims 1-14 on a substrate; b) contacting the IgM containing sample with the immobilized peptide; and c) removing the bound IgM from the immobilized peptide to obtain purified IgM.

21. The method of claim 20, wherein the removing step is effected by washing the substrate-immobilized peptide of step (b) with a solution having a pH effective to cause the removal of bound IgM.

22. The method of claim 20, wherein the removed IgM is at least about 95% pure relative to other classes of immunoglobulin.

23. A method to isolate an antigen specific IgM population comprising the steps of: a) immobilizing an antigen on a substrate; b) contacting the immobilized antigen with a sample containing IgM; and c) further contacting the substrate-immobilized antigen of step (b) with a peptide according to any of claims 1-14 to remove IgM specifically bound to the antigen.

24. The method of claim 23, wherein the specifically bound IgM is at least about 95% antigen specific relative to other IgM molecules.

25. An IgM population produced by the method of claim 23.

26. The method of claim 23 wherein the IgM containing sample is selected from the group consisting of ascites, serum and hybridoma cell culture supernatant.

27. A method to isolate an antigen bound by a specific IgM population, comprising the steps of: a) immobilizing the antigen-specific IgM on a substrate; b) contacting the immobilized antigen-specific IgM with a sample containing the antigen; and c) further contacting the substrate-immobilized antigen-specific IgM of step (b) with a peptide according to any of claims 1-14 to remove antigen specifically bound to the IgM.

28. A method of isolating B cells in a sample, the B cells having at least one IgM molecule integrally present on the cellular membrane, comprising the steps of: a) immobilizing an effective amount of a peptide according to any of claims 1-14 on a substrate; b) contacting said immobilized peptide with the sample for a time and under conditions effective to permit binding of the B cells to the immobilized peptide; and c) isolating peptide bound B cells.

29. The method of claim 28 wherein the B cell-containing sample is selected from the group consisting of peripheral blood lymphocytes or heparinized blood.

30. The method of claim 28 wherein the sample contains human B cells.

31. A method of treating a human disease associated with IgM antibodies comprising the step of administering a peptide according to any of claims 1-14, in an amount effective to disrupt the binding of antigen to IgM antibodies.

32. The method of claim 31 wherein the disease associated with IgM antibodies is selected from the group consisting of rheumatoid factor binding to IgG, isohemagglutinin binding to red blood cells, autoimmune hemolytic anemia, and paraneoplastic syndromes.

33. A composition comprising a peptide according to any of claims 1-14 in association with a therapeutic agent.

34. A method of treating a human disease associated with IgM antibodies comprising the step of administering a composition according to claim 33, wherein the peptide of the composition selectively localizes the activity of the therapeutic agent to IgM-expressing cells in an amount effective to modulate an immune response.

35. A method of treating a human disease associated with IgM antibodies comprising the step of administering a composition according to claim 33, wherein the peptide of the composition is conjugated to the therapeutic agent, wherein the peptide selectively localizes the activity of the therapeutic agent to IgM-expressing cells in an amount effective to mediate an immune response.

36. The method of claims 34 wherein the disease associated with IgM antibodies is cancer or a multiple myeloma.

37. The method of claims 35 wherein the disease associated with IgM antibodies is cancer or a multiple myeloma.

38. A method of treating a human patient for a disease associated with IgM antibodies by removing IgM from serum obtained from said patient, comprising the steps of: a) immobilizing on a substrate, a peptide according to any of claims 1-14; b) contacting the immobilized peptide with serum obtained from the patient; c) repeating step (b) as necessary; and d) returning the IgM-depleted serum to the patient.

39. The method of claim 38 wherein the disease associated with IgM antibodies is cancer or an autoimmune disease.

40. A multimeric form of a peptide according to claim 1.

41. The multimeric peptide of claim 40 attached to a carrier molecule.

42. A method of modulating the immune response comprising administering a therapeutically effective amount of a multimeric peptide according to claim 40.

43. The method of claim 42 wherein the therapeutically effective amount and mode of administration are selected such that the B cell activation is initiated or enhanced in a patient in need thereof.

44. The method of claim 42 wherein the amount and mode of administration are selected such that the B cell activation is inihibited in a patient in need thereof.

45. The method of claim 42 wherein the therapeutically effective amount and mode of administration are selected such that the B cell apoptosis is initiated or enhanced in a patient in need thereof.