WO1999038524A2

WO1999038524A2 - Therapeutic administration of low density lipoprotein receptor for viral infections and immune regulation

Info

Publication number: WO1999038524A2
Application number: PCT/IB1999/000149
Authority: WO
Inventors: Patrick Thomas Prendergast
Original assignee: Patrick Thomas Prendergast
Priority date: 1998-01-29
Filing date: 1999-01-28
Publication date: 1999-08-05
Also published as: ZA99719B; AU2069799A; WO1999038524A3

Abstract

Human and animal low density lipoprotein receptor ('LDL-R' is used herein to refer to low density lipoprotein receptor; 'LDL' is used herein to refer to low density lipoprotein) are administered to provide valuable anti-HIV activity together with anti-Hepatitis viral action. In a preferred aspect, this invention relates to anti-viral and immune regulation activity of the cysteine-rich domain of human low density lipoprotein receptor (LDL-R). As disclosed herein these substances (LDL-R [Human/Animal]) are administered for the treatment of viral infections and as immune regulation agents, and/or these substances are used in the manufacture of a medicament for any such treatment.

Description

THERAPEUTIC ADMINISTRAΗON OF LOW DENSITY LIPOPROTEIN RECEPTOR FOR VIRAL INFECTIONS AND IMMUNE REGULATION

BACKGROUND OF THE INVFNTION

The envelope protein of HIV is a 160-kDa giycoprotein. The protein is cleaved by a protease to give a 120-kDa external protein, gp 120, and a transmembrane giycoprotein, gp 41. The gp 120 protein contains the amino acid sequence that recognizes the receptor on CD4-positive human T-helper cells. We have discovered that human LDL receptor (cysteine rich domain) is a highly specific inhibitor of HIV-1 replication in-vitro. Anti-serum prepared against synthetic peptides corresponding to amino acid residues 307 -330 and 303 - 321 in gp 120 inhibit HIV-induced syncytium formation. Antibody binding to residues 303 - 330 in HIV gp 120 interferes with the binding of the virus to the CD4 receptor and fusion with the plasma membrane. SUMMARY OF THE INVENTION Herein is disclosed that fractions of the human LDL receptor can be obtained by binding to a peptide corresponding to residues 301 - 324 of the HIV gp 120 protein. These fractions have significant potency to prevent syncytium formation and appearance of viral P24 core antigen in the culture medium of HIV-infected CD4 cells and to significantly reduce HIV infectivity. BRIEF DESCRIPTION OF THE DRAWING FIGϋRF

The Figure is a model showing five domains in the structure of the human LDL receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Low density lipoprotein has five structural domains. The deduced amino acid sequence suggests that the human LDL receptor can be divided into five domains which are shown schematically in the Figure. The first domain 1, the cysteine-rich region (ligand binding), consists of the NH₂-terminal 322 amino acids, which include 47 cysteine residues (15% cysteine). All of these cysteine residues appear to be involved in disulfide bonds, since the intact receptor is labeled only poorly with ³H-iodoacetamide unless the disulfide bonds are first reduced (W. Schneider, unpublished data). The boundary for the first domain was arbitrarily set at residue 322 on the basis of the computer analysis described below (see Table 1 , page 2). Table 1 shows alignment of repeats TABLE 1

Residue No.

1-38 - AV G D R - C - E R N E F Q C Q D - - G K C I S Y KWV C D G S A E C Q D G S D E S

41-81 T C L S V T - C - K S G D F S C G G R VN R C I P Q F R C D G Q V D C D N G S D E Q

82-120 G C P P K T - C - S Q D E F R C H D - - G K C I S R Q F V C D S D R D C L D G S D E A

121-159 S C P V L T - C - G P A S F Q C N S - - S T C I P Q L WA C D N D P D C E D G S D E W

170-208 Q G D S S P - C - S A F E F H C L S - - G E C I H S S W R C D G G P D C K D K S D E E

209-247 N C AVA T - C - R P D E F Q C S D - - G N C I H G S R Q C D R E Y D C K D M S D E V

248-288 G C VN V T L C E G P N K F K C H S - - G E C I T L D KV C N MA R D C R D S D E P

291-322 E C G T N E - C - L D N N G G C S - - - H V C - - N D L K I G Y E C L C P D G - - - -

Consensus

Sequence C T C F C G C I C D D C D G S D E

(SEQ. ID NO. 1)

present in the NH₂ terminal region of the LDL receptor. Optimal alignment was made by the computer programs ALIGN and RELATE. Homologous regions between repeats are boxed. Amino acids that are highly conserved among repeats (present at a given position more than 50% of the time) are shown as a consensus sequence on the bottom line. The sequences are shown in the single letter codex which translates to the tree letter code as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe' G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. If the boundary for the first domain were extended to residue 371 , the first domain would include a total of 54 cysteines (15%).

