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Document Type and Number:
WIPO Patent Application WO/1999/032138
Kind Code:
There is disclosed a method of reducing susceptibility of cells to infection by a retrovirus, comprising contacting the cells with an amount of a CD40 binding protein effective to reduce the expression of the chemokine receptor CCR5, and/or induce a post-entry block of the replication of the retrovirus. The method is useful in treating individuals infected with, or at risk of being infected with, HIV. CD40 binding proteins include CD40 ligand, monoclonal antibodies that specifically bind CD40, and combinations thereof. CD40 binding proteins may also be used in combination with other cytokines, including GM-CSF, IL-2 and IL-15.

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Publication Date:
July 01, 1999
Filing Date:
December 18, 1998
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International Classes:
A61K38/00; A61K38/17; A61K38/19; A61K38/20; A61K38/22; A61P31/18; A61P37/02; (IPC1-7): A61K38/19; A61K38/17
Domestic Patent References:
Foreign References:
Other References:
DATABASE AIDSLINE June 1998 (1998-06-01), XP002102579
Attorney, Agent or Firm:
Henry, Janis C. (WA, US)
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We claim:
1. A method for decreasing the infectability of a cell that expresses a CCR5 chemokine receptor for a retrovirus that uses the CCR5 chemokine receptor as a co receptor, comprising contacting the cell with an amount of a CD40 binding protein sufficient to decrease expression of the CCR5 chemokine receptor.
2. The method of claim 1, wherein the CD40 binding protein is a soluble, oligomeric CD40L.
3. The method of claim 1, wherein the cell is also contacte with a cytokine selected from the group consisting of granulocyte macrophage colony stimulating factor, interleukin2, interleukin15, and combinations thereof.
4. The method of claim 2, wherein the cell is also contacte with a cytokine selected from the group consisting of granulocyte macrophage colony stimulating factor, interleukin2, interleukin15, and combinations thereof.
TITLE METHOD FOR REDUCING SUSCEPTIBILITY TO HIV INFECTION TECHNICAL FIELD OF THE INVENTION The present invention relates to a method of reducing the susceptibility of a cell to infection by human immunodeficiency virus (HIV), comprising contacting the cell with an effective amount of a biologically active CD40 binding protein.

BACKGROUND OF THE INVENTION The cluster of differentiation antigen CD4 was originally identifie as a receptor used by human immunodeficiency virus (FIV) in binding to cells (Dalgleish et al., Nature 312: 763; 1984). Subsequent to that time, it became clear that additional molecules, referred to as co-receptors, were necessary for viral entry into cells (Maddon et al., Cell 47: 333; 1986). Eventually, it became clear that certain chemokine receptors could act as co-receptors for HIV. Among these is CCR5, a chemokine receptor that binds N41pla, MIPIB and RANTES. A CCR5 antagonist that is an analog of the chemokine RANTES has been shown to block HIV infection, and may be useful in preventing HIV infection (Simmons et al., Science 276: 276; 1997).

CD40L is a type II membrane polypeptide having an extracellular region at its C- terminus, a transmembrane region and an intracellular region at its N-terminus. Soluble CD40L comprises an extracellular region of CD40L (amino acid 47 to amino acid 261 of human CD40L) or a fragment thereof. CD40L biological activity is mediated by binding of the extracellular region of CD40L with CD40, and inclues B cell proliferation and induction of antibody secretion (including IgE secretion). Soluble, oligomeric CD40L has been shown to induce production of the chemokines MIP-la, MIP-lß and RANTES from monocytes/macrophages.

Accordingly, CD40L (and other CD40-binding proteins that bind CD40 and trigger secretion of these chemokines) may act to reduce the susceptibility of cells to HIV infection by stimulating the production of chemokines that bind to CCR5, and thereby

prevent HIV from using the CCR5 as a co-receptor. However, prior to the instant invention, it was not known that CD40 binding proteins such as CD40L could down regulate CCR5 expression, and render macrophages not permissive for HIV infection.

SUMMARY OF THE INVENTION A recombinant, soluble form of CD40L causes monocytes to down-regulate surface expression of CCR5, and induces expression of the chemokines MIP-la, MIP-lß and RANTES. CD40L-stimulated monocyte-derived macrophages are not permissive for FIV-1 infection, possibly to down regulation of CCR5, and/or a post-entry block of viral replication. Blood monocytes contacte with CD40L are less likely to become infecte with macrophage-tropic virus and may protect bystander cells by producing chemokines that antagonize macrophage-tropic FIV-1 replication in monocytes as well as T cells.

