EP0522081A1

EP0522081A1 - Inhibition of disease associated with immunodeficiency virus infection

Info

Publication number: EP0522081A1
Application number: EP91908056A
Authority: EP
Inventors: Christine Marie Debouck; David Alan 4124 Rittenhouse Lane Brake
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 1990-03-30
Filing date: 1991-03-29
Publication date: 1993-01-13
Also published as: JPH05506031A; AU7677891A; WO1991015224A1; EP0522081A4

Abstract

Compositions pharmaceutiques comprenant des agents inhibant l'adhérence cellulaire à médiation RGD de la protéine tat, utiles dans l'inhibition de la progression d'états pathologiques associés à l'infection due au virus d'immunodéficience chez l'animal.Pharmaceutical compositions comprising agents inhibiting RGD-mediated cell adhesion of the tat protein, useful in inhibiting the progression of disease states associated with infection due to the immunodeficiency virus in animals.

Description

Title

INHIBITION OF DISEASE ASSOCIATED WITH IMMUNODEFICIENCY

VIRUS INFECTION

Field of the Invention

This invention relates to pharmaceutical agents, medicaments and methods for preventing, or treating the effects of abnormalities associated with Immunodeficiency Virus infection.

Background of the Invention

Proteins which interact with integrin cell adhesion receptors frequently contain the amino acid tripeptide RGD sequence within the integrin binding site. RGD sequences are found in fibronectin, vitronectin and collagen and identify extracellular matrix attachment sites utilized for integrin- mediated cell adherence during development and differentiation. Integrin receptors on leukocytes bind to coagulation proteins (von Willebrand factor, fibrinogen, thrombospondin) and complement components (C3b) , and participate in cell-cell adhesion (LFA-1 with I-CAM) . These interactions are involved in homeostatic regulation, phagocytosis, cell migration, cell signaling, cellular trafficking and lymphocyte recognition. In addition, bacterial, parasitic, and viral proteins can contain RGD sequences which recognize integrin receptors and may contribute to pa hogenesis.

Human immunodeficiency virus (HIV) , the etiologic agent of acquired immune deficiency syndrome (AIDS) is a member of the Retroviridae family. There exist several isolates of HIV including human T-lymphotropic virus type-Ill (HTLV-III) , the lymphadenopathy virus (LAV) and the AIDS-associated retrovirus (ARV) which have been grouped in type 1. Related Immunodeficiency Viruses, include HIV type 2, which was shown recently to be associated with AIDS in West Africa (see, Guyader, et al. , Nature 326:662 (1987)). Other Immunodeficiency Viruses include the SIV viruses such as

SIV_mac-BK28 (see, Hirsh et al., Cell 49:307 (1987); Kestler et al.. Nature 331:619 (1988)); the Feline Immunodeficiency Virus (FIV) ; and the Bovine Immunodeficiency Virus (BIV) . Molecular characterization of the HIV genome has demonstrated that the virus exhibits the same overall gag- pol-env organization as other retroviruses. Ratner, et al.. Nature 313:277 (1985); Wain-Hobson, et al.. Cell 40:9 (1985). In addition, Immunodeficiency Viruses contain at least five genes that are not found in other retroviruses: vif , tat, rev, nef and vpr. The gag region encodes 4 core proteins, pl7, p24, p7 and p6, which are prepared by cleavage of a 55 kilodalton gag precursor protein by the HIV protease. The protease is encoded by the pol region.

Immunodeficiency Viruses also have a gene for transactivating protein, termed tat, which contains an RGD sequence. HIV-1 tat is an 86 amino acid long protein which greatly increases viral gene expression and replication. The tripeptide RGD sequence in tat is located in the carboxyl terminal portion of the protein and is highly conserved among HIV-1 isolates. The production of monoclonal antibodies to the tat protein has been disclosed by Brake et al., J. Virol.. 64, 962 (1990) . In spite of the ever-increasing knowledge concerning the pathogenesis of disease states associated with Immunodeficiency Virus infection there continues to be a pressing need for pharmaceutical agents which inhibit Immunodeficiency Virus infection and HIV induced syncytia formation and other abnormalities associated with Immunodeficiency Virus infection including for example, Kaposi's sarcoma.

