EP0571549A1 - NOUVELLE SPECIFICITE DE L'INTEGRINE POUR LA PROTEINE Tat DE L'HIV - Google Patents

NOUVELLE SPECIFICITE DE L'INTEGRINE POUR LA PROTEINE Tat DE L'HIV

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Publication number
EP0571549A1
EP0571549A1 EP92907398A EP92907398A EP0571549A1 EP 0571549 A1 EP0571549 A1 EP 0571549A1 EP 92907398 A EP92907398 A EP 92907398A EP 92907398 A EP92907398 A EP 92907398A EP 0571549 A1 EP0571549 A1 EP 0571549A1
Authority
EP
European Patent Office
Prior art keywords
binding
integrin
tat
hiv tat
tat protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92907398A
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German (de)
English (en)
Other versions
EP0571549A4 (fr
Inventor
Erkki I. Ruoslahti
Bruce E. Vogel
Flossie Y. Wong-Staal
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University of California
Sanford Burnham Prebys Medical Discovery Institute
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University of California
Sanford Burnham Prebys Medical Discovery Institute
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Application filed by University of California, Sanford Burnham Prebys Medical Discovery Institute filed Critical University of California
Publication of EP0571549A1 publication Critical patent/EP0571549A1/fr
Publication of EP0571549A4 publication Critical patent/EP0571549A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention generally relates to the HIV Tat protein and more particularly to the integrin cell surface receptor capable of binding to the HIV Tat protein.
  • HIV-1 Human immunodeficiency virus (HIV-1) encodes a regulatory protein, termed "Tat", that transactivates genes expressed from the long terminal repeat of the virus.
  • Tat exists as either a 72 or 86 amino acid protein, depending on whether it is expressed from one or both coding exons through differential splicing. Both forms are functional transactivators and contain an acidic domain at the amino terminus, a basic region (six arginines and two lysines out of nine contiguous residues) and a cysteine-rich region (seven out of sixteen residues) .
  • the basic domain has been shown to be responsible for nuclear/nucleolar targeting of the Tat protein as well as its capacity to bind to its cognate RNA sequence, TAR.
  • cysteine-rich domain is also critical for transactivation, its function is less defined but has been proposed to mediate metal-linked dimerization of the protein.
  • the 14-amino acid sequence encoded by the second exon has the tripeptide arginine-glycine-aspartic acid (RGD) as its most notable feature.
  • RGD arginine-glycine-aspartic acid
  • An RGD sequence is required for integrin mediated cell adhesion to extracellular matrix proteins such as fibronectin, vitronectin, and fibrinogen as reported in Pierschbacher and Ruoslahti, Nature 309:30-33, (1984) and Ruoslahti and Pierschbacher, Science 238:491-497, (1987).
  • Tat may function as an exogenous factor. Extracellular Tat is internalized by cells and transported to the nucleus, where it retains the ability to transactivate the HIV promoter as discussed in Frankel and Pabo, Cell 55:1189- 1193 (1988) . In addition, Tat can be released from HIV-1 acutely infected cells or Tat-transfected cells. Similar processes of release and uptake have been observed with another retroviral transactivator protein, namely, Tax of HTLV-l.
  • Tat has been shown to modulate cell proliferation, both in the suppression of proliferation of antigen-activated T-cells and in the specific stimulation of proliferation of Kaposi's sarcoma (KS) derived cells as reported in Ensoli et al., Nature 345:84-86 (1990).
  • Tat may contribute to the pathogenesis of HIV via mechanisms that go beyond the activation of virus replication.
  • the details of these mechanisms are not well understood. For example, it is not known how extracellular Tat stimulates KS cell growth or how it is internalized.
  • integrins One mechanism underlying the interactions of cells with one another, with extracellular matrices and with soluble proteins, such as the Tat protein, is binding of cell surface receptors to a cognate ligand on the surface of cells or in the extracellular matrix. In many cases, such a receptor belongs to a class of proteins known as integrins.
  • the integrins are a large family of cell surface glycoproteins that mediate cell-to-cell and cell- to-matrix adhesion as described, for example, in Ruoslahti and Pierschbacher, supra (1987) . All known members of this family of adhesion receptors are heterodimers consisting of an ⁇ and a ⁇ subunit noncovalently bound to each other. Over the past few years, the primary structures of six integrin ⁇ subunits from mammalian cells and one from Drosophila have been deduced from cDNA. Eleven distinct ⁇ subunits have thus far been described.
  • the adhesion of cells to extracellular matrices is mediated in many cases by the binding of a cell surface receptor to an RGD containing sequence in the matrix protein, as reviewed in Ruoslahti and Pierschbacher, supra (1987) .
  • the RGD sequence is a cell attachment site at least in fibronectin, vitronectin, fibrinogen, von Willebrand factor, thrombospondin, osteopontin, and possibly various collagens, laminin and tenascin. Despite the similarity of their cell attachment sites, these proteins can be recognized individually by their interactions with specific receptors.