Computer analysis of the LDL receptor sequence using a dot matrix homology program (Maizel and Lenk, 1981; Dayhoff et al., 1983) shows that the NH₂-terminal, cysteine rich domain is composed of multiple repeat sequences.

When these repeat sequences are optimally aligned, the NH₂- terminal portion of the receptor is composed of a sequence of about 40 residues that is repeated eight times with minor variations (Table 1). With the introduction of a few brief gaps, each of the repeat sequences can be aligned such that the cysteine residues are in register (Table 1 ). At many positions in this sequence, a particular amino acid occurs so frequently that a consensus sequence can be written.

At the COOH-terminal end of seven of the eight repeat sequences in Table 1 , there is a version of the following sequence: Asp-Cys-X-Asp-Gly-Ser-Asp-Glu. Four of eight residues in this sequence have negatively charged side chains. The two ligands for the LDL receptor, apoprotein B and apoprotein E, are known to contain positively charged lysine and arginine residues that are crucial for receptor binding. If these residues are selectively modified by acetylation (Basu et al., 1976), reductive methylation (Weisgraber et al., 1978), or reaction with cyclohexane-dione (Mahley et al., 1977), then ligand binding to the receptor is disrupted and the anti-viral HIV activity is also disrupted. Moreover, mutant forms of apoprotein E with single substitutions of cysteine for arginine residues show reduced binding to the LDL receptor (Schneider et al., 1981 ; Innerarity et al., 1984). In apoprotein E, the crucial arginine and lysine residues are believed to cluster in a small region of the 33 Kilodalton molecule. The three most crucial residues for binding are Arg 142, Arg 145, and Lys 146, all of which are believed to face the same side of a single α-helix (Innerarity et al., 1984). It seems reasonable to speculate that these arginines and lysines interact with the clustered negatively charged residues in the cysteine-rich region of the LDL receptor (Innerarity and Mahley, 1978; Pitas et al., 1980). Therefore two of the repeats shown in Table 1 may constitute one ligand binding site. The finding of a cysteine-rich domain in the LDL receptor explains two important properties of the receptor. First, the receptor is extremely stable as long as the disulfide bonds are not reduced. It can be boiled in SDS or in guanidine and retain the ability to bind LDL as long as reducing agents are not present (Daniel et al., 1983). Thus the presence of disulfide bonds approximately once every seven residues imparts great stability to the receptor binding site. This stability may protect the molecule against extreme changes in pH when it recycles between coated pits on the cell surface and acidic endosomes within the cell, several times within the cell, several times during its life history (Brown et al., 1983). Second, pre-treatment of the receptor with reducing agents markedly retards its mobility on SDS gets (Daniel et al., 1983). The decreased mobility after reduction of disulphide bonds presumably relates to the unfolding of the extensively cross-linked protein.

The second domain 2 (see the Figure) of the human LDL receptor, consisting of approximately 350 residues, begins near the end of the cysteine-rich repeats and extends to amino acid residue 640. Like the cysteine-rich domain, this domain also contains repeat sequences. The optimal alignment for residues 409 to 610 shows that it consists of five repeats of approximately 25 amino acids each; 11 of the amino acids in each repeat are highly conserved. The striking aspect of the second domain is its homology to the amino acid sequence of the polyprotein precursor for mouse EGF. This homology was previously noted when the partial sequence of the bovine LDL receptor was compared with that of the mouse EGF precursor (Russell et al., 1984). The homology between the mouse EGF precursor and the human LDL receptor is even more significant than that previously noted with the bovine receptor. Within a stretch of 350 amino acids (residues 290 to 640 in the LDL receptor), 33% of the amino acids were identical. Maximal homology was noted in a stretch of 139 amino acids (residues 457 to 595 in the LDL receptor) that showed a 40% identity with the EGF precursor.