The present invention relates to a method of reducing the susceptibility of a cell to HIV infection, comprising contacting the cell with an amount of a CD40 binding protein effective to down-regulate CCR5 expression by the cell, or to render the cell non- permissive for HIV infection. CD40 binding proteins are pharmaceutical compositions capable of binding CD40 and transducing a biological signal. CD40 binding proteins are selected from the group consisting of CD40 ligand, monoclonal antibodies that specifically bind CD40, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents results that indicate that CD40L, alone or in combination with GM-CSF, induces production of various chemokines by monocytes.

Figure 2 presents results that indicate that CD40L inhibits the replication of a JRFL pseudotyped retrovirus.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of reducing the susceptibility of a cell to HIV infection, comprising contacting the cell with a CD40 binding protein that is capable of binding CD40 and transmitting a biological signal to a CD40-expressing cell. The binding of the CD40 binding protein causes the down-regulation of CCR5, a chemokine

receptor, which decreases the amount of CCR5 available to act as a co-receptor for HIV entry into the cell. Alternatively, or in addition to this effect, CD40 binding proteins may cause a post-entry block of HIV replication. CD40 binding proteins will also stimulate the cells to secrete chemokines, which may protect bystander cells from HIV infection by antagonizing the use of chemokine receptors as viral co-receptors.

The findings described herein also provide data to enable a method of reducing the susceptibility of an individual to HIV infection, by administering a pharmaceutical composition comprising a substance with CD40 binding protein activity to the individual.

CD40 Human CD40 antigen (CD40) is a peptide of 277 amino acids having a molecular weight of 30,600 (Stamenkovic et al., EMBO J. 8: 1403,1989). A cDNA encoding human CD40 was isolated from a cDNA library prepared from Burkitt lymphoma cell line Raji. The putative protein encoded by the CD40 cDNA contains a putative leader sequence, trans-membrane domain and a number of other features common to membrane- bound receptor proteins. CD40 has been found to be expressed on B lymphocytes, epithelial cells and some carcinoma cell lines.

CD40 is a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family, which is defined by the presence of cysteine-rich motifs in the extracellular region (Smith et al., Science 248: 1019,1990; Mallett and Barclay, Immunology Today 12: 220; 1991). This family inclues the lymphocyte antigen CD27, CD30 (an antigen found on Hodgkin's lymphoma and Reed-Sternberg cells), two receptors for TNF, a murine protein referred to as 4-1BB, rat OX40 antigen, NGF receptor, and Fas antigen. CD40 is functionally expressed on monocytes/macrophages, B cells, lymphoma cells, carcinoma cells, dendritic cells, and vascular endothelial cells.

CD40 may be detected on the surface of a cell by any one of several means known in the art. For example, an antibody specific for CD40 may be used in a fluorescence- activated cell sorting technique to determine whether cells express CD40. Other methods of detecting cell surface molecules are also useful in detecting CD40.

CD40 Monoclonal Antibodies Monoclonal antibodies directe against the CD40 surface antigen (CD40 mAb) have been shown to mediate various biological activities on human B cells. For example, CD40 mAb induce homotypic and heterotypic adhesion (Barrett et al., J. Immunol.

146: 1722,1991; Gordon et al., J. Immunol. 140: 1425,1988), and increase cell size (Gordon et al., J. Immunol. 140: 1425,1988; Valle et al., Eur. J. Immunol. 19: 1463,1989).

CD40 mAb also induce proliferation of B cells activated with anti-IgM, CD20 mAb, or phorbol ester alone (Clark and Ledbetter, Proc. Natl. Acad. Sci. USA 83: 4494,1986; Gordon et al., Leukocyte Typing III; A. J. McMichael ed. Oxford University Press.

Oxford, p. 426; Paulie et al., J. Immunol. 142: 590,1989) or in concert with IL-4 (Valle et al., Eur. J. Immunol. 19: 1463,1989; Gordon et al., Eur. J. Immunol. 17: 1535,1987), and produce IgE (Jabara et al., J. Exp. Med. 172: 1861,1990; Gascan et al., J. Immunol. 147: 8, 1991), IgG, and IgM (Gascan et al., J. Immunol. 147: 8,1991) from IL-4-stimulated T cell-depleted cultures.

