Title:  Inhibitors of HIV infection

United States Patent:  6,358,511

Assignee:  Mount Sinai School of Medicine of the City University of New York (New York, NY)

Filed:  May 3, 1999

The present invention is directed to novel inhibitory compounds which are capable of reducing, eliminating, or preventing human immunodeficiency virus (HIV) infection. These compounds may be polypeptides or peptides comprising particular sequences that inhibit HIV-1 infection. These compounds may be derived from CD8+ lymphocytes. The invention is also directed to novel CD8+ cell lines which secrete these novel inhibitors. The invention is further directed to compositions comprising an inhibitor of the invention and to methods for the use of such compositions in the prevention and/or treatment of HIV infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel inhibitors of HIV replication which are capable of reducing, eliminating or preventing HIV infection and which are isolated from CD8+ lymphocytes. The invention is also directed to cell lines which secrete these novel inhibitors. The invention is further directed to compositions comprising an inhibitor of the invention and to methods for the use of such compositions in the prevention and/or treatment of HIV infection.

An inhibitor of the invention is defined as a peptide or polypeptide secreted by CD8+ cells which is capable of reducing, eliminating or preventing infection by HIV or other retroviruses. The invention is also directed to nucleic acids encoding such inhibitors, and to nucleic acids which hybridize to a known coding sequence at high stringency and encode an inhibitor of the invention. The invention further encompasses analogs, homologs, derivatives and truncated fragments of an inhibitor of the invention which retain these defined functional properties.

As used herein, the term "peptide" refers to an oligomer of at least two contiguous amino acids, linked together by a peptide bond, and not greater than fifty amino acids. As used herein, the term "polypeptide" refers to an oligomer of at least fifty amino acids.

As used herein, "substantially corresponds" means an amino acid sequence having approximately 70% homology in amino acid sequence to an inhibitor of the invention. For example, conservative amino acid substitutions which do not alter the chemical type of amino acid residue in an inhibitor can be introduced into the inhibitor provided that its functional activity is retained. By "homolog" is meant the corresponding peptides or polypeptides which are derived from an inhibitor of the invention so long as the functional properties of the inhibitor are retained.

By "analog" is meant substitutions, rearrangements, deletions, truncations and additions to the amino acid sequence of an inhibitor, so long as its functional properties are retained. Analogs also include inhibitors which contain additional amino acids added to either end of the peptides that do not affect biological activity, e.g., the presence of inert sequences added to a functional inhibitor which are added to prevent degradation. An algorithm can be used in the identification of homologs and analogs, such as the BLASTP program (Altschul, J.Mol.Evol. 36:290, 1993; Altschul, J.Mol.Biol. 215:403, 1990). In a preferred embodiment, a preferred length for a truncated, functional derivative of an inhibitor of the invention ranges from 4 amino acids to 35 amino acids.

The criticality of particular amino acid residues in an inhibitor may be tested by altering or replacing the residue of interest. For example, the requirement for a cysteine residue, which can be involved in the formation of intramolecular or intermolecular disulfide bonds, can be tested by mutagenesis of the cysteine to another amino acid, for example, tyrosine, which cannot form such a bond.

Inhibitors of the invention are described with reference to the following amino acid nomenclature wherein

A=Ala=Alanine

R=Arg=Arginine

N=Asn=Asparagine

D=Asp=Aspartic acid

B=Asx=Asparagine or aspartic acid

C=Cys=Cysteine

Q=Gln=Glutamine

E=Glu=Glutamic acid

Z=Glx=Glutamine or glutamic acid

G=Gly=Glycine

H=His=Histidine

I=IIe=Isoleucine

L=Leu=Leucine

K=Lys=Lysine

M=Met=Methionine

F=Phe=Phenylalanine

P=Pro=Proline

S=Ser=Serine

T=Thr=Threonine

W=Trp=Tryptophan

Y=Tyr=Tyrosine

V=Val=Valine.

In preferred embodiments, an inhibitor of the invention comprises a polypeptide having an amino acid sequence subtantially corresponding to one of the following sequences as listed:

EQVEASVAS (SEQ. ID NO. 1)

EQVEASVASVRSLY (SEQ. ID NO. 2)

Chimeric inhibitors which combine one or more of the preferred peptides or polypeptides or segments or fragments thereof are within the scope of the invention. Inhibitors of the present invention also include cyclic or derivatized peptides, and further include peptides containing D-amino acids as well as L-amino acids.