The third domain 3 (see the Figure) of the human LDL receptor, which was previously identified in the bovine sequence (Russell et al., 1984), is a region (residues 700 to 747, see the Figure) rich in serine and threonine that is just outside the membrane spanning region of the receptor (see the Figure). In the human LDL receptor, this stretch of 48 amino acids contains 18 serine or threonine residues located immediately outside the membrane-spanning region. In the bovine receptor , this sequence was shown by biochemical studies to contain O-linked carbohydrate chains (Russell et al., 1984). The conservation of this serine and threonine-rich region in the human and bovine LDL receptors suggests that it is also the site to which O-linked carbohydrate chains are added in the human receptor sequence.

The external portion of the human LDL receptor contains five potential sites for N-linked glycosylation that exhibit the consensus sequence Asn-X-Ser or Asn-X-Thr (residues sites are in the cysteine-rich domain, and two of the sites are in the second domain that is homologous with the EGF precursor. Quantitative studies have indicated that only two N-linked chains are present in the bovine LDL receptor. The human LDL receptor also contained two N-linked carbohydrate chains (Cummings et al., 1983). It is possible that the three potential N-llnked glycosylatlon sites in the cysteine-rich region are not actually glycosylated because of their occurrence in such an unusual protein environment.

The fourth domain 4 (see the Figure) is a sequence of 22 hydrophobic amino acids that is believed to be the membrane-spanning region of the receptor (see the Figure). A similarly located hydrophobic sequence in the bovine LDL receptor was shown by proteolysis experiments to span the membrane (Russell et al., 1984).

The fifth domain 5 (see the Figure) of the human receptor consists of 50 amino acids at the COOH-terminal end of the protein that are located on the cytoplasmic side of the plasma membrane. This sequence is strongly homologous with the cytoplasmic domain of the bovine LDL receptor (Russell et al., 1984). The 50 residue cytoplasmic domains of the human and bovine LDL receptors are identical with the exception of four residues: serine substituted for threonine at position 825 in the human LDL receptor; asparagine for serine at position 820; histidine for arginine at position 819; and asparagine for serine at position 796.

An affinity chromatography was performed on LDL Human Receptor using a resin-bound peptide of Sequence 'A'.

Sequence 'A'-NNTRKSIRIQRGPGRAFVTIGKIG [SEQ. ID NO. 2] The fraction of LDL that binds to the resin-bound Sequence 'A' peptide is useful in preventing syncytium formation in HIV-infected CD4 cells and reduces HIV infectivity. The cysteine-rich domain of the human LDL receptor according to the present invention is useful in the treatment of AIDS and ARC and in the therapy of Hepatitis B and C virus infections. Procedure 1

Fractionation of LDL-R(Human) on Sequence-A-AffiGel-10.200 mg (dry weight) of LDL-R(Human) were applied to the peptide column (1 x 10 cm bed volume) equilibrated in 10 mM Hepes, pH 7.4, 0.05 M NaCI. The column was extensively washed to remove the unbound or unreactive LDL-R (Human); 1 ml fractions were collected. The column was then eluted in equilibration buffer containing 1 M NaCI to obtain high reactive portions of the LDL(h) receptor. An addition of 240 liters of column equilibration buffer. For the assay of 1 M NaCI eluted fractions, 100 liters of sample were admixed with 250 liters of 1 mg/ml of protamine sulphate. Turbidity was measured at 420 nm. Uronic acid was determined by the carbazole reaction, T. Bitter, H.M. Muir, Anal. Biochem. 4, 330 (1967). Procedure 2 Dose-dependent inhibition of HIV-1 infection of JM cells by

LDL-R-(human) measured by: (i) virus-induced syncytia formation and (ii) levels of P24 virion core antigen in supernatant culture fluid. Virus stock of the GB8 strain prepared from cell-free medium of acutely infected JM cells was diluted in growth medium (RPMI 1640, 10% fetal calf serum) containing different concentrations of test compound. After 15 minutes at room temperature, cells were added and virus adsorption carried out at this temperature for 2 hours to provide a multiplicity of infection (MOI) of 0.001 syncytia-forming units per cell. Infected cells were pelleted, washed three times in phosphate buffered saline, resuspended in fresh growth medium containing test peptide (LDL receptor) at appropriate concentrations and distributed into 24 well tissue culture plates. After 3 days incubation at 37° C, numbers of syncytia were scored in quadruple. The supernatant culture fluid was sampled and clarified by centrifugation (2,000 rpm/5 minutes). The level of P24 antigen was determined by the Abbott core antigen Elisa test after treatment with 0.1% Triton X-100.