In addition, CD40 mAb have been reporte to enhance IL-4-mediated soluble CD23/Fc£RII release from B cells (Gordon and Guy, Immunol. Today 8: 339,1987; Cairns et al., Eur. J. Immunol. 18: 349,1988) and to promote B cell production of IL-6 (Clark and Shu, J. Immunol. 145: 1400,1990). Recently, in the presence of CDw32+ adherent cells, human B cell lines have been generated from primary B cell populations with IL-4 and CD40 mAb (Banchereau et al., Science 241: 70,1991). Furthermore, germinal center centrocytes can be prevented from undergoing apoptosis if they are activated through CD40 and/or receptors for antigen (Liu et al., Nature 342: 929,1989).

Each of the above publications describes CD40 mAb that stimulate a biological activity of B cells.

U. S. S. N. 08/130,541, filed October 1,1993, the relevant disclosure of which is incorporated by reference, discloses two monoclonal antibodies that specifically bind CD40, referred to as hCD40m2 and hCD40m3. Unlike other CD40 mAb, hCD40m2 (ATCC HB11459) and hCD40m3 bind CD40 and inhibit binding of CD40 to cells that constitutively express CD40L. Greater than 95% inhibition of binding was observe with hCD40m2 or with CD40 mAb M3, at concentrations as low as 12.5, ug/ml, as compare to irrelevant IgG or a control CD40 mAb, G28.5. hCD40m2 was also able to inhibit CD40L-induced TNF-a production.

Additional CD40 monoclonal antibodies may be generated using conventional techniques (see U. S. Patent Nos. RE 32,011,4,902,614,4,543,439, and 4,411,993 which are incorporated herein by reference; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference).

Briefly, an animal is injecte with a form of CD40 suitable for generating an immune response against CD40. The animal may be reimmunized as needed until levels of serum antibody to CD40 have reached a plateau, then be given a final boost of soluble CD40, and three to four days later sacrificed. Organs which contain large numbers of B cells such as the spleen and lymph nodes are harvested and disrupted into a single cell suspension by passing the organs through a mesh screen or by rupturing the spleen or lymph node membranes which encapsulate the cells.

Alternatively, suitable cells for preparing monoclonal antibodies are obtained through the use of in vitro immunization techniques. Briefly, an animal is sacrifice and the spleen and lymph node cells are removed. A single cell suspension is prepared, and the cells are placed into a culture which contains a form of CD40, which is suitable for generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.

Cells which are obtained through the use of in vitro immunization or from an immunized animal as described above may be immortalized by transfection with a virus.

For example, the Epstein Barr virus (EBV; see Glasky and Reading, Hybridoma 8 (4): 377- 389,1989) can transfos-n human B cells. Alternatively, the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma"which secrets monoclonal antibody. Suitable myeloma lines are preferably defective in the construction or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal. Many such myeloma cell lines are well known in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (see Catalogue of Cell Lines & Hybridomas, 6th ed., ATCC, 1988).

CD40 Ligand Activated CD4+ T cells express high levels of a ligand for CD40 (CD40L).

Human CD40L, a membrane-bound glycoprotein, was cloned from peripheral blood T- cells as described in Spriggs et al., J. Exp. Med. 176: 1543 (1992), and in United States Patent Application number 07/969,703, filed October 23,1992, the disclosure of which is incorporated by reference herein. The cloning of murine CD40L is described in Armitage et al., Nature 357: 80,1992. CD40L induces B-cell proliferation in the absence of any co- stimulus, and can also induce production of immunoglobulins in the presence of cytokines. In addition, CD40 ligand-transfected cells can stimulate monocytes to become tumoricidal (Alderson et al., J. Exp. Med. 178: 669,1993).

CD40L is a type II membrane polypeptide having an extracellular region at its C- terminus, a transmembrane region and an intracellular region at its N-terminus. Soluble human CD40L comprises an extracellular region of CD40L (amino acid 47 to amino acid 261) or a fragment thereof that binds CD40 and tranduces a signal thereby. CD40L biological activity is mediated by binding of the extracellular region of CD40L with CD40, and inclues B cell proliferation and induction of antibody secretion (including IgE secretion).