Summary of the Invention

This invention lies in the discovery that the tat protein of Immunodeficiency Viruses plays a role in progression of disease states associated with infection by such virus and that the progression of such disease states can be inhibited by interfering with the cell adhesion function of the tat protein.

More specifically, this invention is, in one embodiment, a method for inhibiting progression of a disease state associated with infection in an animal by an Immunodeficiency Virus which comprises administering to the animal an inhibitor of the RGD cell adhesion function in the Immunodeficiency Virus tat protein.

In another aspect, this invention is the use of an inhibitor of the RGD cell adhesion function in the

Immunodeficiency Virus tat protein in the manufacture of a medicament for treating infection by Immunodeficiency Virus .

In a related embodiment, this invention is a pharmaceutical composition comprising an inhibitor of the RGD cell adhesion function of an Immunodeficiency Virus tat protein and a pharmaceutically acceptable carrier.

Detailed Description of the Invention

It has now been found that the tat protein of

Immunodeficiency Viruses functions in cell adhesion of extracellular tat protein and that, by inhibiting such cell adhesion function, transcriptional activation by the tat protein can be inhibited. It has further been found that such inhibition of tat mediated transcriptional activation inhibits, i.e., prevents or slows, progression, including initiation, of disease states resulting from abnormal gene expression. Such disease states include immune dysfunction resulting in immmunodeficiency as well as other disease states associated with Immunodeficiency Virus infection such as Kaposi's Sarcoma. See, e.g., Vogel et al.. Nature 335:606 (1988) . Agents which can be used to interfere with tat cell adhesion in accordance with the method of this invention include peptide mimics of the tat RGD sequence or a tat RGD binding agent. Peptide mimics of the tat RGD mimics may be chemical compounds, peptides or proteins which bind to the RGD cell adhesion receptor, or otherwise inhibit the binding of tat to cells. Such mimics may be chemical compounds which bind to the tat receptor and/or functionally approximate the conformation of the RGD sequence in tat . Such mimics may also be peptides or oligopeptides. Such mimics may contain the partial sequence -RGD- in their structure. Such peptide mimics include tat mutants, i.e., derivatives of the tat protein in which one or more amino acids have been added, deleted, substituted or rearranged, as well as tat proteins which have been chemically modified, so as to diminish cell adhesion.

Peptide mimics, which are useful in the method of the invention, also include oligopeptides, i.e., peptides having two to about 20 amino acids, which oligopeptides compete with the RGD sequence in the tat proteins for binding to the receptor to which the tat RGD sequence binds.

Many RGD peptide mimics are known from research into the role of cell adhesion in, e.g., wound healing, tissue repair and thrombosis. Such previously discovered mimics of RGD- mediated cell adhesion can be used in the method of this invention. See, e.g., U.S. Patents 4,683,291; 4,661,111; 4,614,517; 4,589,881; 4,578,079; 4,517,686; 4,544,500; 4,397,842; 4,857,508; 4,879,313; and U.S. Patent Applications 07/650,527, 07/630,124 and 07/433,933; as well as PCT WO 89/05150 (US 88/04403), EP-A 0 341 915, EP-A 0 275 748, EP-A 0 372 486, EP-A 0 381 033, EP-A 0 410 537, EP-A 0 410 539, EP-A 0 410 540, EP-A 0 410 541, EP-A 0 410 767, and EP-A 0 411 833, all of which are incorporated by reference herein as though fully set forth.

Another embodiment of an inhibitor of RGD-mediated cell adhesion by tat proteins, which is useful in this invention, is a pharmaceutically acceptable tat RGD binding agent which binds to the tat protein of an Immunodeficiency Virus so as to prevent or diminish the affinity of the RGD sequence for cell surface receptors. One example of such RGD binding agent is an antibody to the tat protein, preferably an antibody specific to the RGD sequence. Such antibodies can be prepared by standard techniques for polyclonal or monoclonal antibody production. See, e.g., Brake et al., J. Virol., 64:962 (1990), which is incorporated herein by reference as though fully set forth. Alternatively, it is now possible to prepare chimeric antibodies by recombinant DNA techniques. A further variation of this embodiment is the use of fragments or derivatives of such antibodies. Another example of such RGD binding agent is a soluble derivative of the cell surface receptor for the RGD sequence. Such receptor can be isolated by standard techniques and converted into a soluble form by deleting transmembrane and cytoplasmic domains of the receptor. See, e.g., EP-A 0 240 975, WO 87/05912, WO 87/07302, EP-A 0 257 114, WO 87/06938 and WO 88/01304.

