WO2010078329A1 - Methods and compositions for the treatment of pathogenic diseases - Google Patents

Abstract

The invention features methods of inducing antibody-mediated virolysis in a subject infected with an hCD59 or hCD55 expressing pathogen (e.g., HIV-1).

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF PATHOGENIC DISEASES

Field of the Invention

This invention relates to the treatment of pathogenic diseases.

Background of the Invention

The complement regulatory protein CD59 is expressed on the surface of mammalian cells to protect host cells from the bystander effects of complement activation. CD59 activity inhibits formation of the membrane attack complex of complement (MAC) by binding to complement proteins C8 and C9 and preventing C9 incorporation and polymerization. During maturation by budding, a number of enveloped viruses, such as human cytomegalovirus, HCMV, human T-cell leukemia virus type 1 (HTLV-I), HIV-I, simian immunodeficiency virus, Ebola virus, influenza virus, and vaccinia virus, capture CD59 and use it to evade the complement system (Stoiber et al.

42:153-160 (2005), Bernet et al. J Biosci 28:249-264 (2003), Rautemaa et al. Immunology 106:404-411 (2002), Nguyen et al. J Virol 74:3264-3272 (2000), Saifuddin et al. J. Exp. Med. 182:501-509 (1995), Spiller et al. J Infect Dis 176:339-347 (1997)). Other viruses (e.g., Herpesvirus saimiri) express a CD59-like molecule that aids the virus in avoiding the complement system. Additionally, microbial parasites have been identified that also express a CD59-like molecule (e.g., Naegleήafowleri and Schistosoma manosni (Parizade et al. J Exp Med 179: 1625-1636 (1994), Fritzinger et al. Infect Immun 74: 1189-1195 (2006))). These parasites, many of which are intracellular, are protected from human complement mediated lysis by CD59 and also use CD59 for infectivity (ibid). Summary of the Invention

In one aspect, the invention features a method of potentiating an immune response against a pathogen in a subject by administering to the subject an inhibitor of a surface-bound complement regulatory protein (e.g., a GPI anchor protein inhibitor).

In another aspect, the invention features a method of inducing antibody mediated virolysis in a subject by administering to the subject an inhibitor of a surface-bound complement regulatory protein in an amount sufficient to induce antibody mediated virolysis (e.g., an inhibitor of hCD59). In this aspect, the antibodies are native to the subject, and the subject is infected with a virus expressing hCD59 or hCD55.

In any of the foregoing aspects, the pathogen can be either a virus or a pathogen expressing hCD59 or an hCD59-like molecule or hCD55 or hCD55- like molecule (e.g., human cytomegalovirus (hCMV), human T-cell leukemia virus type 1 , HIV- 1 , simian immunodeficiency virus, Ebola virus, influenza virus, vaccinia virus, Herpesvirus saimiri virus, Naegleήa fowleri, and Schistosoma manosni).

An inhibitor of surface-bound complement regulatory proteins can include an inhibitor of any of the proteins set forth in Table 1 (e.g., hCD59 and hCD55) and can be an antibody, or antigen binding fragment thereof (e.g., a Fab), a small molecule, or a peptidomimetic.

Any of the above methods can also include administering a vaccine or therapeutic antibody against the pathogen to the subject.

By "subject" is meant any mammal that can be infected with a pathogen expressing hCD59, or an hCD59-like molecule, or can be infected with a pathogen expressing hCD55, or an hCD55-like molecule, e.g., a human.

By "GPI anchor protein" is meant glycosylphosphatidylinositol- anchored, type I cell surface proteins including hCD55 and hCD59. hCD55 and hCD59 protect host cells from complement mediated lysis. By "GPI anchor protein inhibitor" is meant a compound that binds a GPI anchor protein and/or disrupts the interaction of another protein with a GPI anchor thereby inhibiting the functions of GPI anchor protein. In every case, GPI anchor protein inhibitors of the invention sensitize viruses expressing hCD59 or hCD55 to complement mediated virolysis.

By "hCD59" is meant a protein having the sequence: MRGLSAEAARGWKRILGAARFCGQSQWESKEGLSCSGCCSSWLSSAIQ VSHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFE HCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLV TPFLAAAWSLHP (SEQ ID NO: 1); or a protein encoded by the cDNA sequence of: atgcgggggctgagcgcagaagcggctcgaggctggaagaggatcttgggcgccgccaggttctgtggacaa tcacaatgggaatccaaggagggtctgtcctgttcgggctgctgctcgtcctggctgtcttctgccattcaggtcat agcctgcagtgctacaactgtcctaacccaactgctgactgcaaaacagccgtcaattgttcatctgattttgatgc gtgtctcattaccaaagctgggttacaagtgtataacaagtgttggaagtttgagcattgcaatttcaacgacgtcac aacccgcttgagggaaaatgagctaacgtactactgctgcaagaaggacctgtgtaactttaacgaacagcttga aaatggtgggacatccttatcagagaaaacagttcttctgctggtgactccatttctggcagcagcctggagccttc atccctaa (SEQ ID NOZ:2) By "hCD59 inhibitor" is meant any compound that binds hCD59 and disrupts hCD59 binding to complement proteins C8 and C9. hCD59 inhibitors of the invention bind to the same portion of hCD59 as ILY domain 4 (ILYd4). Such binding can be determined, for example, through a competitive binding assay between the hCD59 inhibitor and ILYd4. By "antibodies native to a subject" is meant antibodies that are produced by a subject's immune system. Such antibody production can be induced, for example, by vaccination. "Antibodies native to a subject" may also be present due to previous or current exposure to a particular pathogen.

By "amount sufficient" is meant an amount that when administered to a subject is safe and efficacious for the potentiation of an immune response against a particular pathogen.

By "potentiating an immune response" is meant increasing the amount of the formation of a subject's membrane attack complex in the presence of a pathogen expressing hCD59 using a therapy of the invention in comparison to the amount observed in an untreated subject. By "inducing antibody mediated virolysis" is meant increasing the amount of virolysis of a pathogen expressing hCD59 using a therapy of the invention in comparison to the amount observed in an untreated subject.

By "intermedilysin" or "ILY" is meant a polypeptide having the activity of a Streptococcus intermedins intermedilysin polypeptide. ILY can be purified from Streptococcus intermedius or can be produced recombinantly. An exemplary Genbank Accession number corresponding to the nucleic acid sequence of ILY is AB029317, and an exemplary Genbank Accession number corresponding to the polypeptide sequence of ILY is BAE 16324. By ILY is also meant a polypeptide with at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% percent sequence identity to the ILY polypeptide. Additionally and alternatively, ILY is defined as a polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of ILY. ILY can be isolated from any Streptococcus intermedius strain (e.g., strains 1208-1, UNS35, UNS46, and ATCC27335).