  • the present invention satisfies this need and provides related advantages as well.
  • the present invention relates to methods of inhibiting HIV Tat protein binding to a cell expressing an HIV Tat binding integrin cell surface receptor ("integrin") .
  • the methods are accomplished by blocking the binding of the HIV Tat protein to the integrin by binding the HIV Tat protein with a reagent having reactivity with the integrin binding site of the HIV Tat protein or by binding the integrin with a reagent having reactivity with the HIV Tat binding site of the integrin.
  • the integrin can be, for example, human ⁇ 5 or rat cX ⁇ B> 8 .
  • the reagent can be an antibody or active fragments of either the integrin or the HIV Tat protein.
  • the present invention further provides fragments of the HIV Tat protein that can be used in the claimed methods.
  • Such fragments containing an integrin binding site include, for example, the basic domain of the HIV Tat protein.
  • a secondary binding site for cell adhesion may be an RGD-containing region of the Tat protein.
  • the fragments of the present invention can also include an RGD-containing domain of the HIV Tat protein.
  • Isolated nucleic acids encoding the HIV Tat protein, a Tat binding integrin or reactive fragments of either are also provided.
  • the invention further relates to vectors having a nucleic acid encoding such polypeptides or fragments and to host cells containing these vectors.
  • Methods of detecting ligands of the Tat binding integrins in a sample include contacting a Tat binding integrin with the sample and determining the binding of the integrin to the sample. Binding of the integrin to the sample indicates the presence of an integrin-reactive ligand.
  • the methods can be used to detect or purify HIV Tat proteins, active fragments of such proteins or HIV Tat peptide mimetics.
  • the present invention additionally relates to methods of increasing the binding of HIV Tat proteins to cells expressing the integrins of the present invention by overexpressing the integrins.
  • Figure 1 depicts the structure of the Tat protein and Tat-derived peptides. Shown are the cysteine rich domain and the basic domain in exon 1 and the RGD sequence in exon 2.
  • Figure 2 shows the results of cell adhesion to Tat peptides.
  • Figure 2A shows the results of rat L8 cells;
  • Figure 2B shows the results of human SK-LMS cells.
  • Wells of microtiter dishes were coated with various concentrations of Tat 1-86 ( ⁇ ) , Tat 45-86 (f) , or Tat 57- 86 (A) .
  • L8 cells ( ⁇ 10 cells per well) were added to each well and incubated at 37"C for one hour. The attached cells were fixed and stained with crystal violet. The dye was eluted and the absorbance at 600 nm measured in an ELISA reader.
  • SK-LMS cells were tested for attachment to the same peptides, including a 12 amino acid peptide consisting of the basic domain, Tat 45-57 ( ⁇ ) .
  • Figure 3 shows the results of studies relating to the inhibition of cell attachment to Tat with anti- ⁇ v ⁇ 3 VNR antibodies.
  • Wells of microtiter dishes were coated with either fibronectin, vitronectin or Tat peptide 45-86 at 5 ⁇ g/ml.
  • SK-LMS or L8 cells ( ⁇ 10 cells per well) were added to each well in the presence of anti- ⁇ v ⁇ 3 (VNR) or anti- ⁇ 5 B 1 (FNR) integrin antibodies.
  • the attached cells were fixed and stained with crystal violet. The dye was eluted and the absorbance was measured in an ELISA reader.
  • Figure 4 shows the affinity chromatography on Tat peptide.
  • An extract of surface iodinated L8 cells was fractionated on Tat peptide 45-86 coupled to
  • Sepharose Sepharose. After washing, the column was eluted with either 1 mg/ l of the Tat peptide or 200 ⁇ g/ml of the full-length Tat protein. The fractions were analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis under non- reducing conditions.
  • Figure 5 shows the results of immunoprecipitation of material affinity-purified on Tat peptide.
  • An extract of surface-iodinated SK-LMS cells was fractionated on Tat 45-88 coupled to Sepharose as in Figure 4.
  • Tat 57- 86 and Tat 45-57 (basic 12-mer)
  • peak fractions of each eluate were immunoprecipitated with antibodies to the ⁇ v , ⁇ u ⁇ j or ⁇ 5 subunit cytoplasmic domain, and analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis under non- reducing conditions. Shown are the immunoprecipitations of the flow-through (unbound) , GRGDSP eluate and Tat 45- 57 eluate (basic 12-mer) .
  • Figure 6 shows the results of immunoprecipitation of material affinity-purified on GRGDSPK-Sepharose.
  • An extract of surface-iodinated SK- LMS cells was fractionated on GRGDSPK-Sepharose. After elution with GRGDSP (1 mg/ml) , the flow-through (unbound) and eluate (bound) was immunoprecipitated with antibodies to the ⁇ v , B 1# B 3 or ⁇ 5 subunit cytoplasmic domains, and analyzed by SDS-polyacrylamide gel (7.5%) electrophoresis under non-reducing conditions.