Tat protein gene coding sequences for mutagenesis to prepare tat mutants can be prepared from Immunodeficiency Viruses or from any of a number of publicly available Immunodeficiency Virus genomic libraries. Mutagenesis, preferably to substitute one or more of the RGD residues, can be prepared by standard techniques .

Representative compounds which interfere with RGD adhesion are:

Ac-Arg-Gly-Asp-Ser-NH2; Ac-Arg-Gly-D-Asp-Ser-NH2; Ac-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro-Ile-Ser-Ile-

Asn-Tyyr-Arg-NH2;

N-alpha-acetyl-cyclo(S,S)-cysteinylarginylglycylaspartyl- serylarginylglycylaspartylserylcysteine amide; N-alpha-acetyl-cyclo(S,S)-cysteiny1-N-alpha- methylarginylglycylaspartyl-penicillamine amide; cyclo(1,5)-D-alanylarginylglycylaspartylserine;

Ac-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val-OH;

3S, 6S-3-carboxymethyl-6-(3-guanidinoρropyl)-2,5- piperazinedione;

3S, 6R-3-carboxymethyl-6-(3-guanidinoρropyl)-2,5- piperazinedione; cyclo(1,10)-prolylarginylglycylaspartyl-D-phenylalanyl- prolylarginylglycylasparatyl-D-phenylalanyl; cyclo(1,5)-alanylarginylglycylasparty1-D-serine; cyclo (1,6)-glycylarginylglycylaspartylserylproline; cyclo(1,6)-glycylprolinylarginylglycylaspartyl-D-proline; cyclo(1,6)-prolinylglycinylarginylglycinylaspartyl-D-proline;

N-alpha-acetyl-cyclo(S,S)-L-cysteinyl-L-arginyl-glycyl-L- aspartyl-L-tryptophyl-L-penicillamine amide; cyclo(1,8)-methylarginylglycylaspartylphenylalanylarginyl- glycylaspartyl phenylalanine;

Thr-Arg-Tyr-Arg-Gly-Asp-Gln-Asp-Ala-Thr-Met-Ser-OH; cyclo (1 (alpha) , 6 (delta) ) -glycyl-N (alpha) methylarginylglycyl- aspartylserylglutamic acid amide;

N-alpha-benzoyl- (alpha) -methylarginylglycinylaspartyl anilide;

N-alpha-acetyl-cyclo(S,S) cysteiny1(N-alpha-methyl)arginyl glycylaspartyl-(2R,3R)-3-phenylcysteineamide; cyclo(1,6)-prolinylarginylglycinylaspartylglycinyl-D-proline; and cyclo(1,6)-prolinylarginylglycinylaspartylglycinyl-D- phenylalanine.

Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of this invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical

Nomenclature as described in Eur. J. Biochem. 158, 9 (1984) .

Agents which are useful in the method of the invention can be readily prepared and selected on the basis of their ability to inhibit tat cell adhesion. Cell adhesion assays can be carried out by standard techniques known in the art, including, for example, the assay techniques disclosed in the Examples provided hereinbelow. See, also, e.g., Frankel et al. , Cell 55: 1189 (1988), which is incorporated herein by reference as though fully set forth.

In the method of the invention, inhibitors of tat cell adhesion are administered internally, e.g., parenterally, such as by intravenous, subcutaneous or intramuscular injection or infusion, orally, rectally, buccally, transdermally or by inhalation to an animal, especially man, susceptible of infection by an Immunodeficiency Virus. The compounds are typically administered in a pharmaceutically acceptable carrier or diluent selected on the basis of the route of administration. For solid formulations such as pills, powders, tablets, capsules and caplets, useful carriers include, among others, lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

Liquid carriers such as for oral, intranasal or parenteral administration include, among others, glycerin, syrup, peanut oil, olive oil, saline, dextrose and water, optionally bufferred with organic or inorganic salts, for example, acetates, adipates, succinates or citrates of ammonium, potassium or sodium. Additional excipients may be added to adjust tonicity or, especially in the case of a formulation to be lyophilized, stabilizers such as, for example, gelatin, polyvinylpyrrolidine, cellulose, acacia, polyethylene glycol, pyrrolidone or mannitol.