The anti-HIV activity LDL-R of this invention includes low density lipoprotein receptor as set forth in Table 1 , functionally equivalent salts thereof, functionally equivalent derivatives thereof, and functionally equivalent muteins thereof, as well as any portions of any such materials which exhibit affinity for the resin-bound HIV Sequence 'A'. Such portions may be of at least 7 residues, more commonly at least 30 residues, or at least 100 residues, or at least 300 residues, and preferably comprises a part or all of the cysteine- rich first domain (322 residues).

The invention further concerns recombinant DNA molecules comprising the nucleotide sequence coding for said LDL receptor or for its active muteins or fused proteins, expression vehicles comprising them and host cells transformed therewith and to a process for producing the soluble LDL receptor, its active muteins or fused proteins, by culturing said transformant cells in a suitable culture medium.

The isolation of LDL-receptor is well known in the art, and such known methods for isolating LDL-receptor may be used to obtain LDL-receptor for use in accordance with the present invention. As used herein the term "muteins" refers to analogues of the soluble

LDL receptor in which one or more of the amino acid residues of the natural soluble LDL receptor, such as 1-30, preferably 1-10 (e.g., 7) and more preferably 1-5 residues or even only a single residue, are replaced by different amino acid residues or are deleted, or one or more amino acid residues, such as 1-20, preferably 1 - 10 (e.g., 7), more preferably 1-5, or only one residue are added to the natural sequence of the soluble LDL receptor, without changing considerably the antiviral activity of the resulting product. These muteins are prepared by known synthesis and/or site-directed mutagenesis techniques, or any other known technique suitable therefor. The substitutions are preferably conservative. See, e.g., Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference.

The types of such substitutions which may be made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (supra) and FIGS. 3-9 of Creighton (supra). Based on such an analysis, conservative substitutions may be defined herein as exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: ala, ser, thr (pro, giy); 2. Polar, negatively charged residues and their amides: asp, asn, glu, gin;

3. Polar, positively charged residues: his, arg, lys;

4. Large aliphatic, nonpolar residues: met, leu, ile, val (cys); and

5. Large aromatic residues: phe, tyr, trp. The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. Pro, because of its unusual geometry, tightly constrains the chain. Cys can participate in disulfide bond formation which is important in protein folding. Note that Schulz et al. would merge Groups 1 and 2, above. Note also that Tyr, because of its hydrogen bonding potential, has some kinship with Ser, Thr, etc.

Conservative amino acid substitutions according to the present invention, e.g., as presented above, are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. One skilled in the art will appreciate that the effect of substitutions can be evaluated by routine screening assays, either immunoassays or bioassays. For example, a mutant typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, or a biological sample containing a soluble LDL receptor protein, for example, by immunoaffinity chromatography using a specific antibody on a column (to absorb the mutant by binding to at least one epitope).

The term "fused protein" refers to a polypeptide comprising the soluble LDL receptor or a mutein thereof fused with another protein which has an extended residence time in body fluids. The soluble LDL receptor may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.

The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the soluble LDL receptor, muteins and fused proteins thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.

"Functional derivatives" as used herein cover derivatives of the soluble LDL receptor and its fused proteins and muteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it. These derivatives may, for example, include polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the soluble LDL receptor in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties. The term "functional derivative" also includes proteins which have an amino acid sequence longer or shorter than the sequence determined, as long as the protein still has the ability to inhibit viral infection.

As "active fractions" of the soluble LDL receptor, its fused proteins and its muteins, the present invention covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has the ability to inhibit viral infection and/or activity. Such active fractions can be readily determined by testing smaller and smaller portions of the entire soluble LDL receptor or mutein to find the smallest fragment which retains the ability to inhibit viral infections. Any fractions containing the smallest active fraction will also be an active fraction. Undue experimentation would not be involved as the required tests for antiviral activity (as described herein) may be routinely carried out.