  • Figure 7 shows the elution of integrin from Tat column with EDTA or NaCl. Extracts of surface-iodinated L8 or SK-LMS cells were fractionated on Tat 45-86 coupled to Sepharose. Sequential elution of the columns with 10 mM EDTA (in 100 mM NaCl; lanes 2,6), 250 mM NaCl (lanes 3,7) and Tat 45-86 (1 mg/ml; lanes 4,8), followed by immunoprecipiTation with a polyclonal anti-vitronectin receptor antibody and analysis by SDS-polyacrylamide gel (7.5%) electrophoresis under, non-reducing conditions are shown. Lanes 1 and 5 are immunoprecipitates of the flow- through material.
  • Figure 8 shows the results of inhibition of SK- LMS cell binding to vitronectin and Tat peptide.
  • SK-LMS cells were added to microtiter wells coated with Tat 45- 86 or vitronectin (VN) in the presence or absence of inhibitory antibodies.
  • P3G2 is a monoclonal antibody shown previously to inhibit the interaction of a v B 5 with vitronectin.
  • LM 609 inhibits the interaction of ⁇ y ⁇ 3 with vitronectin.
  • Anti-VNR is a polyclonal antibody raised against the ⁇ integrin purified from a placental extract on a GRGDSPK-Sepharose column.
  • FIG. 9 shows the effect of antibodies and peptide on Tat transactivation.
  • LTR-CAT was transfected into L8 cells and transactivated by exogenous GST-Tat fusion protein in the presence of anti- ⁇ v B 3 polyclonal antibody (anti-VNR) , rabbit anti- ⁇ 5 ⁇ 1 (anti-FNR) , normal rabbit serum, or Tat basic peptide.
  • anti-VNR anti- ⁇ v B 3 polyclonal antibody
  • anti-FNR rabbit anti- ⁇ 5 ⁇ 1
  • normal rabbit serum or Tat basic peptide.
  • Lane 1 LTR-CAT alone; lane 2, LTR-CAT plus GST-Tat fusion protein; lane 3, LTR-CAT plus GST protein; lanes 4-8, LTR-CAT plus GST- Tat fusion protein; and lane 4, 300 ⁇ g basic peptide; lane 5, 80 ⁇ l anti- ⁇ v B 3 ; lane 6, 8 ⁇ l anti- ⁇ v B 3 ; lane 7, 80 ⁇ l anti- ⁇ g ⁇ , serum; lane 8, normal rabbit serum.
  • the present invention relates to integrin cell surface receptors, referred to herein as "integrins," that bind to the HIV Tat protein.
  • integrins are the known ⁇ y ⁇ 5
  • rat cells may contain a previously unidentified ⁇ subunit, designated herein as ⁇ g , and the known subunit ⁇ v .
  • the ⁇ v subunit is the most versatile of the integrin a subunits. It has been known to combine with three, possibly four, different ⁇ subunits. Thus, the rat ⁇ 8 subunit of the present invention may add at least one additional ⁇ subunit to this group.
  • the human ⁇ 5 subunit was previously known to combine with ⁇ v , although the Tat binding activity of human ⁇ B 5 was not previously known.
  • the present invention particularly relates to a novel interaction between the HIV Tat protein and the integrins to which the Tat protein bind.
  • previous reports suggest an RGD-dependent mechanism in the binding of Tat to rat L8 cells, as described in Brake et al., J. Cell Biol. 111:1275-1281 (1990), it has now been discovered from cell adhesion and affinity chromatography data that the basic region of the Tat protein mediates this interaction.
  • Immunoprecipitation of the protein purified on the Tat columns identified the Tat binding proteins as components of the integrin 0: ⁇ 5 . This result was consistent with data showing that cell adhesion to Tat could be blocked by polyclonal antibodies to the ct y ⁇ j integrin. Antibodies to this integrin would be expected to bind to the ⁇ v subunit shared by ⁇ y ⁇ 5 and ⁇ and inhibit the function of both heterodimers.
  • the C- y ⁇ g integrin has been shown to bind to vitronectin through the RGD sequence.
  • the Tat protein contains an RGD sequence
  • the integrin recognition sequence is the basic domain of Tat. This conclusion is based on the complete correlation found between the ability of Tat-derived peptides to support cell attachment and bind the ⁇ integrin in affinity chromatography with the presence of the basic sequence in the peptide. In contrast, the presence of absence of the RGD sequence had no influence in either type of assay.