By "domain 4 of ILY polypeptide" or "ILY domain 4 polypeptide" is meant a protein including a fragment of ILY having the activity of the ILY domain 4 polypeptide. Specifically excluded from this definition is the full length ILY protein having the Genbank Accession number BAE 16324. This term is meant to include a protein containing a peptide sequence

GALTLNHDGAFVARFYVYWEELGHDADGYETIRSRSWSGNGYNRGA HYSTTLRFKGNVRNIRVKVLGATGLAWEPWRLIYSKNDLPLVPQRNIS TWGTTLHPQFEDKVVKDNTD (SEQ ID NO:3) or RNIRVKVLGATGLAWEPWRLIYSKNDLPLVPQRNISTWGTTLHPQFED KVVKDNTD (SEQ ID NO:4), or a fragment thereof having ILY domain 4 activity. By ILY domain 4 polypeptide is also meant a polypeptide with at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% percent sequence identity to SEQ ID NO:1 or 2. Additionally ILY domain 4 polypeptide is defined as a polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of the ILY domain 4 polypeptide. The terms are also meant to include any conservative substitutions of amino-acid residues in an ILY domain 4 polypeptide. The term "conservative substitution" refers to replacement of an amino acid residue by a chemically similar residue, e.g., a hydrophobic residue for a separate hydrophobic residue, a charged residue for a separate charged residue, etc. Examples of conserved substitutions for non- polar R groups are alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. Examples of substitutions for polar, but uncharged R groups are glycine, serine, threonine, cysteine, asparagine, or glutamine. Examples of substitutions for negatively charged R groups are aspartic acid or glutamic acid. Examples of substitutions for positively charged R groups are lysine, arginine, or histidine. Furthermore, the term ILY domain 4 polypeptide includes conservative substitutions with non-natural amino- acids. This term explicitly excludes full length ILY.

By "fragment" is meant a portion of a polypeptide that contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of the reference polypeptide. A fragment may contain at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 114 amino acids or more.

By "ILY domain 4 activity" is meant the activity of a compound that antagonizes hCD59 but does not directly cause substantial lysis of human red blood cells (RBCs) in the lysis assay described herein (e.g., less than 50%, 40%, 30%, 20%, 10%, or 5% lysis when administered at a concentration of 6.4 x 10^"7 M).

By "hCD59-like molecule" is meant a molecule expressed by a pathogen that binds domain 4 of the ILY polypeptide. Cells expressing hCD59-like molecules are resistant to the lytic effect of complement by inhibiting complete formation of the membrane attack complex of complement.

By a "pathogen expressing hCD59 or an hCD59-like molecule" is meant a microbe (e.g., a virus, bacteria, or microbial parasite) that contains hCD59 or an hCD59-like molecule on its outer membrane. The term is meant to include viruses that capture hCD59 molecules from host cells by budding during the process of maturation, as well as pathogens that contain genes encoding for hCD59 or hCD59-like molecules. By a "pathogen expressing hCD55 or an hCD55-like molecule" is meant a microbe (e.g., a virus, bacteria, or microbial parasite) that contains CD55 or a CD55-like molecule on its outer membrane. The term is meant to include viruses that capture CD 55 molecules from host cells by budding during the process of maturation, as well as pathogens that contain genes encoding for CD55 or CD55-like molecules.

By "protein" or "polypeptide" or "peptide" means any chain of more than two natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein.

As used herein, a natural amino acid is a natural α-amino acid having the L-configuration, such as those normally occurring in natural proteins. Unnatural amino acid refers to an amino acid, which normally does not occur in proteins, e.g., an amino acid having the unnatural D-configuration; or a (D,L)-isomeric mixture thereof; or a homologue of such an amino acid, for example, a β-amino acid, an α,α-disubstituted amino acid, or an α-amino acid wherein the amino acid side chain has been shortened by one or two methylene groups or lengthened to up to 10 carbon atoms, such as an α-amino alkanoic acid with 5 up to and including 10 carbon atoms in a linear chain, an unsubstituted or substituted aromatic (α-aryl or α-aryl lower alkyl), for example, a substituted phenylalanine or phenylglycine.

The present invention also provides derivatives of the peptides of the invention. Such derivatives may be linear or circular, and include peptides having unnatural amino acids. Derivatives of the invention also include molecules wherein a peptide of the invention is non-covalently or preferably covalently modified by substitution, chemical, enzymatic or other appropriate means with another atom or moiety including another peptide or protein. The moiety may be "foreign" to a peptide of the invention as defined above in that it is an unnatural amino acid, or in that one or more natural amino acids are replaced with another natural or unnatural amino acid. Conjugates comprising a peptide or derivative of the invention covalently attached to another peptide or protein are also encompassed herein. Attachment of another moiety may involve a linker or spacer, e.g., an amino acid or peptidic linker. Derivatives of the invention also included peptides wherein one, some, or all potentially reactive groups, e.g., amino, carboxy, sulfhydryl, or hydroxyl groups are in a protected form.

The atom or moiety derivatizing a peptide of the invention may serve analytical purposes, e.g., facilitate detection of the peptide of the invention, favor preparation or purification of the peptide, or improve a property of the peptide that is relevant for the purposes of the present invention. Such properties include binding to hCD59 or hCD55 or suitability for in vivo administration, particularly solubility or stability against enzymatic degradation. Derivatives of the invention include a covalent or aggregative conjugate of a peptide of the invention with another chemical moiety, the derivative displaying essentially the same activity as the underivatized peptide of the invention, and a "peptidomimetic small molecule" which is modeled to resemble the three-dimensional structure of any of the amino acids of the invention. Examples of such mimetics are retro-inverso peptides (Chorev et al., Ace. Chem. Res. 26: 266-273, 1993). The designing of mimetics to a known pharmaceutically active compound is a known approach to the design of drugs based on a "lead" compound. This may be desirable, e.g., where the "original" active compound is difficult or expensive to synthesize, or where it is unsuitable for a particular mode of administration, e.g., peptides are considered unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.

