JP2001526241A - Methods for reducing susceptibility to HIV infection - Google Patents

Abstract

(57) Abstract: A method for reducing the susceptibility of cells to infection by a retrovirus, comprising reducing the expression of the chemokine receptor CCR5 and / or invading the retroviral replication. Disclosed are methods that include contacting with an effective amount of a CD40 binding protein to induce blockade. This method is useful in treating individuals infected with, or at risk of becoming infected with HIV. CD40 binding proteins include CD40 ligand, monoclonal antibodies that specifically bind CD40, and combinations thereof. The CD40 binding protein can also be used in combination with other cytokines including GM-CSF, IL-2 and IL-15.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field of the Invention The present invention provides a method of reducing the cells susceptible to infection by human immunodeficiency virus (HIV), is contacted with the cells an effective amount of a biologically active CD40 binding proteins And a method comprising:

[0002] cluster of the background differentiation antigen CD4 of the invention is originally human immunodeficiency virus (HIV) has been identified as a receptor for use in binding to cells (Dalgleish et al., Nature 312: 76
3; 1984). Subsequently, it became apparent that an additional molecule, called a coreceptor, was required for the virus to enter cells (Maddon et al., Cell 47:
333; 1986). Ultimately, it has become clear that certain chemokine receptors can act as coreceptors for HIV. Among these is CCR5, a chemokine receptor that binds MIP1α, MIP1β and RANTES. CCR5 antagonists, analogs of the chemokine RANTES, have been shown to block HIV infection and may be useful in preventing HIV infection (Simmons et al., Science 276: 276; 1997).

[0003] CD40L is a type II membrane polypeptide having an extracellular domain at the C-terminus, a transmembrane domain, and an intracellular domain at the N-terminus. Soluble CD40L comprises the extracellular region of CD40L (amino acids 47 to 261 of human CD40L) or fragments thereof. The biological activity of CD40L includes the extracellular region of CD40L and CD40
And its activities include the induction of B cell proliferation and antibody secretion (including IgE secretion). Soluble oligomeric CD40L has been shown to induce the production of chemokines MIP-1α, MIP-1β and RANTES from monocytes / macrophages.

[0004] Thus, CD40L (and other CD40 binding proteins that bind CD40 and trigger the secretion of these chemokines) stimulates the production of chemokines that bind to CCR5, whereby HIV binds CCR5 to the coreceptor. By preventing the cells from becoming susceptible to HIV infection. However, prior to the present invention, it was not known that CD40 binding proteins such as CD40L could down-regulate CCR5 expression and render macrophages intolerant to HIV infection.

[0005] Recombinant soluble forms of the Summary of the Invention CD40L is surface expression of CCR5 on monocytes is downregulated, induces chemokine MIP-l [alpha], the expression of MIP-l [beta] and RANTES. CD
Macrophages induced by monocytes stimulated by 40L are infected with HIV, possibly C
It is not tolerant of down-regulation of CR5 and / or post-entry block of viral replication. Blood monocytes in contact with CD40L appear to be less likely to become infected with macrophage chemotactic virus, and in addition to T cells, monocytes also have macrophage chemotactic HIV-
Bystander cells by producing chemokines that antagonize one replication
) Can be protected.

[0006] The present invention is a method of reducing the susceptibility of a cell to HIV infection, the method comprising down-regulating the expression of CCR5 by the cell or converting the cell to HI.
The method comprises contacting with a CD40 binding protein in an amount effective to render intolerant to V infection. A CD40 binding protein is a pharmaceutical composition capable of binding CD40 and transmitting a biological signal. CD4
The 0 binding protein is selected from the group consisting of a CD40 ligand, a monoclonal antibody that specifically binds CD40, and combinations thereof.