This invention further concerns DNA molecules comprising the nucleotide sequence encoding the soluble LDL receptor, fused proteins, muteins or active fractions thereof, replicable expression vehicle expression of such transformed hosts. The term "DNA molecules" includes genomic DNA, cDNA, synthetic DNA and combinations thereof.

The production of the recombinant soluble LDL receptor may be carried out by different techniques. According to one approach, the known cDNA of the entire human LDL receptor is taken from plasmid pLDLR-2 (Yamamoto et al., op cit). The DNA is subjected to site directed mutagenesis with appropriate oligonucleotides so that a termination codon and a polyadenylation site are inserted after codon 292 of the mature LDL receptor. This construct is then inserted into appropriately constructed expression vectors by techniques well known in the art (see Maniatis et al., op cit.). Double-stranded cDNA is linked to plasmid vector by homopolymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques. DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase.

In order to be capable of expressing the soluble LDL receptor, its muteins or the fused proteins, an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulator information linked to the DNA coding for the desired protein in such a way as to permit gene expression and production of the protein. First, in order for the gene to be transcribed, it must be preceded by a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). They are different for prokaryotic and eukaryotic cells.

It should be understood that while Sequence A peptide has been used, other related peptides with up to 5 amino acid substitutions and those related peptides being extended on the amino or carboxy terminal ends or both as well as those related peptides being truncated on the amino or carboxy terminal ends, have been demonstrated to produce substantially similar results and applicants intend that the expression Sequence 'A^* peptide and variants thereof when used herein with regard to the preparation of anti-HIV LDL-R will include such related peptides. In particular, Table 2 lists certain known variances of the Sequence 'A' region of other HIV isolates. Peptides having these sequences as well as other sequence variations may be substituted for the sequence of sequence 'A' in the process of this invention. In addition, other peptide regions in the gp 120/gp 41 that fulfill the criteria for LDL-R binding are included.

The at least one Sequence 'A' peptide or variant thereof is covalently bound to a chromatography resin in the usual manner and the resin-bound Table 2 HIV Isolate Sequence 'A' - Variant Sequence

lll_B(BH10) N N T R K S I R I Q R G P G R A F V T I G K I G

(SEQ. ID NO.3)

lll_B(BH8) K

(SEQ. ID NO.4)

RF - - - - - S - T K - - - - V I Y A T - Q I -

(SEQ. ID NO.5)

MN Y - K - H I - Y - T K N I - (SEQ. ID NO.6)

SC T R S - H I - - - Y A T - D I -

(SEQ. ID NO.7)

WMJ-2 - - V - R S L S I R - R E I -

(SEQ. ID NO.8)

LAV-MAL R G - H F Q - L Y - T - I V

(SEQ. ID NO.9)

SF-2 S - Y I - H - T - R I -

(SEQ. ID NO.10)

NY5 K - G - A l T L Y A R E - I - (SEQ. ID NO.11)

Z3 S D K I - Q S - R I K V - Y A K - G I T

(SEQ. ID NO.12) sequence 'A' peptide or variant thereof is used to isolate anti-HIV LDL-R by affinity chromatography in the conventional manner. The resin must be insoluble in the solvents and buffers to be employed, it must be mechanically and chemically stable with good flow properties, it must be easily coupled to the sequence A peptide or variant thereof, and it should have a large surface area accessible to the substrate to be absorbed.

Anti-HIV LDL-R can be used to prevent syncytium formation in cells infected with HIV-1 virus or other related viruses having gpl20 surface protein. Anti-HIV LDL-R can be used to treat AIDS and ARC and other diseases caused by the retrovirus HIV or other related viruses having gpl20 surface protein. The amount of anti-HIV LDL-R which is needed to prevent syncytium formation in HIV infected cells can be any effective amount. Experimentally, applicants have determined that anti-HIV LDL-R (or cysteine-rich domain of same) when employed at a concentration of 10 μg/ml resulted in complete inhibition of syncytium formation as well as reduced the presence of P24 antigen, an indicator of viral replication, to below 2.0 x 10². The amount of anti-HIV LDL-R to be administered in order to treat AIDS or ARC or other disease caused by HIV infection can vary widely according to the particular dosage unit employed, the period of treatment, the age and sex of the patient treated, the nature and extent of the disorder treated, and other factors well-known to those practicing the appropriate arts. Moreover, anti-HIV LDL-R can be used in conjunction with other agents known to be useful in the treatment of viral diseases and agents known to be useful to treat the symptoms of and complications associated with diseases and conditions caused by retroviruses. The anti-HIV effective amount of anti-HIV LDL-R to be administered will generally range from about 15 mg/kg to 500 mg/kg. A unit dosage may contain from 25 to 500 mg of LDL-R, or the cysteine rich domain of Human LDL receptor, and can be taken one or more times per day. Anti-HIV LDL-R can be administered with a pharmaceutical carrier using conventional dosage unit forms either orally or parentally.