For rectal or vaginal administration, the compounds can be combined in powder form with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycol and molded into a suppository. For transdermal delivery, the compounds can be combined with an oily preparation, gel, cream or emulsion and administered via a transdermal patch.

In the method of the invention, pharmaceutically acceptable compositions of agents which inhibit RGD-mediated tat cell adhesion are administered to an animal, especially a human, who has been or is suspected of having been infected with an Immunodeficiency Virus, especially HIV, in an amount effective to inhibit progression of immune dysfunction and, preferably, to stabilize or improve the immune dysfunction or other disease state.

The amount administered will vary depending upon a variety of factors including the stage of immune dysfunction progression and the age, weight and general health of the animal being treated. The effects of the method of the invention on immune disorders associated with infection by an Immunodeficiency Virus can be monitored by evaluating T4 and T8 cell counts and the T4/T8 ratio and the dosage rate and dose can be modified accordingly. Alternatively, the presence of virus in the bodily fluids of the animal can be monitored by evaluation of presence of virus antigens, of antibodies to virus antigens, or measuring viral nucleic acid, e.g. using polymerase chain reaction (PCR) , in sera. Or, progression of sarcomas can be monitored and dosage adjusted accordingly in the case of treating Kaposi's Sarcoma. It is expected that the method of the invention will be carried out over a lengthy period of time, e.g., several weeks to months, or for the treatment to be repeated periodically, due to the ability of Immunodeficiency Viruses to remain latent within the infected animal.

Encompassed within the method of the invention is co- administration or simultaneous administration of an inhibitor of RGD-mediated tat cell adhesion with other pharmaceutically active agents, e.g., other agents which are also effective in inhibiting virus replication and syncytia formation, such as other nucleoside analogs such as AZT and ddC, HIV protease inhibitors and sCD4 and truncates thereof. The Examples which follow are illustrative of the invention, and of preferred embodiments thereof, but are in no way meant to be limiting.

Examples

A. Construction of HIV-1 Tat Bacterial Expression Vectors

Construction of the full-length tat (HTLV-IIIB isolate) bacterial expression plasmid, pOTS-TATIII, was previously described in Aldovini, et al., Proc. Nat'1 Acad. Sci.. USA

JL3.:6672-6676 (1986). An RGE tat (mutant 1) expression vector was constructed as follows. An Ndel-Xbal 582 base pair fragment from pOTS-TATIII was gel purified and subcloned into the polylinker region of plasmid pUC19 using T4 DNA ligase. The resulting plasmid, pUC19TAT.WT, was digested with Aval and Xbal and then ligated to a 35 base pair Aval-Xbal synthetic oligonucleotide to generate pUC19TAT.RGE. This synthetic oligonucleotide reconstitutes the 3' end of the tat gene with a single base substitution changing Aspso ^to Glu. A BamHI-Xbal 253 base pair fragment containing the full- length mutated tat gene was purified from pUC19TAT.RGE and then ligated into the BamHI-Xbal site of pOTS-TATIII. A KGE tat (mutant 2) expression vector was similarly constructed except that a 35 base pair Aval-Xbal synthetic oligonucleotide containing a double base substitution (changing Arg78 ^{to L}y^{s anc}- ^AsP80 ^to Glu) was used. These mutations were confirmed by dideoxy sequencing (Sanger, et al., Prσc. Nat'1 Acad. Sci. USA 74:5463-5467 (1977)) using an appropriate sequencing primer.

B. Purification of Wild-type and Mutant Tat Proteins

E . coli (strain AR120) bacterial cells containing the respective pOTS expression vectors were grown in LB broth containing ampicillin at 50 ug/ l at 37°C to an optical density (650 nm) of 0.4 and induced by the addition of nalidixic acid to 60 ug/ml as described by Aldovini, et al., Proc. Nat'l. Acad. Sci. USA. £1:6672-6676 (1986). Five hours after induction, sonicated cell lysates were centrifuged (15,000 x g) and supernatants were acidified by slow addition of 1 M HC1 to pH 3.0 to precipitate nucleic acids.