The peptide and polypeptide inhibitors of the invention can be synthesized according to Merrifield solid-phase synthesis techniques (Kotler et al., Proc. Natl. Acad. Sci. 85:4185-4189, 1985; Barany et al., in Gross et al., eds., The Peptides, Vol. 2, Academic Press, 1980) or other techniques of peptide synthesis known to those skilled in the art. After cleavage and deprotection, synthetic peptides or polypeptides can be purified by, for example, gel filtration, chromatography, and any reverse-phase column/HPLC system known to those skilled in the art. Peptide inhibitors derived from an inhibitor of the invention may also be produced by chemical or enzymatic digestion of the full-length protein using techniques that are known to those skilled in the art.

Peptides and polypeptides may also be prepared by standard recombinant DNA technology using techniques well known to those skilled in the art for nucleotide-based based peptide design (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1995). Site-directed mutagenesis using recombinant DNA techniques, for example, may be used to prepare peptide analogs and homologs from parent peptides.

An inhibitor may be recovered by purification from a cell line secreting such an inhibitor, using standard techniques for protein purification which are known to those skilled in the art, including, but not limited to, size fractionation, ion-exchange chomatography, and reverse-phase chromatography.

The amino acid sequences of the peptides and polypeptides can be confirmed and identified by amino acid composition analysis as well as manual and automated Edman degradation and determination of each amino acid, HPLC analysis, or mass spectrometry.

The inhibitors of the invention are isolated from CD8+ cells which can be isolated from uninfected or HIV-infected individuals. Such cells can be prepared by isolating monocytes from peripheral blood mononuclear cells (PBMC) using ficoll-hypague purification. Preferably, such cells are then immortalized using Herpesvirus saimiri, and the surviving cells are purified by limiting dilution and analyzed by fluorescent-activated cell sorting (FACS). Isolation of soluble inhibitors is accomplished by recovering supernatant from cells of interest and using cell-free supernatants in assays to determine the presence of an inhibitor.

Partial amino acid sequences corresponding to an inhibitor molecule isolated from CD8+ cells can be used to isolate a full-length nucleotide sequence encoding the inhibitor using standard techniques of molecular biology known to those skilled in the art. For example, degenerate nucleotide primers can be generated from a known amino acid sequence identified through purification of a cell supernatant which exhibits the property of inihibiting the replication of HIV. Such primers can then be used in reverse-transcription-polymerase chain reaction (RT-PCR) of RNA isolated from active cell lines exhibiting the desired antiviral characteristics. An RNA molecule which is amplified using these primers can be used to probe a Northern blot of RNA from the original cell line from which the amino acid sequence has been isolated in order to identify an mRNA which corresponds to an inhibitory polypeptide.

Alternatively, a partial amino acid sequence corresponding to an inhibitor can be used to generate immunological reagants. A synthetic peptide which corresponds to the identified sequence can be conjugated to keyhole limpet hemocyanin (KLH) and used to immunize animals of interest such as rabbits and/or mice. Polyclonal antisera can be generated and monoclonal antibodies can be derived using standard techniques. These reagants can be used with an expression library generated from a cDNA library obtained from a cell line of interest which exhibits the requisite antiviral properties in order to identify an inhibitory polypeptide. A commercially available vector which allows library construction for expression cloning with an antibody or for screenning with a DNA probe can be used in these protocols, such as the vector Lambda TriplEX (Clontech, Palo Alto, Calif.). Large scale purification of an inibitory polypeptide can be accomplished by cloning a cDNA into a bacterial expression vector, such as the pTrcHis A,B,C vector (Invitrogen, San Diego, Calif.), which allow high-level expression and ready purification according to the manufacturer's protocol.

The inhibitors of the invention are characterized by an ability to decrease or prevent viral replication in an active HIV infection in vivo or in a cellular model system. An inhibitor may also be characterized by its effects in altering, reducing or eliminating viral morphogenesis, replication, or virion infectivity. Where an inhibitor is incubated with HIV-infected cells, the production of infectious virus progeny is determined relative to control experiments without inhibitor.