USSN 08/477,733 and USSN 08/484,624 describe the preparation of various soluble, oligomeric forms of CD40L, including a soluble CD40L/Fc fusion protein referred to as CD40L/FC2. CD40L/FC2 contains an eight amino acid hydrophilic sequence described by Hopp et al. (Hopp et al., BiolTechnology 6: 1204,1988; referred to as Fla), an IgG, Fc domain, a linker sequence (described in U. S. Patent 5,073,627), and the extracellular region of human CD40L. Also described is a soluble CD40L fusion protein referred to as trimeric CD40L., which contains a 33 amino acid sequence referred to as a"leucine zipper,"the eight amino acid hydrophilic sequence described by Hopp et al. (supra), followed by the extracellular region of human CD40L. Both oligomeric forms of CD40L induce human B cell proliferation in the absence of any co-stimuli, and (in conjunction with the appropriate cytokine) result in the production of IgG, IgE, IgA and IgM. These soluble, oligomeric forms of CD40L will be useful in the present inventive methods, as will other forts of CD40L that can be prepared using known methods of preparing recombinant proteins.

Additional CD40 Binding Proteins Binding proteins may also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes an antibody to CD40. (see James W. Larrick et al., "Polymerase Chain Rection Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells," Biotechnology 7: 934-938, September 1989; Reichmann et al., "Reshaping Human Antibodies for Therapy,"Nature 332: 323-327,1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering," Nature 328: 731-734,1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity,"Science 239: 1534-1536,1988; Chaudhary et al.,"A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin,"Nature 339: 394-397,1989).

Briefly, DNA encoding the antigen-binding site (or CD40 binding domain; variable region) of a CD40 mAb is isolated, amplifie, and linked to DNA encoding another protein, for example a human IgG (see Verhoeyen et al., supra; see also Reichmann et al., supra). Alternatively, the antigen-binding site (variable region) may be either linked to, or inserted into, another completely different protein (see Chaudhary et al., supra), resulting in a new protein with antigen-binding sites of the antibody as well as the functional activity of the completely different protein.

Furthermore, DNA sequences which encode smaller portions of the antibody or variable regions which specifically bind to mammalian CD40 may also be utilized within the context of the present invention. Similarly, the CD40 binding region (extracellular domain) of a CD40 ligand may be used to prepare other CD40 binding proteins. DNA sequences that encode proteins or peptides that form oligomers will be particularly useful in preparation of CD40 binding proteins comprising an antigen binding domain of CD40 antibody, or an extracellular domain of a CD40 ligand. Certain of such oligomer-forming proteins are disclosed in U. S. S. N. 07/969,703; additional, useful oligomer-forming proteins are also disclosed in U. S. S. N. 08/107,353, filed August 13,1993, and in U. S. S. N. 08/145,830, filed September 29,1993.

Once suitable antibodies or binding proteins have been obtained, they may be isolated or purifie by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor

Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques. Recombinant CD40 binding proteins can be prepared according to standard methods, and tested for binding specificity to the CD40 utilizing assays known in the art, including for example ELISA, ABC, or dot blot assays, as well by bioactivity assays such as those described for CD40 mAb.

In vitro and in vivo models Murine models of many infectious human diseases are known in the art. For example, Sher (Imm. Rev. 127: 183 204,1992), discusses mutine models of several different human diseases, including acquired immunodeficiency syndrome (AIDS), toxoplasmosis, leishmaniasis, trypanosomiasis, and shistosomiasis. Nathan (in: Mechanisms of Host Resistance to Infectious Agents, Tumors, and Allografts, R. M.

Steinman and R. J. North, eds., Rockefeller University Press, New York, pp. 165-184, 1986) also reviews the use of mice in the study of various human diseases, and further presents results of studies performed in humans that confirm results first observe in murine models.

Other species also provide useful animal models relevant to HIV infection. For example, Wyand (AIDS Res. and Human Retroviruses 8: 349; 1992) discusses the use of SIV-infected Rhesus monkeys for the preclinical evaluation of AIDS drugs and vaccines.

Simian and feline models (Gardner, Antiviral Res. 15: 267; 1991; Stahl-Hennig et al., AIDS 4: 611; 1990) and murine models (Ruprecht et al., Cancer Res. 50: 5618s; 1990) have been propose for evaluating anti-retroviral therapy.