  • the RGD sequence is present in a context not suitable for integrin binding because even the ⁇ v B 3 integrin, which has the greatest binding activity with short RGD-containing peptides as shown in Figure 6 failed to bind appreciably to the RGD-containing Tat peptides. Most integrins require divalent cations for their activity and are dissociated from their ligands in the presence of EDTA. In addition to utilizing the basic domain, the interaction between ⁇ y ⁇ 5 and Tat has the unusual feature that it is stable in the presence of 10 mM EDTA and is therefore not divalent cation-dependent. The interaction of the o y ⁇ 5 integrin with Tat was, however, inhibited by high NaCl concentrations.
  • integrin Although this salt sensitivity is unusual among integrins, it is not unprecedented.
  • the ⁇ 3 ⁇ , integrin also demonstrates a salt-labile, RGD-independent binding to collagen and laminin as discussed in Elices et al., J. Cell Biol. 112:169-181 (1991). It could be that ⁇ 3 ⁇ . also binds to a basic region within these proteins.
  • CT j ⁇ , integrin may have two functionally distinct ligand binding sites.
  • the ⁇ 4 ⁇ , integrin has two distinct ligand binding sites, one for the endothelial cell ligand, V-CAM, and the other for an alternatively spliced segment of fibronectin as reported in Elices et al., Cell 60:577-584 (1990).
  • c- y ⁇ g may have a second ligand binding site for a basic extracellular protein yet to be identified.
  • the integrin Ilb/IIIa may also share some of the basic peptide binding properties of ⁇ ⁇ ⁇ 5 . Peptides containing both an RGD and a basic segment bind more avidly to Ilb/IIIa than peptides containing RGD alone as reported in Savage et al., J. Biol. Chem. 265:11766-11772 (1990).
  • Both rat L8 and human SK-LMS cells remained round when plated on a surface coated with Tat and Tat- derived peptides containing the basic domain. This behavior is consistent with the finding that cells expressing a ⁇ E> 5 attach to vitronectin, but do not spread. According to reports in Wayner et al. , J. Cell Biol. 113:919-929 (1991), spreading on vitronectin is dependent on the presence of ⁇ .
  • Tat was found to bind an integrin containing a ⁇ subunit designated herein as B 8 and the known ⁇ v .
  • the Tat protein was found to bind the known human integrin, a v B 5 .
  • the rat integrin may also be G- y ⁇ g because it behaved identically to the human a ⁇ integrin in the affinity chromatography experiments and migrated similarly in SDS-PAGE.
  • the antibodies prepared against the cytoplasmic peptide of the human ⁇ 5 subunit were weakly reactive against a number of rat cell lines. Poor reactivity of the antibodies with the rat B 5 subunit may therefore explain the lack of immunoprecipitation of the L8 integrin observed.
  • the ⁇ subunit of the Tat binding integrin from the L8 cells may be an alternatively spliced ⁇ 5 variant or a completely different ⁇ subunit altogether.
  • the rat B 8 subunit is distinguishable from known ⁇ subunits.
  • direct comparisons show that the apparent molecular weight of the ⁇ 8 subunit is lower than many of the previously characterized ⁇ subunit.
  • the ⁇ v subunit has been shown to associate with the B 1# ⁇ 3 , or ⁇ 5 subunits and may also combine with B 6 , antibodies to the cytoplasmic tails of each of these known subunits failed to immunoprecipitate the Tat binding receptor.
  • the possibility that the ⁇ subunit differs from a previously characterized ⁇ subunit by alternative splicing or proteolytic processing cannot be excluded.
  • the L8 cells have a ⁇ j subunit that is functional, but does not bind to Tat.
  • the ⁇ v subunit is the most versatile of the integrin subunits in that it is known to combine with three, possibly four, different ⁇ subunits. It now appears that at least one additional ⁇ subunit can be added to this group.
  • the rat ⁇ integrin is likewise distinguishable from the known integrins in its binding specificity.
  • the ⁇ 4 ⁇ , integrin binds to sequences that do not contain the RGD sequences, yet the Tat protein does not contain any sequences significantly homologous to the ⁇ 4 B 1 target sequence.
  • ⁇ subunit in combination with the ⁇ v subunit it forms the integrins of the present invention that binds to the HIV Tat protein.
  • the specificity of the integrins of the present invention is such that it can bind the intact Tat protein or a truncated form lacking the RGD-containing region and, therefore, can be used to control the activities of the Tat protein.
  • the ⁇ component of the Tat binding integrins gives a double band in a non-reduced SDS-gel electrophoresis in the molecular weight range of about 75-95 kD, representing two forms of the same ⁇ subunit or two different subunits.
  • the present invention is contemplated to include both.
  • the full length Tat protein contai s the following amino acid sequence:
  • a basic domain of the Tat protein has been found to be the dominant binding site for the integrin of the present invention. This basic region contains nine amino acid residues of which six are arginine and two are lysine. The amino acid sequence of the basic region is RKKRRQRRR in the HIV strain HXB2. In studies to determine the role of RGD in binding the Tat protein, it was found that a variant peptide in which RGD was replaced with KGE was still capable of binding to the Tat protein with only a small reduction in the binding activity.