Inhibitors can be characterized in tissue culture models of viral infection using cells infected with any lentiviral or retroviral infection, including, but not limited to, those resulting from HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus and all strains and isolates thereof. Specific HIV strains which have tropism for certain cell types can be used, including the macrophage-tropic HIV Ba-1, and the T-tropic HIV IIIB and MN. In general, isolates can include lymphotropic and macrophage-tropic strains, primary strains derived from blood cells or tissues, and North American, European, African and Asian isolates.

Primary cells or cell lines which can be used for inhibitor studies on virus-infected cells are preferably those that are susceptible to such viral infection. Such cells include, for example, peripheral blood lymphocytes (PBL), especially CD4+ cells, and macrophages.

Assay systems which employ a vector-delivered full or partial HIV genome into a eucaryotic cell can be used to simulate the production of viral proteins and virion production, and such cells can be used in the characterization of an HIV inhibitor.

To assess whether an inhibitor reduces or eliminates the generation of infectious viral progeny, the quantity and type of infectious progeny are assayed at suitable times post-infection. Evidence of microscopically observed viral spread, cytopathic effect, and increased amounts of the p24 capsid protein can provide an assessment as to whether infectious progeny are being generated. The assessment of progeny virus infectivity may be determined further by recovery of infected cells and co-cultivation with suitable cells (e.g., PBL or macrophages) or by the recovery of supernatant from the infected cells and cell-free infection of suitable cells. Another method of phenotypic determination involves the observation of progeny virus for morphological analysis, e.g., by electron microscopy.

Quantitative assessment of an HIV infection conducted in the presence of an inhibitor can also be determined using molecular markers, for example, by assaying viral p24 production by ELISA assay, reverse transcriptase activity, or viral DNA synthesis by quantitative PCR using standard techniques known to those skilled in the art.

A DNA encoding an inhibitor can be used to engineer cell lines which constitutively express the inhibitor in order to test the effect of an inhibitor on different isolates of HIV-1 or other HIV strains. Such isolates include lymphotropic and macrophage-tropic strains, primary strains derived from blood cells or tissues, and North American, European, African and Asian isolates. Such methods can allow the selection of an inhibitor which has optimal inhibitory effect on a particular viral isolate of interest.

The inhibitors of the invention can be tested in animal models of HIV infection, including the SCID-Hu mouse model of HIV-1 infection (Aldrovandi et al., J. Virol. 70:1505, 1996) and SIV-infected monkeys. Such models of infection are suitable for testing the inhibitors of the invention for efficacy against challenge with HIV or other lentiviruses and other retroviruses in order to identify those inhibitors which can be used for prevention or treatment of viral infection.

The inhibitors of the invention can be assayed to determine the concentration required to achieve an antiviral effect against a target virus. A convenient variable for measurement is the concentration of an inhibitor required to inhibit 50% of viral replication (IC₅₀), whether assayed in cell culture or with the use of a molecular marker such as the measurement of viral p24 production by ELISA assay, presence of viral RNA, reverse transcriptase activity, or viral DNA synthesis by quantitative PCR using standard techniques known to those skilled in the art.

Inhibitors of the invention can be evaluated for cytotoxic effects using standard assays that measure cell viability. Such assays include ¹⁴C protein hydrolysate, ³ H thymidine uptake, MTT reduction, and cell growth. Such parameters as TD₅₀ (toxic dose to 50% of the tested culture) can be derived from such assays. Comparison of the TD₅₀ so derived with the IC₅₀ (inhibitor concentration required to inhibit 50% of the viral marker being tested or viral replication) can indicate a therapeutic index for a particular compound (TI). Preferably, the IC₅₀ is at least ten times higher than the TD₅₀, and the IC₅₀ is effective at a minimum of 10^-6 M in culture to be considered as a prospective inhibitor of the invention. Most preferably, an inhibitor of the invention exhibits an IC₅₀ of 10^-7 M or 10^-8 M.

The inhibitors of the invention are useful in the isolation of HIV or other lentiviral and retroviral mutants which are resistant to the inhibitor but which can be used in subsequent screens to identify other antiviral agents to which they are susceptible, thereby generate a profile of inhibition for a particular viral isolate.