Following centrifugation and neutralization to pH 7.5 using 1.5 M Tris base (pH 8.5), samples were applied to a

Sephadex G-25F column (Pharmacia, Piscataway, NJ) (1 x 40 cm) equilibrated in 50 M NaMES, pH 6.5. The protein peak was pooled and concentrated 10-fold using an Amicon YM-5 membrane (Amicon, Danvers, MA) . Samples were then applied to individual anti- tat immunoaffinity columns (3 ml bed volume) equilibrated in phosphate-buffered saline (PBS, pH 7.4). Columns were prepared by coupling purified anti-tat monoclonal antibody (see below) to CNBr-activated Sepharose 4B (Pharmacia) using manufacturers' recommended conditions. The columns were washed with PBS and the flow-through material reapplied twice prior to elution. Bound samples were eluted, (100 mM sodium citrate/0.5 M NaCl, pH 3.0), protein peaks were pooled and were immediately neutralized to pH 7.5 using solid Tris base. Immunoaffinity purification was monitored by SDS-PAGE using Coomassie blue staining and Western blot analysis as previously described by Aldovini, et al., Proc. Na '1 Acad. Sci. USA. 83:6672-6676 (1986). Samples were concentrated and protein concentrations were determined by the method of Bradford, Anal. Biochem. ____, 248-254 (1976) and the samples were then stored at 4°C until use.

C. Cell Culture

The human T-lymphocytic HUT-78 and MOLT-4 suspension cell lines, the human myelomonocytic THP-1 suspension cell line and the rat skeletal muscle-derived ~><- adherent myoblasts were obtained from American Type Culture Collection (Rockville, MD) . A G418-resistant A5 HeLa cell line containing a stably integrated HIV-1 LTR-CAT transcription unit was also used. All cell lines, except A5, were grown and routinely subpassaged in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco

Laboratories, Grand Island, NY), 2mM L-glutamine, 10 U/ml penicillin G, and 10 ug/ml streptomycin (DMEM) . Cell line A5 was routinely cultured in DMEM supplemented with 0.4 mg/ml G418 (Gibco) .

D. Peptide Synthesis

The peptides GRGDSPK and GRADSPK were synthesized as carboxyl-terminal amides using FMOC chemistry with the RaMPS system (Du Pont Corp., Wilmington, DE) . Amino acids and other reagents were obtained from Du Pont; all solvents were of the highest grade available. The synthesis was performed according to manufacturers instructions by deblocking the RapidAmide resin with piperidine/DMF; washing alternatively with DMF and methanol; adding the FMOC- amino acid and rocking for 2 hr; washing with DMF and methanol, and then testing by ninhydrin reaction; repeating these steps for each amino acid. The peptide was cleaved from the resin and blocking groups removed with trifluoroacetic acid: 1,2- ethanedithiol: phenol. The eluted peptide was precipitated with diethyl ether, and washed with ether and then with ether: ethyl acetate 1:1. The peptides were purified on a FPLC Mono S column (Pharmacia LKB Biotechnology, Piscataway, NJ) . The sample was applied in 50mM aH2Pθ , pH 3.0, 30% acetonitrile, and eluted with a linear NaCl gradient to 0.35 M. The column eluent was monitored at OD214. The peptides eluted as single peaks, and each contained the appropriate molar ratios of amino acids by compositional analysis

(Protein Structure Laboratory, Wistar Institute, Phil, PA) .

E. Cell Adhesion Assay

For the adhesion assay, which was substantially as described by Ruoslahti, et al., Methods Enzymol. £2.: 803-831 (1982), polystyrene plate (Linbro 76-203; non-tissue culture treated, Flow Laboratories, McClean VA) were coated overnight at 37°C with wild-type and mutant tat proteins, and the matrix protein vitronectin. Vitronectin was purified according to the method of Dahlback, et al., Biochemistry, 21:2368-2374 (1985) . The plate was washed with PBS, with