Additional examples of derivatives within the above general definitions include the following:

(I) Cyclic peptides or derivatives including compounds with a disulfide bridge, a thioether bridge, or a lactam. Typically, cyclic derivatives containing a disulfide bond will contain two cysteines, which may be L-cysteine or D-cysteine. Advantageously, the N-terminal amino acid and the C-terminal amino acids are both cysteines. In such derivatives, as an alternative to cysteine, penicillamine (β,β-dimethyl-cysteine) can be used. Peptides containing thioether bridges are obtainable, e.g., from starting compounds having a free cysteine residue at one end and a bromo-containing building block at the other end (e.g., bromo-acetic acid). Cyclization can be carried out on solid phase by a selective deprotection of the side chain of cysteine. A cyclic lactam may be formed, e.g., between the γ-carboxy group of glutamic acid and the ε- amino group of lysine. As an alternative to glutamic acid, it is possible to use aspartic acid. As an alternative to lysine, ornithine or diaminobutyric acid may be employed. Also, it is possible to make a lactam between the side chain of aspartic acid or glutamic acid at the C-terminus and the α-amino group of the N-terminal amino acid. This approach is extendable to β-amino acids (e.g., β-alanine). Alternatively, glutamine residues at the N- terminus or C-terminus can be tethered with an alkenedyl chain between the side chain nitrogen atoms (Phelan et al., J. Am. Chem. Soc. 119:455-460, 1997).

(II) Peptides of the invention, which are modified by substitution. In one example, one or more, preferably one or two, amino acids are replaced with another natural or unnatural amino acid, e.g., with the respective D- analog, or a mimetic. For example, in a peptide containing Phe or Tyr, Phe or Tyr may be replaced with another building block, e.g., another proteinogenic amino acid, or a structurally related analogue. Particular modifications are such that the conformation in the peptide is maintained. For example, an amino acid may be replaced by a α,α-disubstituted amino acid residue (e.g., α- aminoisobutyric acid, 1-amino-cyclopropane-l-carboxylic acid, 1-amino- cyclopentane-1-carboxylic acid, 1 -amino-cyclohexane- 1 -carboxylic acid, 4- amino piperidine-4-carboxylic acid, and 1-amino-cycloheptane-l -carboxylic acid). (III) Peptides of the invention detectably labeled with an enzyme, a fluorescent marker, a chemiluminescent marker, a metal chelate, paramagnetic particles, biotin, or the like. In such derivatives, the peptide of the invention is bound to the conjugation partner directly or by way of a spacer or linker group, e.g., a (peptidic) hydrophilic spacer. Advantageously, the peptide is attached at the N- or C-terminal amino acid. For example, biotin may be attached to the N-terminus of a peptide of the invention via a serine residue or the tetramer Ser-Gly-Ser-Gly.

(IV) Peptides of the invention carrying one or more protecting groups at a potentially reactive side group, such as amino-protecting group, e.g., acetyl, or a carboxy-protecting group. For example, the C-terminal carboxy group of a compound of the invention may be present in form of a carboxamide function. Suitable protecting groups are commonly known in the art. Such groups may be introduced, for example, to enhance the stability of the compound against proteolytic degradation.

By a "derivative" of a peptide of the invention is also meant a compound that contains modifications of the peptides or additional chemical moieties not normally a part of the peptide. Modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Methods of derivatizing are described below.

Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1 ,3-diazole. Histidyl residues are generally derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK_a of the guanidine functional group. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'~N~C~N~R') such as l-cyclohexyl-3-(2- morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3 (4 azonia 4,4-dimethylpentyl) carbodiimide. Aspartyl and glutamyl residues can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Polypeptides or derivatives thereof may be fused or attached to another protein or peptide, e.g., as a glutathione- S-transferase (GST) fusion polypeptide. Other commonly employed fusion polypeptides include, but are not limited to, maltose-binding protein, Staphylococcus aureus protein A, polyhistidine, and cellulose-binding protein.

By a "peptidomimetic small molecule" of a peptide is meant a small molecule that exhibits substantially the same ILY domain 4 activity as the peptide itself. By "substantially pure polypeptide" is meant a polypeptide or peptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the polypeptide is an ILY domain 4 polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure ILY domain 4 polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding an ILY domain 4 polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.

The "percent sequence identity" of two nucleic acid or polypeptide sequences can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;

Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, Academic Press, 1987; and Sequence Analysis Primer, Gribskov, and Devereux, eds., M. Stockton Press, New York, 1991; and Carillo and Lipman, SIAM J. Applied Math. 48: 1073, 1988. Methods to determine identity are available in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux et al, Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoL Biol. 215:403, 1990). The well known Smith Waterman algorithm may also be used to determine identity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH Bethesda, Md. 20894). Searches can be performed in URLs such as the following: http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html; or http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi. These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By "hybridize" is meant to form a double-stranded complex containing complementary paired nucleobase sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl. and Berger, Methods Enzymol. 152:399 (1987); Kimmel, Methods Enzymol. 152:507 (1987))

By "hybridizes under high stringency conditions" is meant under conditions of stringent salt concentration, stringent temperature, or in the presence of formamide. For example, stringent salt concentration will ordinarily be less than about 750 rnM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C in 750 niM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180 (1977));

Gmnstein and Hogness (Proc. Natl. Acad. Sci. USA 72:3961 (1975)); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York (2001)); Berger and Kimmel (Guide to Molecular Cloning Techniques, Academic Press, New York, (1987)); and Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York). Preferably, hybridization occurs under physiological conditions. Typically, complementary nucleobases hybridize via hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. By "therapeutic antibody" is meant a pharmaceutical composition containing an antibody or antibody derivative formulated to treat a pathogenic disease.

Brief Description of the Drawings

Figs. IA and IB are schematics showing an optimal alignment of the indicated toxin fragments.

Figs. 2A and 2B are histograms showing the amount of hCD59 expressed on the CD59 negative promonocytic cell line UIc (Fig. 2A) and the amount expressed on CD59 positive T CD4+ lymphocytic cell line ACH-2 (Fig. 2B). Fig. 2C is a graph showing percent virolysis as a function of anti-HIV gp-120 antibody concentration.

Fig. 2D is a graph showing the amount of p24 released in the indicated cells treated with the indicated compound. The abrogation of hCD59 with ILYd4 sensitizes HIV from hCD59 positive cells to complement-mediated virolysis.