In preferred embodiments of the invention, an inhibitor of HIV replication comprises a polypeptide defined by the following properties: a) isolated from the CD8+ cell line K#1 50K; b) inhibits the replication of HIV Ba-1 in macrophages and the replication of HIV IIIB in CD4+ cells; c) is not a cytokine selected from the group consisting of RANTES, MIP-1.alpha. and MIP 1-.beta.; d) a molecular weight of approximately 8 kd; e) inhibits the replication of HIV in the viral life cycle following reverse transcription but before integration into the cellular genome; f) is stable at pH 2; g) maintains activity after being subjected to freezing and thawing; and h) comprises a peptide or polypeptide having an amino acid sequence which substantially corresponds to SEQ. ID NO. 1 or SEQ. ID NO. 2.

In other preferred embodiments of the invention, an inhibitor of HIV replication comprises a polypeptide defined by the following properties: a) isolated from the CD8+ cell line K#1 50K; b) inhibits the replication of HIV Ba-1 in macrophages and the replication of HIV IIIB in CD4+ cells; c) is not a cytokine selected from the group consisting of RANTES, MIP-1.alpha. and MIP 1-.beta.; d) a molecular weight of approximately 8 kd; e) inhibits the replication of HIV in the viral life cycle following reverse transcription but before integration into the cellular genome; f) is stable at pH 2; and g) maintains activity after being subjected to freezing and thawing.

The invention is further directed to CD8+ cell lines which are the source of one or more of the soluble factors of the invention which are capable of inhibiting the replication of HIV. CD8+ cells can be recovered from uninfected or HIV-infected individuals. Cells of interest can be transformed by Herpesvirus saimiri (HVS) in order to establish cell lines for further study or to be used as continuing sources of inhibitory factors (Weber et al., Proc. Natl. Acad. Sci. USA 90:11049-11053, 1993; Biesinger et al., Proc. Natl. Acad. Sci. USA 89:3116-3119, 1992). In preferred embodiments, the cell lines of the invention include the CD8+-derived cell lines K#1 50K, and Caf 10. The invention is also directed to any single cell clones which are derived from the cell lines of the invention.

In view of the above noted properties of the inhibitors of the invention, it is further contemplated that the inhibitors of the invention may be used in compositions for the prevention or treatment of an HIV or other lentiviral and retroviral infections, and the treatment of consequent pathologic conditions such as AIDS. Another aspect of the invention, therefore, is directed to methods for preventing and treating an HIV or other lentiviral or retroviral infection by administering a composition containing one or more of the inhibitors of the invention to an individual infected with or exposed to HIV for a time and under conditions to accomplish such result.

The inhibitors, compositions and methods of the invention can be used in the treatment of HIV-positive individuals, including those exhibiting the conditions of AIDS-related complex (ARC) and AIDS, as well as those who are asymtomatic. These inhibitors, compositions and methods can also be used in the prophylaxis of HIV or other lentiviral and retroviral infections, and can also be used the treatment or prophylaxis of veterinary infections caused by lentiviruses and other retroviruses.

The inhibitors of the invention may be used alone or in combination with other known or to be discovered inhibitors of HIV replication, including, but not limited to, other antiviral compounds, immunomodulators, antibiotics, vaccines, chemokines and other therapeutic agents. Particular agents which can be used in combination with the inhibitors of the invention include, but are not limited to, azidothymidine (AZT), dideoxyinosine (DDI), dideoxycytosine (DDC), saquinavir, indinavir, ritonavir, and other antiviral compounds. The inhibitors of the invention may also be used in combination with agents which are used to treat secondary complications of HIV infection, e.g., gancyclovir used in the treatment of cytomegalovirus retinitis. Combination therapy may retard the development of drug-resistant mutants by requiring multiple mutation events for the emergence of a fully drug-resistant isolate.

The inhibitors of the present invention may be administered to a host as a composition in an amount effective to inhibit HIV infection and/or replication in target cells. The compositions contain an effective dosage of at least one of the inhibitors of the present invention, together with an acceptable carrier.

The inhibitors of the invention may be systematically administered for preventing or treating an HIV or other lentiviral or retroviral infection. When used systemically, the inhibitor compositions may be formulated as liquids, pills, tablets, lozenges or the like, for enteral administration, or in liquid form for parenteral injection. The peptides and/or polypeptides (or inhibitor-protein conjugates) may be combined with other ingredients such as carriers and/or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be physiologically acceptable, efficacious for their intended administration and cannot degrade the activity of the active ingredients of the compositions. An inhibitor can also be covalently attached to a protein carrier, such as albumin, so as to minimize diffusion of the inhibitor.