Macrophages/monoovtes and Dendritic Cells Activated macrophages ingest (phagocytose) microbes, produce and release highly reactive intracellular oxygen species, and secrete various cytokines that upregulate immune and inflammatory responses of the mammal to the microbe or microbes. Activation of macrophages is confirme in vitro by various means involving measuring one or more of these activities.

One of the primary functions of peripheral blood monocytes is to regulate an immune or inflammatory response by synthesis and secretion of an array of biologically

active molecules including enzymes, plasma proteins, cytokines and chemokines. Activated macrophages produce and secrete various cytokines and chemokines, including Interleukin-6 (IL-6), Interleukin-1 a and 13 (IL-la, IL-113), Tumor Necrosis Factor a, (TNF-a), Interleukin-8 (IL-8), Macrophage Inhibitory Peptide-la (MIP-la), Macrophage Inhibitory Peptide-113 (MIP-lB), Interleukin-12 (IL-12) and growth regulatory protein (GRO). These molecules have broad immunoregulatory properties, and are useful in modulating an immune or inflammatory response.

Monocytes/macrophages are believed to be a major target of FIV-1 in vivo, and are thought to play an important role in the persistence of infection, serving as a viral reservoir. Immature monocytes in the blood differentiate and infiltrate tissues as more differentiated macrophages. In culture, blood-derived monocytes mimic this differentiation, enlarging and expressing various enzymes and cell-surface antigens characteristic of macrophages (Kaplan and Gaudernack, J. Exp. Med. 156: 1101; 1982).

Correlating with the differentiation of monocytes/macrophages is the differential expression of chemokine receptors. Freshly isolated monocytes express low levels of CCR5 mRNA that increase after in vitro differentiation. The increase in the expression of CCR5 correlates with susceptibility to infection by macrophage-tropic (M-tropic) strains of Hie-1.

Dendritic cells (DC) are often referred to as professional antigen presenting cells; as such, they play a critical role in the development of an immune response. DC express CD40, and are known to be activated for antigen presentation by binding to CD40 ligand or agonistic CD40 antibodies. These cells are also thought to play a role in HIV infection and in developing an anti-HIV immune response. Similar to monocytes/macrophages, DC may also serve as reservoirs of virus, and may potentiate the infection of T cells by HIV. Various methods for isolation of DC are known in the art, including purification from peripheral blood by elutriation or affinity purification using monoclonal antibodies, isolation from cord blood, or from other DC-rich organs such as spleen, followed by growth under appropriate culture conditions.

Administration of CD40 binding proteins The present invention provides methods of using therapeutic compositions comprising an effective amount of a CD40 binding protein and a suitable diluent and

carrier, and methods for reducing susceptibility to infection by retroviruses, including SV. The use of CD40 binding proteins in conjunction with soluble cytokine receptors or cytokines, or other immunoregulatory molecules is also contemplated. For example, CD40 binding proteins can be used in conjunction with factors that are known to activate monocytes/macrophages, such as granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-y), fusion proteins comprising GM-CSF such as those described in U. S. patent 5,073,627, and Interleukins 2 and 15. The CD40 binding proteins and the factor (s) can either be combine in suitable solution, or can be administered separately.

For therapeutic use, purifie CD40 binding protein is administered to a patient, preferably a human, for treatment in a manner appropriate to the indication. Thus, for example, CD40 binding protein compositions administered to reduce susceptibility to retrovirus infection can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique. Typically, a therapeutic agent will be administered in the form of a composition comprising purifie CD40 binding protein in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.

Ordinarily, the preparation of such CD40 binding protein compositions entails combining the CD40 binding protein with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e. g., sucrose) as diluents.

Appropriate dosages can be determined in trials, first in an appropriate animal model, and subsequently in the species to be treated. The amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the individual being treated, and so forth. Typically, therapeutically effective dosages of CD40 binding proteins will be in the range of from about 0.01 to about 1 mg/kg body weight.

The relevant disclosures of all references cited herein are specifically incorporated by reference. The following examples are intended to illustrate particular embodiments, and not limit the scope, of the invention.

Example 1 This example describes the preparation of cells used to determine the effects of soluble, oligomeric CD40 ligand (CD40L) on HIV replication. Monocytes were isolated from fresh peripheral blood mononuclear cells (PMBC) by adherence to plastic at 37°C.