  • ligands which bind to the Tat binding integrins can contain both a basic domain and a nearby RGD sequence. While the basic region appears to be the primary binding site of these integrins than the RGD- containing region, at least some of the RGD-directed integrins recognize basic regions in addition to the RGD- containing sequences. This conclusion is supported in part by studies in which peptides that contain both an RGD and a basic segment bound more avidly to the ⁇ ⁇ b / ⁇ 3 integrin than peptides containing RGD alone as described in Savage et al., J. Biol. Chem. 265:11766-11772 (1990).
  • the present invention also provides isolated fragments specific to the Tat binding integrins, particularly to the ⁇ subunmits such as, for example, human ⁇ 5 and rat ⁇ g .
  • Such fragments can be regions obtained from the native Tat protein or synthetic polypeptides having amino acid sequences of the Tat protein regions. These fragments are necessarily of sufficient length to be distinguishable from fragments of other known ⁇ s and, therefore, are "specific to" or "unique to" the ⁇ subunits of the Tat binding integrins, particularly human B 5 and rat ⁇ 8 , for example.
  • Such fragments specific to B 8 can be determined using methods disclosed herein and known in the art.
  • fragments also retain the Tat binding function and can therefore be used, for example, as inhibitors of the binding of the Tat binding integrins to the Tat protein, or as an indicator to detect the Tat binding integrins of the present invention.
  • One skilled in the art can determine other uses for such fragments.
  • substantially purified or “isolated” mean that the material, either polypeptide or nucleic acid, is substantially free of contaminants normally associated with the native or natural environment.
  • a reactive fragment of the HIV Tat protein refers to a polypeptide having substantially the amino acid sequence of a portion of the HIV Tat protein that retains the integrin binding site so as to remain substantially reactive with the Tat binding integrins.
  • a reactive fragment of a Tat binding integrin refers to a polypeptide having substantially the amino acid sequence of a portion of the integrin that retains the Tat binding function.
  • Amino acid sequences such as those for ⁇ 1 , ⁇ 2 and B 3 , having less than 50% homology with the sequence of ⁇ or ⁇ 5 are not substantially the same sequence and, therefore, do not fall within the definition of these ⁇ subunits.
  • amino acid sequences set forth herein additions, deletions or substitutions can be made and tested to determine their effect on the function of the ⁇ subunits.
  • amino acids can be modified to alter integrin binding function.
  • the invention also provides reagents having specificity for the integrins of the present invention and particularly the rat B 8 and human ⁇ 5 subunits.
  • reagents such as antibodies
  • Such reagents can be used to immunologically distinguish these B subunits from other molecules.
  • Various methods of raising such antibodies are well established and are described, for example, in Antibodies, A Laboratory Manual. E. Harlow and D. Lane, pp. 139-283 (Cold Spring Harbor Laboratory, 1988) , incorporated herein by reference.
  • the invention further provides isolated nucleic acids that encode the Tat protein or reactive fragments thereof having binding sites or regions recognized by the ⁇ -containing integrins of the present invention. Similarly, isolated nucleic acids encoding the Tat binding integrins or reactive fragments thereof are also provided. Following standard methods as described, for example, in Maniatis et al., Molecular Cloning, Cold
  • these nucleic acid sequences can be identified, isolated and then cloned into an appropriate expression vector.
  • the vector can then be inserted into a host, which will then be capable of expressing the Tat recombinant proteins, Tat binding integrins or reactive fragments of each.
  • the invention also relates to vectors containing nucleic acids encoding such sequences and to hosts containing these vectors.
  • the present invention further relates to nucleic acids that can be used as probes for diagnostic purposes.
  • nucleic acid probes can hybridize with a nucleic acid having a nucleotide sequence specific to the Tat binding integrins or to the Tat protein but do not hybridize with nucleic acids encoding non-Tat binding integrins or Tat proteins, particularly other cell surface receptors or non-Tat proteins, respectively.
  • Nucleic acids are also provided which specifically hybridize to either the coding or non-coding DNA of the Tat binding integrins or the Tat protein.
  • Such nucleic acids can be identified and prepared by methods known in the art, such as a standard nucleic acid synthesizer.
  • the binding of the Tat binding integrins can be blocked by various means.
  • the binding of a ⁇ 8 - or ⁇ 5 -containing integrin can be blocked by a reagent that binds the ⁇ subunit or the ⁇ -containing integrin.
  • reagents include, for example, peptides and polypeptides containing the basic region of the Tat protein or antibodies specifically reactive with the ⁇ subunit or a ⁇ -containing integrin.
  • the binding of the Tat protein to the Tat binding integrins can be blocked by binding the integrins with Tat-derived peptides having an amino acid sequence that recognizes the Tat binding site of the integrins.