As used herein, a physiologically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and the like. The use of such media and agents are well-known in the art.

The forms of the compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the ultimate solution form must be sterile and fluid. Typical carriers include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e., biocompatible buffers), ethanol, polyol such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Sterilization can be accomplished by an art-recognized technique, including but not limited to, filtration or addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.

Production of sterile injectable solutions containing the subject inhibitors is accomplished by incorporating these compounds in the required amount in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization, preferably filter sterilization. To obtain a sterile powder, the above solutions are vacuum-dried or freeze-dried as necessary.

The polypeptide compositions also may be impregnated into transdermal patches, plasters and bandages, preferably in a liquid or semi-liquid form.

When the inhibitors of the invention are administered orally, the compositions thereof containing an effective dosage of the peptide may also contain an inert diluent, an assimilable edible carrier and the like, be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like.

The subject inhibitors are thus compounded for convenient and effective administration in physiologically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective dosage.

The precise effective amount of inhibitor to be used in the methods of this invention to prevent or treat an HIV infection cannot be stated because of the nature of the infectious process. It must be noted that the amount of inhibitor to be administered will vary with the degree of infection in an individual, as determined by such parameters as viral load and CD4 cell counts. Individual-specific variables such as age, weight, general health, gender, diet, and intake of other pharmaceuticals can factor into the choice of dosage. The design of an optimal protocol for an infected individual may further consider the identity of the viral isolate(s) isolated from an infected individual with an infection for optimal result. A further consideration in protocol design would be the presence of a viral strain which is already resistant to existing protease or reverse transcriptatse inhibitors.

The amount of an inhibitor of the invention per unit volume of composition for administration depends upon the amount of active ingredients that are afforded directly to the site of infection. However, it can generally be stated that a peptide or polypeptide inhibitor of the invention should preferably be present in an amount of at least about 1.0 nanogram per milliliter of combined composition, more preferably in an amount up to about 1.0 milligram per milliliter.

Systemic dosages depend on the age, weight and condition of the individual and on the administration route. For example, a suitable dosage for the administration to adult humans ranges from about 0.01 to about 100 mg per kilogram body weight. The preferred dosage ranges from about 0.5 to about 5.0 mg per kilogram body weight.

Since the inhibitory compositions of this invention are effective in reducing or eliminating the ability of HIV or other lentiviruses and other retroviruses to generate infectious progeny, periodic readministration of the compositions may be indicated and preferred.

The peptide and polypeptide inhibitors of the invention can also be delivered to an individual by administering a vector that comprises and expresses a nucleic acid encoding the inhibitor. DNAs encoding one or more of the inhibitors of the invention can be delivered to the cells of an individual in need of such an inhibitor by any method of gene transfer known to those skilled in the art, including, but not limited to, viral vectors, lipid-mediated delivery, transfection, electroporation, as well as other methods. Viral vectors which can be used to deliver such inhibitors include those derived from DNA and RNA viruses, including, but not limited to, adenovirus, herpesvirus, poxvirus, retrovirus, and adeno-associated virus.

Parameters, which are used to monitor the effect of an inhibitor of the invention administered to an individual with an established HIV infection or administered to an individual for prophylaxis, include the use of CD4 counts, plasma viral RNA concentration, viral phenotype, p24 antigen concentration, viral phenotype, level of anti-HIV antibodies as well as other markers of the clinical progression of an HIV infection known to those skilled in the art.

It will be recognized that the inhibitors and methods of the invention can be used in the treatment or prevention of any other lentiviral or retroviral infection, including, but not limited to, those resulting from HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), visna virus and all strains and isolates thereof.

The practice of the invention employs, unless otherwise indicated, conventional techniques of protein chemistry, molecular virology, microbiology, recombinant DNA technology, and pharmacology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc., New York, 1995; Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985; and Molecular Cloning: A Laboratory Manual, Sambrook et al., eds., 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.

Claim 1 of 2 Claims

What is claimed is:

1. An inhibitor of HIV replication, comprising a peptide or polypeptide having the amino acid sequence of SEQ. ID NO. 1.

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