Following one hour of culture, the adherent cells were washed extensively with medium and then removed from the flask by gentle scraping. Residual T-and B-lymphocytes were further depleted using anti-CD2 and anti-CD19 coated beads (Dynal Inc.). These monocytes were typically 95% CD14 positive as assayed by flow cytometry. Mature macrophages were derived by culturing purifie monocytes 7 days in RPMI 1640 containing 10% fetal calf serum (FCS) in 5% C02 incubator at 37°C with or without GM- CSF. At this time, 95% of the cells had become enlarged or spindle shaped with extended processes.

For RNA extraction and RT-PCR analysis of chemokine receptors of freshly isolated monocytes, cells were further purifie using immunomagnetic microbeads conjugated with monoclonal anti-CD14 antibody (MACS Magnetic Cell Sorting Kit, Miltenyi Biotec Inc.). Monocyte purity was assessed by staining with anti-CD14 and by forward and side scatter measurement on flow cytometry. By this analysis the cell populations appeared to contain >99% monocytes.

Example 2 This example describes an immunofluorescence assay for analyzing cell purity and the presence or absence of selected cell surface markers. Monocytes/macrophages were dislodged from plates by treatment with PBS/0. SmM EDTA and incubated at room temperature for 15 minutes with PBS/4% Human AB Serum to block Fc receptors. The cells were then incubated for 15 minutes on ice in the presence of an appropriate dilution (in PBS containing 2% Human AB serum) of the following monoclonal antibodies

(mAb): anti-CD4 (Leu-3A, Becton Dickinson); anti-CD14 (LeuM3, Becton Dickinson); anti-CXCR4 (12G5, kindly supplie by Dr. J. Hoxie, University of Pennsylvania); anti- CCR5 (2D7 and 2F9, kindly supplie by Dr. C. R. MacKay, LeukoSite, Inc.). Reactivity was compare to an isotype-matched control mAb (Zymed).

After three washes with cold PBS, the cells were incubated for 15 minutes on ice with 0.1 ml of 1: 50 dilution of goat anti-mouse IgG (H+L) F (ab') 2 labeled with phycoerythrin (PE) (Boehringer Mannheim). After three additional washes with PBS, cells were resuspended in PBS containing 4% formaldehyde and then analyzed by FACS on a Becton Dickinson FACSsort. For each determination 10,000 cells were analyzed.

Fluorescence was analyzed by using the CellQuest software (Becton Dickinson). To evaluate the level of nonspecific binding of goat anti-mouse IgG to monocytes- macrophages, cells were incubated with an isotype matched IgG control mAb before adding the secondary PE-conjugated antibody and processed as indicated above.

A marked decrease was observe in the expression of the chemokine receptor CXCR4 and CD4 after 24 hours of culture, and by day 7 of culture, CXCR4 and CD4 were undetectable. This finding was reproduced in cells from at least five different donors. Cells of some donors maintained expression of CXCR4, together with CCR5 and CD4, probably due to the culture conditions, cell type or differentiation stage of monocytes/macrophages.

Example 3 This example describes RNA-PCR of chemokine receptor MARNA from cells isolated as described previously. Total cellular RNA was prepared using Triazol (GibcoBRL), treated with RNase-free DNase (Boehringer-Mannheim) and used as a template in RT-PCR. RNA was reverse transcribed in a 20 pI reaction containing 1 pg of total RNA, 1 llg oligo-dT, 200U Superscript reverse transcriptase (GibcoBRL) and 0.5 mM of each dATP, dCTP, dGTP, dTTP. After incubating at 37°C one hour, 1: 10 of the cDNA product was amplifie in a 20 p1 of rection containing 0.1 pg of each primer hybridizing to the 5'-and 3'-ends of the chemokine receptors (CCR1-5 and CXCR4) or to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and IU of a Taq/Pwo polymerase mixture (Boehringer Mannheim).

CCRs were amplifie by denaturing at 94°C, followed by a 30 cycles (94°C, 40 s; 60°C 40 s; 72°, 1 min). To control for contamination of the cellular RNA with genomic DNA, control cDNA rections in which reverse transcriptase was omitted were prepared in parallel. These were uniformly negative. The PCR products were separated by electrophoresis through 1 % agarose. To test for the linearity of amplification, a 10-fold dilution series, starting at lpg of chemokine receptor plasmid DNA was amplifie under conditions identical to those described above.