  • blocking can be carried out by binding the Tat protein or a reactive fragment thereof with a reagent specific for the Tat protein at a site that inhibits the Tat protein from binding with the integrin, such as an anti-Tat antibody or a reactive fragment of the integrin.
  • the ability to block the binding of the Tat protein by the Tat-binding integrins can be used to control HIV mediated conditions.
  • the activities of the Tat protein as a growth factor for Kaposi's sarcoma cells and as a transactivator of the HIV promoter when internalized by cells can be inhibited or otherwise controlled.
  • the binding of the Tat binding integrins to the Tat protein can mediate cell adhesion, preventing this binding can prevent the adhesion of cells to the endogenous ligand of the integrin.
  • Other activities of the integrin can be similarly inhibited by the use of the Tat mimicking compounds of the present invention.
  • cell adhesion can be promoted by increasing the expression of these integrins by a cell.
  • the activities of the endogenous ligand or ligands can be mimicked with the compounds of this invention.
  • the binding can be enhanced by methods in which the Tat binding integrin is overexpressed in such cells.
  • Methods of enhancing the overexpression of such integrins can be accomplished, for example, by introducing a nucleic acid encoding for the integrin into the genome of a cell by methods known to those skilled in the art.
  • the present invention further provides methods of detecting ligands that bind the Tat binding integrins.
  • the methods include contacting the integrin or binding fragment thereof with a solution containing ligands known to or suspected of binding the Tat binding integrins. Ligands that bind such integrins are then detected. Assays useful to carry out these methods are well known in the art and are described, for example, in Hautanen et al., J. Biol. Chem. 264:1437-1442 (1989) and Smith et al., J. Biol. Chem. 265: 11008-11013 (1990), both of which are incorporated herein by reference.
  • Such methods can be used to identify or screen for additional compounds that are bound by the Tat binding site on the integrins of the present invention.
  • Such compounds can be naturally occurring ligands, such as derivatives of the Tat binding site or other compounds capable of being bound by the integrin Tat binding site.
  • Such compounds having the desired binding function can be peptides, peptide derivatives or mimetics, or other compounds, so long as they are capable of being bound by the Tat protein's integrin binding site.
  • the intact Tat protein was synthesized using t- butoxycarbonyl (BOC)-protected amino acids for stepwise synthesis on an Applied Biosystems 431A (Foster City, CA) solid phase automated peptide synthesizer. Amino acids were added as hydroxybenzotriazole (HOBt) esters using n- methylpyrrolidone as the coupling solvent. The synthesis was accomplished starting with 0.5 mmol of Boc-Glu(OBzl)- O-phenylacetamidomethyl resin (0.69 g substituted at 0.72 mmol) with a minimum of two couplings for each amino acid. The average repetitive coupling efficiency was 99.32% as determined by a qualitative ninhydrin assay.
  • BOC t- butoxycarbonyl
  • cysteine sulfhydryls were protected with a p- methylbenzyl group to yield a fully reduced form after low/high HF cleavage using suitable scavengers. All other peptides used were synthesized with an Applied Biosystems Model 430A synthesizer using similar chemistry.
  • a band of 150 kD and a doublet of 75-95 kD were identified that bound to a full length Tat protein coupled to a Sepharose column and eluted with 1 mg/ml of a Tat peptide (amino acid residues 45-86) containing the basic region and the RGD region but lacking the cysteine rich region of the protein.
  • a second peptide representing the cell adhesion site of fibronectin, GRGDSP was not effective at eluting the Tat binding proteins.
  • the peptide covering residues 45-86 coupled to Sepharose bound the same proteins.
  • Identical bands were eluted with the truncated peptide (residues 45-86) or the full length Tat protein, demonstrating that the peptide and protein had similar binding sites.
  • An additional band of approximately 120 kD was eluted from the full length Tat protein column, but not from the shorter Tat peptides. This band has not been further identified, but may represent a subunit of a second binding integrin.
  • the peptide that contained residues 45-86 was synthesized with the KGE sequence substituted for RGD.
  • the same proteins were bound to the KGE variant peptide and were eluted from the Tat column with this variant peptide indicating that the role of RGD in binding Tat is not a primary one.
  • affinity chromatography on GRGDSP-Sepharose was performed with the unbound material from the Tat peptide column.
  • a band with a similar electrophoretic mobility to the 150 kD Tat binding protein together with a band distinct from the 85 and 95 kD bands obtained from the Tat column were eluted from the GRGDSP-Sepharose column. These bands were identified as the subunits of the ⁇ y ⁇ 3 integrin.
  • Figure 5 shows an immunoprecipitation of proteins eluted from the Tat 45-86 peptide column.
  • the column was eluted sequentially with the peptide GRGDSP followed by the Tat 57-86 peptide and finally the 12 amino acid peptide containing the basic domain of Tat. Shown is the material eluted from the column with the indicated peptide after immunoprecipitation with antibodies to the specified integrin subunits.