For oral administration the anti-HIV LDL-R can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions. The solid unit dosage forms can be a capsule, which can be of the ordinary hard- or soft-shelled gelatine type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and cornstarch. In another embodiment, the compounds of this invention can be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, lubricants intended to improve the flow of tablet granulations and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium, or zinc stearate, dyes, coloring agents, and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include dilutents such as water and alcohols, for example, ethanol, benzyl alcohols, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptably surfactant, suspending agent, or emulsifying agent.

The anti-HIV LDL-R of this invention may also be administered parentally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally, as injectable dosages of the compound in a physiologically acceptable dilutent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketols such as 2,2-dimethyl-1,3-dioxolane-4-methanol, others such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent and other pharmaceutical adjuvants. Illustrative of oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Suitable fatty acids include oleic acid, steric acid, and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamines acetates; anionic detergents, for example, alkyl. aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides. and polyoxyethylenepolypropylene copolymers, and amphoteric detergents, for example, alkyl-beta-aminepropionates and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures. The parenteral compositions of this invention will typically contain from about 0.5 to about 75% by weight of anti-HIV LDL-R in solution. Preservatives and buffers may also be used advantageously. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 10 to about 20. The quantity of surfactant in such formulations ranges from about 2 to about 20% by weight. Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol, administered in liposomes for oral or parental.

The present invention is also directed to the use of human low-density lipoprotein receptor, a functionally equivalent salt thereof, a functionally equivalent derivative thereof, a functionally equivalent mutein thereof, or a part of any such receptor, salt, derivative or mutein in the manufacture of a medicament for use in the treatment of a condition selected from the group consisting of: (a) immunodeficiency resultant from a viral infection;

(b) immunodeficiency resultant from at least one of the following: bacterial, mycoplasmic, fungal and parasitic infections;

(c) immunodeficiency resultant from the growth of neoplastic tissue; (d) immunodeficiency resultant from any cytokine or hormone imbalance or imbalance of any natural product within the patient;

(e) myalgic encephalomyelitis (ME);

(f) post inoculation or viral infection fatigue syndrome; (g) tuberculosis infection; and

(h) hepatitis.

Preparation of the Sequence 'A' Peptide

The peptide sequence 'A' was synthesized by solid-phase methods using an Applied Biosystems Model 430A synthesizer on 0.5 mmol of a Boc Gly(PAM)-resin (0.8 mmol/g). The N^α-t-Boc-protected amino acids were double coupled as their preformed symmetrical anhydrides, first in N,N-dimethylformamide then in dichloromethane using protocols supplied by the manufacturer. Asn, Arg and Gin were double coupled as their 1-hydroxybenzotriazole esters. An acetic anhydride capping step was included between each successive amino acid. The side chain protection was as follows: Lys(2-CIZ, Arg(Tos), Ser(Bzl), Thr(Bzl). The peptide was deprotected and cleaved from the resin (0.25 mmol) by treatment with liquid hydrofluoric acid (HF) containing 5% anisole at -5° C (salt-ice bath) for 40 minutes. After removal of the HF in vacua the peptide was extracted from the resin with 30% acetic acid followed by 30% acetonitrile. The filtrates were lyophilized and the residue dissolved in 6 M urea. The peptide was purified on a Beckman 2 inch x 150 mm C18 column at 80 mi/min with a 20-25% linear gradient of acetonitrile in 0.1% trifluoroacetic acid over 15 min. The main peak was isolated and lyophilized leaving 137.9 mg of the desired product. Amino acid analysis (6N HCI, 48 hours, 106°C) Asx 1.79(2); Thr 1.98(2); Ser 0.81(1); Glx 0.98(1), Pro 0.94Q; Gly 4.14(4); Ala 1.01(1); Val 1.09(1); He 3.85(4); Phe 0.98(1); Lys 2.07(2); Arg 3.75(4).