DMEM and then 0.1 ml of DMEM was added to each well. The adherent L3 myoblasts were suspended by trypsinization and washed three times with trypsin-inhibitor (0.5 mg/ml sterile PBS) ; HUT-78, MOLT-4, and THP-1 cells were washed three times in PBS; 100 ul of each cell suspension was added to wells in quadriplicate (1 X 10^β cells/ml in DMEM) . The HIV-1 replication competent human T-lymphocytic cell lies HUT-78 and MOLT-4, and the human myelomonocytic cell line THP-1 were selected because of the known HIV-1 tropism for T-lymphocyte and monocyte/macrophage cell. (See, e.g., Kikukawa et al., J. Virol. 57, 1159 (1986); Levy et al. , Science (Wash. DC) ., 235, 840 (1984); Tsuchiya et al., Int. J. Cancer. 26, 171 (1980) .) The Ls cell line, a rat skeletal muscle cell line was utilized because of its discriminating adhesion properties. The cells were incubated in the coated wells for 1 hr, fixed with 4% formalin and stained with 1% toluidine blue. Cell adherence was quantitated using a microtiter plate reader (Dynatech Laboratories, Chantilly, VA) set at

570 nM. In other experiments, the cells were incubated with cytochalasin B and colchicine, separately or in combination, at 10 ug/ml each for 30 min at 4°C, and then added to the protein-coated wells.

Tat was highly efficient in mediating cell adherence of all four cell lines tested. The tat-mediated adhesion was dose dependent and the tat coating concentration which gave half-maximal cell adhesion was between 1 and 5 ug/ml. For each cell line the level of adherence to tat-coated wells was equivalent to the highest level of binding obtained with control matrix proteins. The single amino acid Dso~^E substitution (aspartic acid substituted by glutamic acid) introduced in tat was sufficient to significantly reduce or completely eliminate tat-mediated cell binding. The D-E change was selected since the overall net charge of tat would not be affected and an RGD-RGE substitution has been shown to abrogate cell binding to fibronectin (Obara, et al. , Cell 53:649-657

(1988) ) . The tat D-E mutation completely eliminated the adherence of Ls cells and reduced the binding (40-90%) of the HUT-78, MOLT-4 and THP-1 cells. The L₈ myoblasts did not adhere to the mutant RGE tat protein even when wells were coated at concentrations up to 120 ug/ml, whereas wild-type RGD tat protein coated at 1 ug/ml or more mediated efficient cell adhesion.

The KGE mutant tat protein also completely lacked cell attachment for L3 cells. In addition, the adhesion of the other cell lines to the KGE tat mutant was only slightly above background. The adhesion was performed at a single protein concentration (5 ug/ml) since insufficient quantities of the KGE mutant were available to perform a full dose response. The lack of cell adhesion to the RGE and KGE mutant tat proteins could not be attributed to lower levels of mutant tat bound to the wells, since similar amounts of anti-tat MAb bound to wells coated with either wild-type tat, mutant RGE, or mutant KGE tat protein as judged by ELISA. The influence of mutations at the RGD site on tat cell adhesion suggested that cell binding could involve participation of an integrin. To further investigate this possibility, the effect of EDTA, cytoskeletal blockers and an RGD-containing peptide on cell adhesion was investigated. For these investigations the skeletal muscle derived L₈ cell line was used because this line has been characterized for integrin expression and has been found to adhere primarily to vitronectin, e.g., Biesceker, J. Immunol.. 145, 209 (1990) . The restricted integrin distribution of L₈ cells allows for more straightforward interpretation of the results.

The interaction between tat and L3 cells was dependent on divalent cations. The addition of EDTA abrogated cell attachment and adherence was restored by the addition of either Ca²⁺ or Mg²⁺. In addition, cell attachment required an intact cyotskeleton, as cell binding was eliminated in the presence of cytochalasin B plus colchicine. These results are consistent with integrin-mediated cell attachment (Ruoslahti, et al. , Methods Enzymol . 82:803-831 (1982) and Ruoslahti, et al. , Science (Wash. DC), 22£:491-497 (1987) . The L3 cell attachment to tat was also abrogated by addition of the synthetic peptide GRGDSPK. This peptide, which contains the RGD tripeptide, reduced the cell adherence to background levels when present at concentrations greater than 100 ug/ml. L₈ cell attachment to the control matrix protein vitronectin was inhibited at similar levels of the peptide. In contrast, the control synthetic peptide, which contained an RAD sequence, did not block cell attachment at concentrations up to 5 mg/ml. These results are also consistent with integrin-mediated cell attachment. Ruoslahti, et al., Methods Enzvmol. 82:803-831 (1982) and Ruoslahti, E. et al., Science (Wash. DC), 2 :491-497 (1987) .