[0007] The invention relates to a method of reducing the susceptibility of the cells to HIV infection, can be transmitted to the cell, bind CD40, and the biological signals to CD40 expressing cells And B. contacting with a CD40 binding protein.
Binding of the CD40 binding protein results in down-regulation of the chemokine receptor CCR5, which reduces the amount of CCR5 available to serve as a coreceptor for HIV to enter cells. Alternatively, or in addition to this effect, the CD40 binding protein may cause a post-entry block of HIV replication. Also CD
The 40 binding protein stimulates cells to secrete chemokines, which may protect bystander cells from HIV infection by antagonizing the use of chemokine receptors as viral co-receptors.

[0008] The findings described herein also provide data that allow a method of reducing an individual's susceptibility to HIV infection by administering to the individual a pharmaceutical composition comprising a substance having CD40 binding protein activity. provide.

[0009] The CD40 human CD40 antigen (CD40) has amino acids 22 with a molecular weight of 30,600.
Seven peptides (Stamenkovic et al., EMBO J. 8: 1403, 1989). Human CD40
CDNA encoding cDN was prepared from Burkitt's lymphoma cell line Raji
A library isolated. The putative protein encoded by this CD40 cDNA contains a putative leader sequence, a transmembrane domain and some of the 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.

[0010] CD40 is a member of the tumor necrosis factor (TNF) / nerve growth factor (NGF) receptor family, which is defined by the presence of a cysteine-rich motif in the extracellular domain (Smith et al., Science 248: 1019, 1990; Mallett and Barcla
y, Immunology Today 12: 220; 1991). This family includes the lymphocyte antigens CD27 and CD30 (antigens found in Hodgkin's lymphoma and Reed-Sternberg cells)
Two receptors for TNF, a murine protein called 4-1BB, rat O
X40 antigen, NGF receptor, and Fas antigen are included. CD40 is functionally expressed on monocytes / macrophages, B cells, lymphoma cells, carcinoma cells, dendritic cells, and vascular endothelial cells.

[0011] CD40 can be detected on the surface of cells by any one of several means known in the art. For example, an antibody specific for CD40 is detected using a fluorescence-activated cell sorting technique (fluorescence-activated cell sorting technique).
e) can be used to determine whether the cells express CD40. Other methods of detecting cell surface molecules are also useful in detecting CD40.

CD40 Monoclonal Antibody Monoclonal antibody against the CD40 surface antigen (CD40 mAb) is a human B
It has been shown to mediate various biological activities on cells. For example, CD
The 40 mAb induces homotypic and heterotypic adhesion (Barrett et al., J. Immunol. 146: 1722, 1
991; Gordon et al., J. Immunol. 140: 1425, 1988), increasing the size of cells (Go
rdon et al., J. Immunol. 140: 1425, 1988; Valle et al., Eur. J. Immunol. 19: 1463, 19
89). In addition, CD40 mAb was used as an 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; AJ McMichael, Oxford University
Press. Oxford, p.426; Paulie et al., J. Immunol. 142: 590, 1989), or with IL-4 (Valle et al., Eur. J. Immunol. 19: 1463, 1989; Gordon et al., Eur. J. Imm
unol. 17: 1535, 1987). Induces proliferation of activated B cells and stimulates IgE from T cell depleted clusters stimulated with IL-4 (Jabara et al., J. Exp. Med. 172: 1861, 199).
0; Gascan et al., J. Immunol. 147: 8, 1991), IgG, and IgM (Gascan et al., J. Immunol.
Immunol. 147: 8, 1991).

[0013] In addition, CD40 mAb is capable of IL-4 mediated soluble CD23 / Fc from B cells.
Enhances εRII release (Gordon and Guy, Immunol. Today 8: 339, 1987; Cairns et al., Eur. J. Immunol. 18: 349, 1988) and promotes B-cell IL-6 production (Clar
k and Shu, J. Immunol. 145: 1400, 1990). Recently, in the presence of CD w32 + adherent cells, the human B cell line has been transformed into IL-4 and CD40 mA.
b, produced from a primitive B cell population (Banchereau et al., Science 241: 70,
1991). Furthermore, apoptosis of germinal center central cells can be prevented if they are activated at the receptor for CD40 and / or antigen (Liu et al., N.
ature 342: 929, 1989). Each of the above publications describes a CD40 mAb that stimulates B cell biological activity.