Preparation of AFFIGEL-10 bound Sequence 'A' Peptide

To 10 ml AffiGel-10 (BIORAD) in 0.1 M MOPS, pH 6.0-10.0 coupling buffer is added 100-300 mg of SEQUENCE 'A' PEPTIDE in coupling buffer.

The coupling reaction is carried out at 40 degrees C. with gentle rocking for 4 hrs. Remaining active esters on the resin are blocked by adding 0.1 ml of 1 M glycine ethyl ester (pH 8) or 0.1 ml of 1 M ethanolamine HCI (pH 8) per ml of gel and then incubated for 1 hour. The resin is then transferred to a column and extensively washed with 0.01 Hepes, 0.05 M NaCI, pH 7 4.

Table 3

Putative gpl20 LDL-R binding regions of HIV gp120 - ^'65 ISTSKRGKVQKEYAFFYK gp120 - ³⁰⁶ NNNTRKSIRIQRGPGRAFO gp120 - ⁴⁷⁷ SELYKYKWKIEPLGVAP gp120 - ⁴⁸⁴ PTKAKRRWQREKRAVGI

Putative gp41 LDL-R binding regions of HIV gp41 ²⁷ GERDRDRSI RLVNGSLAL gp41 ^27" VELLGRRGWEALKYWWNL gp41 ³²⁵ AYRAIRHIPRRIRQGLER Table 3 shows putative LDL-R binding regions of gp120 and gp41.

Abilitv of anti-HIV LDL-R to prevent svncytia formation and expression of P24 viral core antigen using JM cells and GBB virus strain

To show that the LDL-R that binds Sequence 'A' blocks HIV Infection, CD4 T-cells (JM) were exposed to a clinical isolate of HIV-1 , GBB. The virus was first incubated with LDL-R for 15 minutes and then the cells were added . After 2 hours adsorption, the virus inoculum was removed and the cells were washed three times to remove traces of input virus. Antiviral activity was determined after 3 days incubation by plotting the mean number of syncytia found in quadruple cultures against logic concentration of LDL-R. The 50% effective dose ED₅₀ of LDL-R for inhibition of syncytia was estimated as 0.4 μg/ml. The potency of LDL-R was also measured by assaying viral core antigen (P24 test-Abbott) in the supernatant fluid. ED₅₀ values of 0.5 μg/ml were obtained for LDL-R.

Claims

1. A method of enhancing immune response in a patient suffering from a condition selected from the group consisting of:

(a) immunodeficiency resultant from a viral infection; (b) immunodeficiency resultant from at least one of the following: bacterial, mycoplasmic, fungal and parasitic infections;

(c) immunodeficiency resultant from the growth of neoplastic tissue;

(d) immunodeficiency resultant from any cytokine or hormone imbalance or imbalance of any natural product within the patient; (e) myalgic encephalomyelitis (ME);

(f) post inoculation or viral infection fatigue syndrome;

(g) tuberculosis infection; and (h) hepatitis, comprising administering to said patient an immune response enhancing effective amount of a pharmaceutical formulation comprising recombinant human low-density lipoprotein receptor or part thereof.

2. A method according to Claim 1 wherein the part of the low-density lipoprotein is the cysteine rich domain or part thereof.

3. A method of enhancing immune response in a patient suffering from a condition selected from the group consisting of: a. immunodeficiency resultant from a viral infection; b. immunodeficiency resultant from at least one of the following: bacterial, mycoplasmic, fungal and parasitic infections; c. immunodeficiency resultant from the growth of neoplastic tissue; d. immunodeficiency resultant from any cytokine or hormone imbalance or imbalance of any natural product within the patient; e. myalgic encephalomyelitis (ME); f. post inoculation or viral infection fatigue syndrome; g. tuberculosis infection; and h. hepatitis, comprising administering to said patient an immune response enhancing effective amount of a pharmaceutical formulation comprising a mimic molecule to the cysteine rich domain of recombinant human low density lipoprotein receptor.