  • a small amount of material eluted with the peptide GRGDSP could be immunoprecipitated with ⁇ , and ⁇ 3 antibodies. However, the majority of the ⁇ , and ⁇ 3 subunit-associated material did not bind to the column and was detected in the unbound fraction.
  • a y B 5 integrin binds to the basic domain of Tat and not the RGD sequence, it appears to be an unusual interaction of an integrin with its ligand. To test this further, the EDTA-sensitivity of the integrin association with Tat was determined. It is known that integrins typically require divalent cations to bind their ligands and can be eluted from ligand " affinity columns with EDTA. The interaction between ⁇ y ⁇ 5 and Tat was, however, insensitive to elution with 10 mM EDTA in affinity chromatography experiments ( Figure 7) .
  • the NaCl concentration in the solution containing 10 mM EDTA was lowered from 150 mM to 100 mM to insure elution would result from divalent cation chelation and not an overall increase in the salt concentration of the solution.
  • Immunoprecipitations of the peak fractions eluted with 10 mM EDTA, 250 mM NaCl, or the Tat 46-86 peptide revealed that the receptor was eluted from the Tat column with high salt or with the peptide, but chelating the divalent cations which are normally required for integrin function was not effective in disrupting this unusual integrin-ligand interaction.
  • a monoclonal antibody recognizing ⁇ y ⁇ 3 did not inhibit the binding of the cells to either substrate.
  • the results suggest that ⁇ y ⁇ 5 mediates the attachment of SK-LMS cells to both vitronectin and Tat and that distinct regions of the receptor may be utilized for the binding to each ligand.
  • the anti-VNR polyclonal antibodies against the vitronectin receptor inhibited the adhesion of these cells to both vitronectin and to the Tat peptide.
  • Anti- FNR antibodies were not effective in preventing the adhesion of these cells to either substrate.
  • Tat derived peptides were used to investigate the mechanism of cell interaction with the Tat protein.
  • L8 rat skeletal muscle cells were chosen for these experiments because they had been shown to bind to Tat in a previous study described in Brake et al., supra. In agreement with this earlier study, L8 cells readily attached to the Tat protein ( Figure 2A) . L8 cells also attached to the Tat 45-86 peptide that contained the RGD sequence and the basic domain, but not the cysteine rich domain.
  • Polyclonal antibodies against the cytoplasmic tails of v , ⁇ 3 , and ⁇ 5 were raised in rabbits by immunization with synthetic peptides coupled to keyhole limpet hemocyanin.
  • the peptides used were KRVRPPQEEQEREQLQPHENGEGNSET from the C-terminus of ⁇ v as described in Suzuki et al., Proc. Natl. Acad. Sci. USA 83:8614-8618 (1986), KFEEERARAKWDTANNPLYKEATSTFTNITYRGT from the C-terminus of B 3 as described in Fitzgerald, J. Biol. Chem.
  • LM 609 Monoclonal antibodies (LM 609) were a gift of Dr. David Cheresh and are described in Cheresh and Spiro, J. Biol. Chem. 262:17703-17711 (1987) or Bristol Myers-Squibb Pharmaceutical Research Institute (P3G2) which is described in Wayner et al., J. Cell Biol. 113:919-929 (1991) .
  • Immunoprecipitations were performed by incubating material in the presence of 5 ⁇ l of immunized rabbit serum and 50 ⁇ l protein A-Sepharose for 1 hour.
  • the receptor-antibody-protein A complex was spun down and washed three times with 0.5% Triton X-100, 150 ⁇ M NaCl, 50 ⁇ M Tris, pH 7.4. The complex was then boiled in electrophoresis sample buffer and loaded on 7.5% SDS- polyacrylamide gels.
  • the Tat-binding proteins were isolated from surface-iodinated cells essentially as described in Pytela et al., PNAS fUSA. 82:5766-5770 (1985) and Pytela et al., Cell 40:191-198 (1985), both incorporated herein by reference. Cells were detached from culture plates in 100 ⁇ g/ml trypsin (Sigma) as in the cell adhesion assays, and washed three times in 500 ⁇ g/ml soybean trypsin inhibitor (Sigma) .
  • Cells were surface iodinated and extracted with a buffer containing 150 ⁇ m octyl glucoside, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 ⁇ g/ml aprotinin, 1 ⁇ g/ml leupeptin, 0.4 ⁇ g/ml pepsTatin, 150 mM NaCl and 50 mM Tris, pH 7.4.
  • the extracts were clarified at 15,000 x g and passed over a column containing various peptides coupled to cyanogen bromide activated Sepharose 4B (Pharmacia).
  • the column was washed with several volumes of extraction buffer containing 50 mM octyl glucoside.