Figs. 3A and 3B are graphs showing percent virolysis as a function of concentration of the indicated compound. Viral preparations (20 μl containing 5 ng HIV-I p24/ml), derived from H9 cells infected with HIV-IMN or HIV-I chronically infected cell line OMlO, were pre-incubated with IL Y4 (Fig. 3A) or anti-hCD59 monoclonal Ab (BRIC229, Bristol, Great Britain) (Fig. 3B) at various concentrations as indicated for 30 min at 37⁰C. Fig. 3C is a series of histograms showing the level of hCD59 in two cell lines that express hCD59 at a high level. Solid grey curves are stained with isotype-matched Ab + FITC-labeled secondary Ab. Blank black curves are anti-hCD59 + FITC-labeled secondary Ab. Fig. 4A is a graph showing percent HIV-I virolysis in several patients.

Two plasma samples from HIV-I -infected were tested and the each sample was repeated once. Open bars, black bars, and gray bars represent ILYd4 treatment, anti-CD59 treatment, and medium alone.

Fig. 4B is a graph showing percent HIV-I virolysis in samples treated with the indicated compound IL Y4 pre-incubation triggers significantly higher complement-mediated virolysis than pretreated with anti-hCD59 antibody or PBS pre-incubation. Pooled data of ILY4 or anti-CD59 Ab treatment experiments from all participants are shown. Horizontal bars represent means of pooled responses. Fig. 4C is a graph showing percent HIV- 1 virolysis in samples treated with the indicated sera and ILYd4.

Fig. 4D is a graph showing the amount of p24 production in cells exposed for 10 days to conditioned medium from virions pretreated with the following conditions: medium alone, anti-CD59 Ab (BRIC 229), rILYd4, and Triton X or originally exposed to heat-inactivated serum. The experiments were repeated twice for each test. The results are represented by mean 6 SD

Fig. 5 is a series of graphs showing percent virolysis in samples treated with the indicated compound of virons isolated from patient serum. HIV-I primary isolates were derived from six HIV-I -infected patients. PBMCs preincubated with rILYd4 (20 mg/ml), medium only, or anti-hCD59 monoclonal Ab (BRIC 229) were treated with heat-inactivated plasma from 5 HIV-I -positive individuals containing anti-HIV-1 envelope Abs (patients 1-5 shown in Table I) followed by exposure to 10% normal human serum as a source of complement (heat-inactivated normal serum was used as a negative control). Each panel represents the sensitivity of HIV-I virons derived from one patient to complement-mediated virolysis activated by the endogenous anti-HIV-1 Abs developed in five HIV-I -infected patients who were naive for antiretroviral therapy. Horizontal lines represent the mean. Statistical significance (p , 0.01 versus medium treatment group) is indicated by an asterisk. Fig. 6 is a series of graphs showing percent virolysis in samples treated with the indicated compound as induced by anti-HIV-1 antibodies isolated from patients. In the presence of rILYd4, the endogenous anti-HIV-1 Abs lyse the HIV-I virions through complement-mediated virolysis. In the presence of rILYd4, the endogenous anti-HIV-1 Abs developed in six HIV-I -infected patients are shown to destroying HIV-I virions through complement-mediated virolysis. Each panel represents the ability of the endogenous anti-HIV-1 Abs developed in one patient to destroy the HIV-I -infected PBMC- derived virions. Horizontal lines represent the mean. Statistical significance (p , 0.01 versus medium treatment group) is indicated by an asterisk.

Detailed Description

In general, the invention features methods of inducing antibody- mediated virolysis in a subject infected with an hCD59 or hCD55 expressing pathogen (e.g., HIV-I). CD59 and CD55 receptor activity has been associated with decreased sensitivity to endogenously antibodies. Previously it was unknown whether a subject's endogenously produced antibodies were sufficient to induce virolysis of HIV-I . We have discovered that a subject's endogenously produced antibodies, in combination with an inhibitor of GPI anchor proteins (e.g., an inhibitor of hCD59), are sufficient to produce virolysis. The invention also features the potentiation of an immune response in a subject infected with an hCD59 expressing pathogen. These methods also optionally include the prior or simultaneous treatment of the subject with a vaccine and/or therapeutic antibodies. I. Targets

The invention features the inhibition of components of a pathway responsible for complement-mediate virolysis in order to potentiate an immune response against hCD59 or hCD55 expressing viruses. Mammalian cells are provided with surface-bound complement regulatory proteins that protect them from uncontrolled complement-mediated lysis (Table 1). Two of these regulators in humans, CD55 (also known as DAF) and CD59, are glycosylphosphatidylinositol-anchored, type I cell surface proteins (GPI), which inhibit formation of the C3 convertases and prevent the terminal polymerization of the membrane attack complex, respectively. These proteins can be incorporated into the envelope of a virus, thereby shielding the virus from complement mediated virolysis. In one embodiment, the invention features inhibition of the GPI proteins, including hCD59 and hCD55. Compounds and methods for inhibiting these proteins are provided.

Table 1

II. Inhibitors

The invention features inhibitors of the above described target proteins involved in complement mediated virolysis. Such inhibitors can be, for example, toxins, antibodies (or antibody fragments), and/or small molecule inhibitors.

Toxins

The invention features the administration of modified toxins that antagonize hCD59 or other molecules in the complement pathway (e.g., Table 1 protiens). Such toxins are modified to reduce the toxicity of the toxins to non-infected cells.

ILY Streptococcus intermedins intermedilysin antagonizes hCD59 while causing toxicity in human cells. We have previously shown that domain 4 of ILY (ILYd4) (and a truncated form of ILYd4) can antagonize hCD59 without general cellular toxicity (see, e.g., International Application No. PCT/US2008/004191, which is hereby incorporated by reference in its entirety).

ILYd4 has the following sequence:

GALTLNHDGAFVARFYVYWEELGHDADGYETIRSRSWSGNGYNRGA HYSTTLRFKGNVRNIRVKVLGATGLAWEPWRLIYSKNDLPLVPQRNIS TWGTTLHPQFEDKVVKDNTD (SEQ ID NO:3)

A truncated form of ILYd4 has the following sequence: RNIRVKVLGATGLAWEPWRLIYSKNDLPLVPQRNISTWGTTLHPQFED KWKDNTD (SEQ ID NO:4)

ILY-r elated toxins

Based on their sequence similarity to ILY and binding to hCD59, the toxins perfringolysin O (PFO) and vaginolysin (VLY) are also useful for potentiating an immune response in HIV positive patients. These toxins can be modified to reduce cellular toxicity by any method known in the art. In particular, truncated forms of PFO and VLY are useful in the methods of the invention.