The CXCR4 MARNA levels, although still high in cells after 24 hours of cultivation, decreased upon in vitro differentiation. In contrast, the CCR5 mRNA levels, barely detectable in freshly isolated monocytes, increased after in vitro differentiation, consistent with the higher infectability of these cells by M-tropic FIV-1 isolates.

CCR2A, CCR2B and CCR3 were up-regulated following 24 hours adhesion to plastic; MARNA levels for these chemokine receptors remained high until day 3, at which time they dropped dramatically (CCR3 MARNA was still detectable). In contrast, CCR1 is constitutively expressed in both freshly isolated and cultured monocytes, while CCR4 is not. These results suggest that CXCR4 and CCR5 receptors are differently regulated during macrophage differentiation. Thus, CCR5 mRNA and protein levels increase upon cultivation, allowing only the M-tropic strains of FIV-1 to enter these cells.

Example 4 This example describes the preparation of luciferase reporter viruses and an infectivity assay designed to measure the ability of the viruses to infect cells. NL4-3-Luc- R-E-virus stocks pseudotyped by various Envs are generated by transfecting 293T cells as described previously (Connor et al., Virology 206: 936; 1995) with 10 pg each of NL4- 3-Luc-R-E-and pcDNA-I/AMP-based expression vectors (Invitrogen) encoding FIV-1 JRFL, ADA, HXB2 or amphotropic MLV (A-MLV) Env. Virus-containing supernatants are collecte 48 hours post-transfection and frozen at-70°. Viruses are quantifie by p24 gagELISA.

For single cycle infections with NL-Luc viruses pseudotyped with different Envs, lXl06 cells in 24-well plates, stimulated as described for individual experiments, are infecte for four hours with 100 ng of p24 gag of virus. This amount of virus is equivalent to 5-7 x10'TCmso (determined by limiting dilution analysis of the virus stocks on activated PBMC) corresponding to an MOI of about 0.4. After the four hour period, cells are washed again, and cultured for an additional eight days. At designated time points the cells are lysed in 200 VI lysis buffer (Promega) and stored at-70°C. Luciferase activity is determined in 20 p1 of each lysate using commercially available reagents (Promega) in a Lumat LB 9501 luminometer.

In one such experiment, cells were stimulated with CD40L (300 ng/ml), CD40L + GM-CSF (300 ng/ml and 1,000 U/ml, respectively) or SDF-la (10 nM) for 7 days.

Luciferase activity was high for both the M-tropic and amphotropic FIV-1 reporter viruses in the absence of CD40L. However, in the presence of CD40L, the luciferase activity was lower for FIV-1 reporter viruses pseudotyped by HIV-1, suggesting that CD40L suppresses FIV-1 entry by down regulating the MARNA and protein levels of CCR5. In addition, the luciferase activity for the amphotropic reporter virus was also depressed, suggesting that CD40L treatment of macrophages/monocytes may also result in a post-entry block of replication of retroviruses.

In another experiment, monocytes were treated with GM-CSF (either 100 U/ml or 10 U/ml; Immunex Corporation, Seattle, WA), CD40L (100 ng/ml), or a combination of GM-CSF and CD40L. The cells were stimulated with GM-CSF for 72 hours, then with

CD40L for 48 hours, analyzed by FACS for expression of chemokine receptors, and subjected to infectivity assay. In addition, supernatant fluids were harvested and assayed for the presence of chemokines. Results of the infectivity assay are shown in Table 1 below.

Table 1: Effect of Cytokines on Entry and Replication of FIV-1 Luciferase Pseudotype Viruses GM-CSF, 100 U/ml JRFL 4,45 1 A-MLV 11,818 No ENV 40 GM-CSF, 10 U/ml JRFL 6,495 A-MLV 7,497 No ENV 15 CD40L JRFL 4,273 A-MLV 11,051 No ENV 21 GM-CSF, 100 U +CD40L JRFL 267 A-MLV 1474 No ENV 16 GM-CSF, 10 U, + CD40L JRFL 2,530 A-MLV 9,334 No ENV 15 * Luciferase activity, in counts per second Moreover, as shown in Figure 1, the combination of CD40L and GM-CF induced production of greater amounts of several chemokines than either cytokine alone. In addition, FACS analysis indicated that the chemokine receptor CCR5 was down-regulated in cells treated with GM-CSF, as wells as those treated with CD40L.