An interesting observation of the tat-mediated cell adherence was that L3 myoblasts remained round after attachment, whereas when bound to vitronectin the L₈ cells were spread, flattened and had numerous projections. Cell spreading was also found for L3 cells bound nonspecifically to tissue culture-treated plastic or poly-lysine. Absence of cell spreading on tat was also noted for the T-lymphocytic and myelomonocytic cell lines. The distinct morphology of L₈ cells bound to tat suggests that post- binding events leading to cytoskeletal reorganization differ for cells bound to tat compared with cells bound to extracellular matrix proteins such as vitronectin.

F. Cellular Uptake and CAT Assays Purified wild-type, RGE, or KGE mutant tat protein (2 ug, 4.1 X 10^"8M) were added directly to the media (DMEM/lOOuM chloroquine) of Petri dishes containing 10^ A5 cells. After 24 hr incubation, the cells were washed with PBS, replenished with fresh media, and harvested 24 hr later. Cells were washed twice in PBS, and resuspended in lysis buffer (ImM DTT, 0.1% Triton X-100, 10% glycerol, 10 mM Tris, pH 8.0) . After three cycles of freeze-thawing, samples were centrifuged, supernatants were removed, and the protein content was determined as described by Bradford, et al. , Anal. Biochem 11, 248 (1976) .

Equal concentrations of A5 samples were assayed for chloramphenicol acetyl transferase (CAT) activity as previously described (Valerie, et al.. Nature (Lond.), £11:78-81 (1988)) . Unacetylated ¹ c-chloramphenicol and acetylated forms separated by thin layer chromatography were excised after overnight exposure to film and the amount of radioactivity was quantitated in a liquid scintillation counter (Beckman Instrument, Irvine, CA) . The relative CAT activity (+/- standard deviation) was calculated from the ratio of the percent conversion of mutant tat divided by the percent conversion of wild-type tat from at least three independent assays.

The addition of purified wild-type tat protein to these cells resulted in a pronounced increase in CAT activity. The transactivating activity of the RGE mutant in this assay, although significant, was reproducibly one-half the level produced using wild-type tat protein. This 50% reduction was consistently observed over a wide dose range and was also observed for L₈ cells transiently transfected with a HIV-1 LTR-CAT containing plasmid. Furthermore, the double mutant containing a KGE sequence had only one-tenth the transactivating activity of wild-type tat . These results demonstrate that RGD-mediated tat cell adhesion is required for exogenous tat-induced transactivation.

G. Additional RGD Oliσopeptide Mimics In similar experiments, three RGD peptide mimics were similarly tested for the ability to inhibit cell adhesion to tat protein. A typical protocol is as follows:

1. Two days prior to the assay, cells are split to insure high viability and typical morphology. One microtiter plate requires 1 x 10^ cells.

2. One day prior to the assay a flat, nontissue culture treated microtiter ELISA plate is coated with tat protein. Typically, the RGD-containing protein or peptide of interest is diluted (PBS/0.005% Na 3) to a final concentration of 5 ug/ml and 100 ul is added to each well. A multichannel micropipetter is recommended for this procedure to insure reproducibility of protein concentration in each well. It should be noted that a larger or smaller concentration of protein (empirically determined) may be necessary to yield adequate binding. In addition at least three wells should receive PBS/NaN alone to serve as a negative control.

Positive control wells are also recommended. The plate is incubated at 37° C overnight.

3. The cells are carefully dislodged from the flask following brief exposure (i.e., 30 seconds) to 1.0 ml of 0.4% trypsin-EDTA solution. Ten mis. of 0.5 mg/ml trypsin inhibitor is quickly added and cells spun at 3000 rpm in a clinical microfuge. The cell pellet is washed two additional times in trypsin-inhibitor (10 mis/wash) . Cells are resuspended in the appropriate volume of sterile PBS and total viable cell count determined. After spinning, the cell concentration is adjusted to 106 cells/ml in DMEM without serum.