The sequences for domain 4 of VLY and PFO (and truncated domain 4) are shown in Fig. IA (and Fig. IB). Non-ILY related toxins

Additional toxins that bind other proteins in the complement mediated pathway are also useful to potentiate an immune response in HIV positive subjects. Such toxins can, for example, antagonize other GPI anchor proteins 5 associated with complement mediated virolysis. Aerolysin binds the GPI anchor regions of GPI-linked proteins including CD55 and CD 59. Non-toxic forms of aerolysin are therefore useful in the methods of the invention. FLAER is an inactive variant of aerolysin that does not cause lysis of cells (Cytometry B Clin Cytom. 2007 May; 72:167). Clostridium septicum alpha 10 toxin is homologous to aerolysin and also specifically binds GPI-anchored proteins. The alpha toxin m45 mutant with two amino acid changes, S189C/S238C, lost cytotoxicity but still possessed binding activity for GPI- anchored proteins (J MoI Mocrobiol Biotechno, 2006; 11 :20).

15 Antibodies

The invention includes the production of antibodies that antagonize GPI anchor proteins (e.g., hCD59 and hCD55). The invention provides for the production of antibodies, including, but not limited to, polyclonal and monoclonal antibodies, anti-idiotypic antibodies, murine and other mammalian antibodies, antibody fragments, bispecifϊc antibodies, antibody dfoδhers or tetramers, single chain antibodies (e.g., scFv's and antigen-binding antibody fragments such as Fabs, diabodies, and Fab' fragments), recombinant binding regions based oi antibody binding regions, chimeric antibodies, primatized antibodies, humanized and fully human antibodies, domain deleted antibodies, and antibodies labeled with a detectable markei or coupled to a toxin or radionuclide. Such antibodies are produced by conventional method kfiϋ>wn in the art.

Polyclonal Antibodies

Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate 30 intervals. The animals are bled, and the sera is assayed against purified protein usually by ELISA. Polyclonal antibodies that specifically bind to GPI anchor proteins (e.g., hCD59 and hCD55) can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the antigen and an adjuvant. It may be useful to conjugate the antigen or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent (e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, or succinic anhydride) .

For example, animals can be immunized against GPI anchor proteins (e.g., hCD59 and hCD55), immunogenic conjugates, or derivatives, by combining 1 μg to 1 mg of the peptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled, and the serum is assayed for antibody titer to the antigen or a fragment thereof. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with a different conjugate of the same polypeptide, e.g., conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. Chimeric, humanized, or fully human polyclonals may be produced in animals transgenic for human immunoglobulin genes, or by isolating two or more GPI anchor protein reactive B-lymphocytes from a subject for starting material.

Polyclonals may also be purified and selected for (such as through affinity for a conformationally constrained antigen peptide), iteratively if necessary, to provide a monoclonal antibody. Alternatively or additionally, cloning out the nucleic acid encoding a single antibody from a lymphocyte may be employed.

Monoclonal Antibodies In another embodiment of the invention, monoclonal antibodies are obtained from a population of substantially homogeneous antibodies (i.e., the individual antibodies including the population are identical except for possible naturally occurring mutations that may be present in minor amounts). Thus, the term monoclonal indicates the character of the antibody as not being a mixture of discrete antibodies .

Monoclonal antibodies can be prepared by methods known in the art, such as the hybridoma method of Kohler and Milstein by fusing splenocytes from immunized mice with continuously replicating tumor cells such as myeloma or lymphoma cells. (Kohler and Milstein Nature 256:495 1975; Gulfre and Milstein Methods in Enzymology: Immunochemical Techniques 73:1 1981, Langone and Banatis eds., Academic Press). The hybridoma cells are then cloned by limiting dilution methods, and supernates are assayed for antibody production by ELISA, RIA, or bioassay. In another embodiment, monoclonals may be made by recombinant DNA methods. For preparation of monoclonal antibodies (Mabs) that specifically bind

GPI anchor proteins (e.g., hCD59 and hCD55), any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein ((1975) supra. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the Mabs in the invention may be cultivated in vitro or in vivo. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing technology known in the art.

In general, a mouse or other appropriate host animal, such as a hamster, is immunized with a polypeptide that includes GPI anchor proteins (e.g., hCD59 and hCD55) to induce lymphocytes that produce or are capable of producing antibodies that can specifically bind to the antigen or fragment thereof used for immunization. Alternatively, lymphocytes are immunized in vitro.

The splenocytes of the immunized host animal (e.g., a mouse) are extracted and fused with a suitable myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding (1986) Monoclonal Antibodies: Principles and Practice, pp. 59 - 103, Academic Press). Any suitable myeloma cell line may be employed in accordance with the present invention; however, preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC- 11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.

The hybridoma cells thus prepared may be seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. The hybridoma cells obtained through such a selection and/or culture medium in which the hybridoma cells are being maintained can then be assayed to identify production of monoclonal antibodies that specifically bind GPI anchor proteins (e.g., hCD59 and hCD55). Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunoabsorbent assay (ELISA) or using a surface plasmon resonance. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Rodbard Anal Biochem. 107:220 1980.

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting all or part of the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (Morrison et al. Proc Natl Acad Sci. U.S.A. 81 :6851 1984) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. In that manner, chimeric or hybrid antibodies are prepared that have the binding specificity of an anti-GPI anchor protein monoclonal antibody. Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody including one antigen- combining site having specificity for GPI anchor proteins according to the invention and another antigen-combining site having specificity for a different antigen. Modified Antibodies

Modified antibodies of the invention include, but are not limited to, chimeric monoclonal antibodies (for example, human-mouse chimeras), human monoclonal antibodies, and primatized monoclonal antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb (see e.g., U.S. Patent Nos. 4,816,567 and 4,816,397). Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin, such as one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see e.g., U.S. Patent No. 5,585,089).

Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; US 4,816,567; and EP 125,023. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called best-fit method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies. It is also desired that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are prepared through an analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues may be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Completely human antibodies are useful for therapeutic treatment of human subjects. Such antibodies may be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice may be immunized in the normal fashion with a selected antigen. See for examples, PCT Publication Nos. WO 94/02602, WO 00/76310; U.S. Patent Nos. 5,545,806; 5,545,807; 5,569,825; 6,150,584; and 6,512,097.

Human monoclonal antibodies can also be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been previously described.

Completely human antibodies which recognize a selected epitope can also be generated using a technique referred to as guided selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in US 4,946,778 and 5,258,498.

Alternatively, phage display technology (McCafferty et al. Nature 348:552 1990) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from non-immunized donors.