Example 5 Monocytes were obtained, and stimulated with two different preparations of CD40L, in the presence or absence of a neutralizing monoclonal antibody to CD40L (M90; Immunex Corporation, Seattle, WA), for seven days. The cells were then infecte as previously described, and the effect of CD40L on infectivity assessed by luciferase assay. Results are shown in Figure 2. Both preparations of D40L inhibited replication of the JRFL pseudotyped virus; the inhibition was reverse by the addition of neutralizing antibody to CD40L.

In addition, the cells were analyzed for expression of various surface markers, including chemokine receptor CCR5. Both preparations caused a decrease in the expression of CCR5. These results indicate that CD40L may decrease the replication of retroviruses that use CCR5 as a co-receptor for infection of macrophages/monocytes in several ways: increase in chemokine production will result in a block of viral entry because the necessary co-receptors are not available for viral binding; down-regulation of the CCR5 chemokine receptor further decreases the number of available co-receptor molecules. In addition, CD40L, alone or in combination with GM-CSF, may induce a post-entry block of replication, as shown by the decrease in infectivity of the amphotropic murine retrovirus in previous Examples.

Example 6 Peripheral blood mononuclear cells were obtained, and stimulated with phytohemaglutinin (PHA) for 72 hours. The cells were then washed, and medium containing Interleukin-2, CD40L, or IL-2 + CD40L, was added and the cells were incubated for 24 or 48 hours. At these time points, the cells were infecte with FIV-1 Luciferase pseudotyped with JRFL or A-MLV or no ENV as described above. After five days, the cells were harvested and a luciferase assay was performed. The results for 48

hour time point are shown in Table 2 below; similar results were observe for the 24 hour time point.

Table 2: Effect of Cytokines on Entry and Replication of HIV-1 Luciferase Pseudotype Viruses in PBMC JRFL IL-2 7,473 IL-2/CD40L 44 CD40L 1,237 A-MLV IL-2 43,666 IL-2/CD40L 1533 CD40L 52,890 No ENV IL-2 38 IL-2/CD40L 13 CD40L 11 These results demonstrate that CD40L causes a reduction in JRFL entry, and that the combination of IL-2 and CD40L provides a powerful method of inhibiting FIV-1 entry and replication. Because IL-15 shares many activities with IL-2, similar results would also be expected from this combination of cytokines.

Example 7 This example demonstrates that CD40L has similar effects on chemokine and chemokine receptor expression in dendritic cells (DC) to those observe for monocytes.

Peripheral blood mononuclear cells are obtained, and cultured for five to seven days in GM-CSF and IL-4. Both monocytic cells and DC (cultured in GM-CSF and IL4) exhibit

high levels of CCR5. When CD40L is added to the culture for the final 24 to 48 hours, the levels of CCR5 are significantly decreased. Moreover, CD40L-stimulated DC also express high levels of the 13-chemokines MIP-la, MIP-113, and RANTES. CD40L also results in diminished levels of some strains of HIV (as measured by analyzing reverse transcriptase (RT) activity) in co-cultures of T cells and DC infecte with the BAL strain of HIV. However, in T cell/DC co-cultures infecte with the EB strain of HIV, CD40L did not result in decreased levels of RT activity; CD40L stimulation appeared to result in slightly higher RT activity in these co-cultures.

Example 8 This example demonstrates that CD40L inhibits replication of simian immunodeficiency virus (SIV) in rhesus macaque cells. Peripheral blood mononuclear cells were obtained, and stimulated with CD40L, which induced proliferation in a dose- dependent fashion. Viral antigen production by SIVmac239-infected PBMCs was reduced by about 70% (2712 719 pg/ml; p<0.05) in PBMCs activated in the presence of CD40L (50, ug/ml). RT-PCR of total RNA from the PBMCs showed a 50.4% reduction in the expression of SIV gag and a 226% increase in the expression of Interleukin-16 (IL- 16) MARNA. The viral inhibition was not due to increased secretion of IL-16, as supernatant fluids from CD40L-stimulated and control cultures contained similar amounts of IL-16 (26.4 2.5 versus 25.3 1.7 pg/ml, respectively). Thus, CD40L leads to diminished SIV replication in rhesus macaque cells in a manner that involves IL-16 MARNA expression.