4. The microtiter plate is washed three times in PBS to remove any unbound material. These washings can be carried out using an automated plate ELISA plate washer or alternatively wells can be rinsed using a hand-held squirt bottle. 100 ul of DMEM without serum is then added to each protein-coated well.Peptides are diluted in H₂O/O.IM acetic acid, or in DMEM without serum. Peptides are preincubated with cells for 30-60 minutes at 4°C on a rotating device.

5. 100 ul of cell suspension is then added to desired wells and plate is placed in a tissue culture incubator (5% CO2, 37°C) for 1 hour. Incubation can proceed for as long as

2 hours.

6. Plate is quickly inverted and firmly shaken 2 times and wells gently rinsed 2 times with PBS. A 100 ul of fixative (4% formalin in PBS) is quickly added to each well and incubated at room temperature for 15 minutes.

7. Plate is quickly inverted and firmly shaken 2 times and wells gently rinsed 2 times with PBS. A 100 ul of staining solution (1% toluidine blue in 4% fixative) is added to each well and incubated for 20 minutes at room temperature.

8. Plate is quickly inverted and firmly shaken 2 times and wells rinsed 3-5 times with dH2θ until all excessive stain has been removed. Plate is tilted and allowed to air dry.

9. Adherence is quantitated at an optional density set to 570 nm using an ELISA plate reader.

1. Cyclo(1,6)glycylprolinylarginylglycylaspartyl-D-proline;

2. Cyclo(1, 6)prolinylglycinylarginylglycinylaspartyl- D-proline;

3. Cyclo(1,5)D-alanylarginylglycinylaspartylserine.

The OD readings from control wells (i.e., no peptide) were 0.11 in the case of L₈ cell and 0.18 in the case of MOLT-4 cells. In CAT assays similar to those described above, in which RGD peptide mimics were incubated with cells and with tat for at least 6 hours, and prefereably overnight, prior to washing, Peptide No. 1 was shown to significantly inhibit exogenous transactivation. Partial inhibition was observed with the other peptides. With certain other peptides, including the peptide GRGDSPK, no inhibition was observed.

The above Examples demonstrate that inhibition of RGD- mediated tat cell adhesion is an effective method for inhibiting tat-induced transactivation and, hence, of inhibiting progression of disease states associated with Immunodeficiency Virus infection.

The above description and examples disclose how to make and use the invention, including preferred embodiments thereof, but is in no way limiting of the invention which emcompasses all variations and modifications emcompassed within the scope of the claims which follow.

Claims

Claims :

1. A method for inhibiting progression of a disease state associated with infection in an animal by an Immunodeficiency Virus which comprises administering to the animal an inhibitor of the RGD cell adhesion function in the Immunodeficiency Virus tat protein.

2. The method of claim 1 wherein the inhibitor inhibits RGD-mediated tat cell adhesion.

3. The method of claim 2 wherein the inhibitor is an RGD-containing peptide mimic of the tat RGD sequence or a tat RGD binding agent .

4. The method of claim 3 wherein the inhibitor has the partial sequence -RGD-.

5. The method of claim 3 wherein the inhibitor is an antibody to the tat RGD binding site.

6. The method of claim 1 wherein the Immunodeficiency Virus is a Human Immunodeficiency Virus.

7. The method of claim 5 wherein the disease state is Kaposi's Sarcoma.

8. A pharmaceutical composition comprising an inhibitor of the RGD cell adhesion function of an Immunodeficiency Virus tat protein in a pharmaceutically acceptable carrier.

9. The use of an inhibitor of the RGD cell adhesion function in the Immunodeficiency Virus tat protein in the manufacture of a medicament for treating infection by Immunodeficiency Virus.

10. The use according to claim 9 wherein the inhibitor is an RGD-containing peptide mimic of the tat RGD sequence or a tat RGD binding agent.

11. The use according to claim 10 wherein the inhibitor has the partial sequence -RGD-.

12. The method of claim 10 wherein the inhibitor is an antibody to the tat RGD binding site.

13. The use according to claim 9 wherein the Immunodeficiency Virus is a Human Immunodeficiency Virus,