The invention provides functionally-active fragments, derivatives or analogues of the immunoglobulin molecules which specifically bind to a Table 1 protein. Functionally active in this context means that the fragment, derivative or analogue is able to induce anti-anti-idiotype antibodies (i.e. tertiary antibodies) that recognize the same antigen that is recognized by the antibody from which the fragment, derivative or analogue is derived. Specifically, in a preferred embodiment, the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.

The present invention provides antibody fragments such as, but not limited to, F(ab')₂, F(ab)₂, Fab', Fab, and scFvs. Antibody fragments which recognize specific epitopes may be generated by known techniques, e.g., by pepsin or papain-mediated cleavage.

The invention also provides heavy chain and light chain dimers of the antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent No. 4,946,778; Bird Science 242:423 1988; Huston et al. Proc Natl Acad Sci. U.S.A. 85:5879 1988; and Ward et al. Nature 334:544 1989), or any other molecule with the same specificity as the antibody of the invention. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may be used (Skerra et al. Science 242: 1038 1988).

Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries for clones of Fab fragments that bind the specific antigen or by screening antibody libraries.

In other embodiments, the invention provides fusion proteins of the immunoglobulins of the invention, or functionally active fragments thereof. In one example, the immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20, or 50 amino acid portion of the protein) that is not the immunoglobulin. Preferably, the immunoglobulin, or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain. As stated above, such fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system. In another embodiment, the invention provides for the compositions and use of pooled antibodies, antibody fragments, and the other antibody variants described herein. For example, two or more monoclonals may be pooled for use.

Small molecules

In general, novel drugs for the prevention or treatment of infection by pathogens expressing hCD59, hCD55, hCD59-like molecules, or hCD55-like molecules can be identified from large libraries of natural products, synthetic (or semi-synthetic) extracts, and chemical libraries using methods that are well known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening methods of the invention and that dereplication, or the elimination of replicates or repeats of materials already known for their therapeutic activities against pathogens, can be employed whenever possible.

Candidate compounds to be tested include purified (or substantially purified) molecules or one or more components of a mixture of compounds, and such compounds further include both naturally occurring or artificially derived chemicals and modifications of existing compounds. For example, candidate compounds can be polypeptides, synthesized organic or inorganic molecules, naturally occurring organic or inorganic molecules, nucleic acid molecules, and components thereof.

Numerous sources of naturally occurring candidate compounds are readily available to those skilled in the art. For example, naturally occurring compounds can be found in cell (including plant, fungal, prokaryotic, and animal) extracts, mammalian serum, growth medium in which mammalian cells have been cultured, protein expression libraries, or fermentation broths. In addition, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographic Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). Further, libraries of natural compounds can be produced, if desired, according to methods that are known in the art, e.g., by standard extraction and fractionation.

Artificially derived candidate compounds are also readily available to those skilled in the art. Numerous methods are available for random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, for example, saccharide-, lipid-, peptide-, and nucleic acid molecule-based compounds. In addition, synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemicals (Milwaukee, WI). Libraries of synthetic compounds can also be produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation. Further, if desired, any library or compound can be readily modified using standard chemical, physical, or biochemical methods. The techniques of modern synthetic chemistry, including combinatorial chemistry, can also be used (reviewed in Schreiber, Bioorganic and Medicinal Chemistry 6:1172-1152, 1998; Schreiber, Science 287: 1964-1969, 2000).

When a crude extract is found to have an effect on the survival of pathogens expressing hCD59, further fractionation of the positive lead extract can be carried out to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having a desired activity. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives of these compounds. Methods of fractionation and purification of such heterogeneous extracts are well known in the art. If desired, compounds shown to be useful agents for treatment can be chemically modified according to methods known in the art. III. Methods of Administration

Therapy according to the invention may be performed alone or in conjunction with another therapy and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment optionally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed, or it may begin on an outpatient basis. The duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient responds to the treatment. Routes of administration for the various embodiments include, but are not limited to, topical, transdermal, transcranial, nasal, and systemic administration (such as, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, or oral administration). As used herein, "systemic administration" refers to all nondermal routes of administration, and specifically excludes topical and transdermal routes of administration.

Therapy can be in combination with therapeutic antibodies. Furthermore, therapy can also include vaccination. Vaccination can occur prior to, during, and or after administration of GPI anchor protein inhibitors. The invention also features the administration of the inhibitors of the invention in combination with other anti-pathogen therapies. For example, current treatment for HIV infection consists of highly active antiretroviral therapy, or HAART. Current HAART options are combinations (or "cocktails") including at least three drugs belonging to at least two types, or "classes," of antiretroviral agents. Typically, these classes are two nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs) plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI). New classes of drugs such as entry inhibitors provide treatment options for patients who are infected with viruses already resistant to common therapies, although they are not widely available and not typically accessible in resource- limited settings. Examples of current anti-HIV therapies include AZT, efavirenz, zidovudine, lamivudine, tenofovir, emtricitabine, and ritonavir or combinations thereof. Dosages

The dosage of compounds of the invention depends on several factors, including: the administration method, the disease to be treated, the severity of the disease, whether the disease is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic) information about a particular patient may affect dosage used.

As described above, the compounds of the invention may be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. The compounds may also be administered topically in the form of foams, lotions, drops, creams, ointments, emollients, or gels. Parenteral administration of a compound is suitably performed, for example, in the form of saline solutions or with the compound incorporated into liposomes

IV. Indications The compositions and methods of the invention are useful for treating any disease characterized by undesired hCD59 or hCD55 activity, including those set forth below.

The compounds and methods of the invention are useful for the treatment of pathogens characterized by hCD59 expression or expression of hCD59-like molecules. For example, the compounds and methods of the invention are useful to treat viruses containing hCD59 in their envelope, where the hCD59 is captured during maturation by budding from a host cell expressing hCD59 (e.g., human cytomegalovirus, HCMV, human T-cell leukemia virus type 1, HIV-I, simian immunodeficiency virus, Ebola virus, influenza virus, and vaccinia virus (a poxvirus); (Stoiber et al. MoI. Immunol. 42:153-160 (2005), Bernet et al. J Biosci 28:249-264 (2003), Rautemaa et al. Immunology 106:404-411 (2002), Nguyen et al. J Virol 74:3264-3272 (2000) ,Saifuddin et al. J. Exp. Med. 182:501-509 (1995), Spiller et al. J Infect Dis 176:339-347 (1997))). The invention features the treatment of subject having or at risk of developing an infection with any enveloped virus. Such viruses include Positive sense (+) RNA viruses (e.g., Togaviruses, Flaviviruses,

Picornaviruses, Caliciviruses, Norwalkviruses, and Coronaviruses), Negative sense (-) RNA viruses (e.g., Rhabdo viruses, Orthomyxoviruses, Paramyxoviruses, Bunyaviruses, and Arenaviruses), Double strand (+/-) RNA viruses (e.g., Reoviruses), retroviruses (e.g., Oncornavirinae (HTLV-I, HTLV- 2), Lentivirinae (HIV-I and HIV-2), and Spumavirinae), and DNA viruses (e.g., Poxviruses (Vaccinia virus), Herpesviruses, Hepadnaviruses, Papovaviruses, Adenoviruses, and Parvoviruses).