4. An anti-viral therapy where a protease inhibitor is administered in combination with recombinant human low density lipoprotein receptor or part thereof to alleviate symptoms of CRIX Belly, or elevated cholesterol and triglyceride levels.

5. An anti-viral therapy where one or more anti-viral agents is administered in combination with recombinant human low-density lipoprotein receptor or part thereof for the treatment of a viral infection.

6. An anti-viral therapy according to claim 5 or 6 wherein the part of the recombinant human low-density lipoprotein receptor is the cysteine-rich domain or part thereof.

7. A process for isolating the anti-viral portion of recombinant human LDL which comprises: a. binding of LDL receptor to resin-immobilized Sequence 'A' peptide

NNTRKSIRIQRGPGRAFVTIGKIG or its sequence variants; b. segregating the resin-bound Sequence 'A' peptide or its sequence variants from the bound LDL receptor having anti-viral activity; c. removing the bound anti-viral LDL receptor from the resin-bound Sequence 'A' peptide or its sequence variants by washing the resin with a salt solution; and d. isolating the anti-viral recombinant human LDL receptor from the salt solution.

8. A pharmaceutical composition for the treatment of AIDS or ARC, containing an anti-HIV effective amount of recombinant human LDL-R or part thereof.

9. A pharmaceutical composition for reducing syncytium formation in HlV-infected CD4 cells, containing an anti-HIV effective amount of LDL-receptor or part thereof.

10. A pharmaceutical composition according to claim 9 or 10 wherein the part of the recombinant human low-density lipoprotein is the cysteine-rich domain or part thereof.

11. A pharmaceutical composition for the treatment of blood or body fluids or organs to neutralize and remove immunosupressive peptides and/or viruses containing recombinant human LDL-receptor or part thereof.

12. A pharmaceutical composition for the treatment of hepatitis 5 containing recombinant human LDL-receptor or part thereof.

13. A treatment according to claim 12 wherein said hepatitis is selected from the group consisting of hepatitis A, hepatitis B and hepatitis C.

14. A pharmaceutical composition according to claim 11 ,12 or 13 wherein the part of the recombinant human low-density lipoprotein receptor is 0 the cysteine-rich domain or part thereof.

15. Treatment of a viral infection in a host wherein the host is administered a pharmaceutical composition containing recombinant human low-density lipoprotein receptor or part thereof.

16. A treatment according to claim 15 wherein the host is a human or 5 animal.

17. A pharmaceutical composition according to claim 15 wherein the part of the recombinant human low-density lipoprotein receptor is the cysteine-rich domain or part thereof.

18. A method of enhancing immune response in a patient suffering o from a condition selected from the group consisting of:

(a) immunodeficiency resultant from a viral infection;

(c) immunodeficiency resultant from the growth of neoplastic tissue; 5 (d) immunodeficiency resultant from any cytokine or hormone imbalance or imbalance of any natural product within the patient;

(e) myalgic encephalomyelitis (ME);

(f) post inoculation or viral infection fatigue syndrome;

(g) tuberculosis infection; and 0 (h) hepatitis, comprising administering to said patient an immune response enhancing effective amount of a pharmaceutical formulation comprising human low-density lipoprotein receptor, a functionally equivalent salt thereof, a functionally equivalent derivative thereof, a functionally equivalent mutein thereof, or a part of any such receptor, salt, derivative or mutein.

19. A method as recited in claim 18, further comprising administering to said patient a protease inhibitor.

20. A method as recited in claim 18 or 19, further comprising administering to said patient at least one anti-viral agent.

21. A method according to claim 18 wherein said hepatitis is selected from the group consisting of hepatitis A, hepatitis B and hepatitis C.

22. Use of human low-density lipoprotein receptor, a functionally equivalent salt thereof, a functionally equivalent derivative thereof, a functionally equivalent mutein thereof, or a part of any such receptor, salt, derivative or mutein in the manufacture of a medicament for use in the treatment of a condition selected from the group consisting of: (a) immunodeficiency resultant from a viral infection; (b) immunodeficiency resultant from at least one of the following: bacterial, mycoplasmic, fungal and parasitic infections;

(c) immunodeficiency resultant from the growth of neoplastic tissue;

(f) post inoculation or viral infection fatigue syndrome;

(g) tuberculosis infection; and (h) hepatitis.