  • the bound receptor was then eluted with the appropriate peptide at a concentration of 1 mg/ml in the buffer used to wash the column. Aliquots were boiled in electrophoresis sample buffer and run on 7.5% SDS-polyacrylamide gels or used for immunoprecipitation.
  • Polyclonal antibodies can be prepared by any method known in the art using as the immunogen an appropriate protein or a synthetic peptide derived therefrom.
  • the integrin of the present invention the Tat protein or equivalent compounds can be used as the immunogen.
  • Such methods as described, for example, in Argraves et al., J. Cell Biol. 105:1163-1173 can be used.
  • a synthetic peptide containing at least one lysine residue is coupled to Keyhole Limpet Hemocyanin (KLH) as follows: by stirring 5.6 ml KLH (10 mg/ml in phosphate buffered saline) with 0.5 ml m- maleimidobenzoyl-N-hydroxysuccinimide ester (25 mg/ml in dimethyl formamide) for 30 minutes at room temperature. The mixture is filtered and 25 g of the peptide is added followed by three hours of stirring at room temperature. After dialysis against phosphate buffered saline, the mixture containing the coupled peptide is emulsified in Freund's complete adjuvant, mixed and injected into a New Zealand White female rabbit.
  • KLH Keyhole Limpet Hemocyanin
  • Cells were seeded in 10 cm dishes (10 cells/dish) . The following day, cells were transfected with 5 ⁇ g LTR-CAT and/or 5 ⁇ g LTR-Tat plasmids by DEAE dextran precipitation as described in David et al. , Basic Methods in Molecular Biology, p. 388 (Elsevier 1986) , incorporated herein by reference. Two days posttransfection, cells were incubated with 80 ⁇ g or 8 ⁇ l rabbit anti-human vitronectin receptor (anti- ⁇ y ⁇ 3 ) , anti- fibronectin receptor (anti- ⁇ - g ⁇ ,,) or normal rabbit serum in 3 ml of complete DMEM for 30 minutes before the addition of 0.5 ⁇ l of E.
  • ⁇ y ⁇ 5 serves as the receptor for Tat internalization
  • Antibodies to ⁇ 5 ⁇ 1 were used as a control.
  • a recombinant GST-Tat fusion protein added to L8 cells was able to efficiently and specifically transactivate an LTR reporter gene transfected into these same cells.
  • CAT activity was not diminished in the presence of anti- ⁇ y ⁇ 3 antibodies at concentrations sufficient to inhibit cell adhesion (lane 5) nor was it inhibited in the presence of 300 ⁇ g of basic peptide (lane 4) .

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Abstract

L'invention se rapporte aux protéines Tat de l'HIV (virus de l'immunodéficience humaine) et au récepteur de surface de cellules d'intégrine qui reconnaît spécifiquement la protéine Tat de l'HIV. L'intégrine est un hétérodimère contenant une sous-unité alphav et une ou plusieurs sous-unités beta tel que le complexe alphavbeta5 dérivé de cellules humaines et le complexe alphavbeta8 dérivé de cellules de rat. L'invention se rapporte en outre à des fragments réactifs comportant des séquences qui correspondent aux régions de liaison de la protéine Tat de l'HIV et aux intégrines de liaison avec la protéine Tat. Des procédés permettant de réguler la liaison de la protéine Tat de l'HIV à des cellules exprimant ces intégrines sont également décrits.
EP92907398A 1991-02-14 1992-02-13 NOUVELLE SPECIFICITE DE L'INTEGRINE POUR LA PROTEINE Tat DE L'HIV. Withdrawn EP0571549A4 (fr)

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WO1991015224A1 (fr) * 1990-03-30 1991-10-17 Smithkline Beecham Corporation Inhibition de la maladie associee a l'infection due au virus de l'immunodeficience
US6258550B1 (en) 1993-04-23 2001-07-10 Virginia Commonwealth University Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site
CA2215483C (fr) * 1995-03-08 2012-01-03 Jean-Francois Zagury Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
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US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them
US5891994A (en) * 1997-07-11 1999-04-06 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
US6399067B1 (en) 2000-04-28 2002-06-04 Thymon L.L.C. Methods and compositions for impairing multiplication of HIV-1
WO2006088740A2 (fr) 2005-02-15 2006-08-24 Thymon, L.L.C. Methodes et compositions permettant d'alterer la multiplication de vih-1
WO2016118433A1 (fr) 2015-01-20 2016-07-28 Shayakhmetov Dmitry M Variants d'adénovirus déciblés et procédés associés
US9982276B2 (en) 2015-03-12 2018-05-29 Adcure Biotechnologies, Llc. Penton-mutated, integrin-retargeted adenovirus vectors with reduced toxicity and their use

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JOURNAL OF VIROLOGY, vol.64, no.2, February 1990 pages 962 - 965 BRAKE, D.A. ET AL.; 'Characterization of murine monoclonal antibodies to the tat protein from human immunodeficiency virus type 1' *
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