These methods and compositions of the invention are also useful for the treatment of patients infected with parasites or viruses expressing hCD59 or hCD59-like molecules, such as Herpesvirus saimiri virus, Schistosoma manosni, and Naegleria fowleri (expressing hCD59-like molecules) (Parizade et al. J Exp Med 179:1625-1636 (1994), Fritzinger et al. Infect Immun 74: 1189-1195 (2006)).

Many of the above pathogens also express hCD55 or hCD55-like molecules. Therefore, the methods and inhibitors of the invention are also useful for the treatment of pathogens expressing hCD55 or hCD55-like molecules.

In any of these embodiments, GPI anchor protein inhibitors can be administered directly to a tissue infected with an hCD59 or hCD55-expressing pathogen, or systemically to a subject infected with an hCD59 or hCD55- expressing pathogen. Preferably, the inhibitors are administered with an antibody specific for the hCD59 expressing pathogen.

Therapy may be performed alone or in conjunction with other antimicrobial therapies. Other anti-microbial therapies include antibiotics and therapeutic antibodies. The duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient responds to the treatment. Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to recovery from any as yet unforeseen side-effects.

V. Therapeutic antibodies

Methods of developing therapeutic antibodies for use in combination with hCD59 or hCD55 inhibitors of the invention are well known in the art. An example of such antibodies, for treating HIV, are the humanized antibody hNM-01 (Nakamura et al., Hybridoma, 19:427 (2000)) and the humanized KD- 247 antibody (Matsushita et al., Hum Antibodies 14:81-88 (2005)). Other antibodies (preferably humanized antibodies) can be developed using any epitope of HIV or other hCD59-expressing pathogen using standard methods.

VI. Experimental Results We have discovered that hCD59 expression in HIV is a critical regulator for protecting HIV from complement-mediated virolysis. The following experiments demonstrate that inhibitors of hCD59, in combination with antibodies provided by the serum of infected subjects, are sufficient to induce HIV virolysis. We prepared isolates of HIV from ACH-2 cells that express hCD59 at a high level and of HIV from Ul, which lack hCD59 expression. The hCD59 expression of the two cell lines was demonstrated by fluorescent activated cell sorting (FACS) analysis (Fig. 2 A and 2B). When exposed to complement and anti-HIV-gpl20 antibody, HIV from hCD59 negative cells was sensitive to complement mediated virolysis, while HIV from hCD59 positive cells was resistant to complement-mediated virolysis (Fig. 2C). This result indicates that hCD59 in HIV is a critical regulator for protecting HIV from complement- mediated virolysis.

Pre- incubation of ILYd4 with hCD59 positive HIV from two cell lines expressing hCD59 at high levels blocked hCD59 function and sensitized HIV to anti-HIV gp 120/ 160 antibody dependent complement-mediated virolysis (Fig. 2D) in a dose-dependent manner (Fig. 3A). In contrast, pre-incubation of anti-hCD59 antibody BRIC229 with hCD59 positive HIV from two cell lines expressing hCD59 at high levels did not induce virolysis in a dose dependant manner (Fig. 3B). The level of hCD59 was demonstrated using FACS analysis (Fig. 3C).

Next, we tested whether the anti-HIV antibodies of HIV positive subjects can induce complement mediated virolysis in the presence of an inhibitor of hCD59 (Fig. 4A). Using the sera from HIV patients as a source of anti-HIV antibodies, we demonstrated that IL Y4 abrogates hCD59 function and enhances HIV-I patient antibody-dependent complement-mediated virolysis (Fig. 4B). The sera from several patients induced less complement-mediated virolysis, indicating that these sera may contain lower titers of anti-HIV complement activating antibodies. Preincubation with rILYd4 dramatically increased complement-mediated virolysis of CD59-positive virions exposed to HIV-I plasma, but not to the control plasma (Fig. 4C). Fig. 4D shows that p24 was undetectable in the supernatant from H9 cells exposed to conditioned medium from Triton X-100 treatment (total lysis), indicating that potentially infective particles were totally lysed and no infectious viral particles remained. Additionally, we generated primary HIV-I isolates from six HIV-I- seropositive individuals who were naive for antiretroviral therapy, and we tested whether rILYd4 sensitizes these virions to complement-mediated virolysis. In the same experiment, we assessed the relative potency of endogenous anti-HIV- 1 Abs developed by HIV-I -infected patients to promote complement-mediated virolysis of PBMC-derived HIV- 1 primary isolates in the presence and absence of rILYd4. To this end, we pretreated the primary HIV-I isolates with or without rILYd4 and exposed them to heat-inactivated HIV-I plasma (patients 1 to 5 in Table 2), followed by incubation with pooled normal human serum as a source of complement. The results showed that rILYd4 sensitized each of the six primary HIV-I isolates to complement- mediated virolysis activated by HIV-I plasma (Fig. 5). In the presence of rILYd4, each of the five different HIV-I plasma samples tested significantly increased complement-mediated lysis of each of the six primary HIV-I isolates (Fig. 6). These effects of rILYd4 were comparable with, albeit much stronger than, those mediated by the anti-hCD59 monoclonal Ab BRIC229 (Figs. 5 and 6). These results confirm that rILYd4 sensitizes HIV-I to complement- mediated virolysis not only under experimental conditions using cell lines and commercially available Abs, but also of primary HIV-I isolates sensitized by the endogenous anti-HIV-1 Abs naturally present in the blood of HIV individuals. These results also indicate that inhibition of hCD59 with rILYd4 unprotects HIV-I, unleashing the ability of complement to lyse the virions sensitized by anti-HI V- 1 Abs present in the circulation of patients with HIV- 1.

Table 2

Methods

Preparation of HIV

Suspension cell lines were grown in RPMI 1640 (Invitrogen) with 10% fetal bovine serum (Invitrogen), 50 U/mL penicillin, 50 μg/mL streptomycin (Invitrogen), and 2 niM glutamine (Invitrogen). Cells were treated with 10 ng/mL of PMA (Sigma). After 24 h PMA treatment, supernatant was harvested for measuring HIV-1 p24 by ELISA. Viral preparations (20 μl containing 100 0 ng HIV-1 p24/ml) derived from the supernatant of PMA-activated ACH-2 or Ul cell cultures.

gpl20/160 mediated virolysis

HIV virus was pre- incubated with ILY4 at 20 μg/ml for 30 min at 37°C. 5 After pre-incubation, anti-HIV-1 gp 120/ 160 polyclonal antibodies (Abcom, Cambridge, MA) and complement or heat-inactivated serum were added. HIV- 1 structural protein p24 was then measured by ELISA to determine the extent of virolysis. Treatments with growth medium and Triton X-IOO were also included in each experiment to determine background and 100% viral lysis, respectively. Each value represents the mean ± SD of three experiments. Data were compared using the paired two-tailed Student t test.

Native antibody mediated virolysis

Viral preparations (20 μl containing 5 ng HIV-I p24/ml) derived from OM 10, an HIV-I chronically infected cell line, were pre-incubated with ILY4 or anti-hCD59 monoclonal Ab (BRIC229, Bristol, Great Britain) at 300 μg/ml for 30 min at 37⁰C in a 5% CO₂ incubator. After pre-incubation, plasma from HIV-I -infected individuals (1:5 at final dilution) and complement or heat- inactivated serum (1 : 10 at final dilution) were added. Treatments with growth medium and Triton X-IOO were also included in each experiment to determine 0 and 100% viral lysis, respectively. Percentage of virolysis was calculated by measuring the release of HIV-I p24 caused by complement activation compared to total p24 content released by detergent.

Measurem ent of HIV-I p24 in plasm a samples from HIV-1-infected patients

Plasma specimens were tested for HIV- 1 p24 Ag using the Perkin Elmer HIV-I ELISA kit as described above. Each plasma sample was treated with the lysis buffer included in the ELISA kit to lyse the viral particles for releasing HIV-I core protein p24, which was then measured.

Preparation of HIV-I Isolates from Patients

HIV-I primary isolates were generated by coculture of PBMCs from HIV-I- infected and healthy donors. PBMCs were prepared from heparinized peripheral blood donated by six HIV-I -seropositive patients naive for antiretroviral therapy (patients 1-6 in Table I) and by HIV-1-seronegative donors. PBMCs from seronegative and seropositive individuals were stimulated separately for 2 days with PHA (5 mg/ml) and cocultured at a 1:3 ratio in the presence of IL-2 (10 ng/ml) in complete RPMI 1640 medium (200 ml per well) in 96- well round- bottom plates. After 7 days of coculture, supernatants were harvested, aliquoted, and stored at -8O⁰C as HIV-I primary isolate stocks for virolysis assay.

Complement-mediated virolysis activated by anti— HIV-I Λbs in plasma ofHIV-1-infected patients Viral preparations (20 ml; 5 ng HIV- 1 p24/ml) derived from the chronically-infected cell line OMlO or from primary HIV-I isolates were preincubated for 30 min at 37°C with either rILYd4 (20 mg/ml) or neutralizing anti-hCD59 monoclonal Ab (30 mg/ml; BRIC229). After preincubation, heat- inactivated plasma from either HIV-I -infected or healthy individuals (1 :5 at final dilution) were individually added as a source of endogenous Abs, followed by exposure to either complement-competent or heat-inactivated human serum diluted in GVB++ buffer. Triton X-100 was used for determining the total virolysis. Experiments were conducted in duplicates and the paired two-tailed Student's t test was used to compare the means 6 SD.

Other Embodiments

Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, immunology, pharmacology, endocrinology, or related fields are intended to be within the scope of the invention. All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually incorporated by reference.

What is claimed is:

Claims

1. A method of potentiating an immune response against a pathogen in a subject, said method comprising administering to said subject a GPI anchor protein inhibitor; wherein said pathogen expresses hCD59 or an hCD59-like molecule.

2. The method of claim 1, wherein said pathogen is selected from the group consisting of: of human cytomegalovirus (hCMV), human T-cell leukemia virus type 1, HIV-I, simian immunodeficiency virus, Ebola virus, influenza virus, vaccinia virus, Herpesvirus saimiri virus, Naegleriafowleri, and Schistosoma manosni.

3. The method of claim 2, wherein said pathogen is HIV- 1.

4. The method of claim 2, wherein said pathogen is hCMV.

5. The method of claim 1 , wherein said GPI anchor protein inhibitor is an hCD55 inhibitor.

6. The method of claim 1, wherein said GPI anchor protein inhibitor is an hCD59 inhibitor.

7. The method of claim 1, wherein said GPI anchor protein inhibitor is a small molecule.

8. The method of claim 1, wherein said GPI anchor protein inhibitor is an antibody, or functional fragment thereof.

9. The method of claim 8, wherein said GPI anchor protein inhibitor is a Fab.

10. The method of claim 1, wherein said GPI anchor protein inhibitor is a peptidomimetic.

11. The method of claim 1 , wherein said GPI anchor protein inhibitor is not ILY domain 4.

12. The method of claim 1, further comprising administering a vaccine against said pathogen to said subject.

13. The method of claim 1, further comprising administering a therapeutic antibody against said pathogen to said subject.

14. A method of inducing antibody mediated virolysis in a subject, said method comprising administering to said subject GPI anchor protein inhibitor in an amount sufficient to induce antibody mediated virolysis; wherein the antibodies are native to said subject; and wherein said subject is infected with a virus expressing hCD59.

15. The method of claim 14, wherein said virus is HIV-I .

16. The method of claim 14, wherein said virus is hCMV.

17. The method of claim 14, wherein said GPI anchor protein inhibitor is an hCD55 inhibitor.

18. The method of claim 14, wherein said GPI anchor protein inhibitor is an hCD59 inhibitor.

19. The method of claim 14, wherein said GPI anchor protein inhibitor is is a small molecule.

20. The method of claim 14, wherein said GPI anchor protein inhibitor is an antibody, or functional fragment thereof.

21. The method of claim 20, wherein said GPI anchor protein inhibitor is a Fab.

22. The method of claim 14, wherein said GPI anchor protein inhibitor is a peptidomimetic.

23. The method of claim 14, wherein said GPI anchor protein inhibitor is not ILY domain 4.

24. The method of claim 14, further comprising administering a vaccine against said pathogen to said subject.

25. The method of claim 14, further comprising administering a therapeutic antibody against said pathogen to said subject.