USSN 08 / 130,541, filed October 1, 1993, the relevant disclosure of which is incorporated by reference, discloses two monoclonal antibodies that specifically bind CD40, termed hCD40m2 and hCD40m3. I do. Unlike other CD40 mAbs, hCD40m2 (ATCC HB11459) and hCD40m3 bind CD40 and convert C40 into cells that constitutively express CD40L.
Inhibits D40 binding. Irrelevant IgG or control CD40 mAb, G28.5
12.5 μg / ml for hCD40m2 or CD40 mAb M3 compared to
At concentrations as low as> 95% inhibition of binding was observed. Also, hCD40m
2 was also able to inhibit CD40L-induced TNF-α production.

[0015] Additional CD40 monoclonal antibodies can be produced using conventional techniques (US Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411).
No., 993, which are incorporated herein by reference;
clonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, P
lenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies
: A Laboratory Manual, Harlow and Lane (eds.). Cold Spring Harbor Labora
See also tory Press, 1988 (also incorporated herein by reference).
.

[0016] Briefly, forms of C suitable for generating an immune response against CD40
Animals are injected with D40. The animals can be immunized as needed until the concentration of serum antibodies to CD40 reaches a plateau, and then sacrificed 3-4 days after a final boost with soluble CD40. Organs containing a large number of B cells, such as spleen and lymph nodes, are harvested and dissected by passing them through a mesh screen or by disrupting the membrane of the spleen or lymph nodes that enclose the cells, resulting in a single cell suspension. Use as a suspension.

[0017] Alternatively, cells suitable for preparing monoclonal antibodies are obtained by using in vitro immunization techniques. Briefly, animals are sacrificed to remove spleen and lymph node cells. A single cell suspension is prepared and placed in a culture containing CD40 in a form suitable to generate the immune response. Subsequently, the lymphocytes are harvested and fused as follows.

[0018] Cells obtained from animals immunized as described above or using immunization as described above can be immortalized by transfecting with a virus.
For example, Epstein-Barr virus (EBV; Glasky and Reading, Hybrido)
ma 8 (4): 377-389, 1989) can transform human B cells. Alternatively, the harvested spleen and / or lymph node cells are fused with appropriate myeloma cells to produce "hybridomas" that secrete monoclonal antibodies. Suitable myeloma lines are preferably defective in structure and expression of the antibody, and additionally cooperate with cells from the immunized animal. Many such myeloma cell lines are known in the art and include the American Type Culture Collection (ATCC), Rockville, MD.
(Catalogue of Cell Lines & Hybridomas, 6t
h ed., ATCC, 1988).

CD40 ligand activated CD4 + T cells express high levels of ligand for CD40 (CD40L). Human CD40L, a membrane-bound glycoprotein, is available from Spriggs et al., J. Exp. Med.
176: 1543 (1992) and U.S. Patent Application 07 / 969,70 filed October 23, 1992.
No. 3 (the disclosure of which is incorporated herein by reference) was cloned from peripheral blood T cells. Murine CD40L is cloned by Armitag
e et al., Nature 357: 80,1992. CD40L can induce B cell proliferation in the absence of any costimulation and can also induce immunoglobulin production in the presence of cytokines. In addition, cells transfected with the CD40 ligand can stimulate monocytes to become tumoricidal (Alderson et al., J. Exp. Med. 1
78: 669, 1993).

CD40L is a type II membrane polypeptide having an extracellular region at the C-terminus, a transmembrane region, and an intracellular region at the N-terminus. Soluble human CD40L is CD40
The extracellular region of CD40L (amino acid 47) that binds and thereby signals
Or amino acid 261) or a fragment thereof. The biological activity of CD40L is mediated by binding of the extracellular region of CD40L to CD40, and its activities include B cell proliferation and 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 called CD40L / FC2. CD40L / FC2 is an eight amino acid hydrophilic sequence described by Hopp et al. (Hopp et al., Bio / Technology 6: 1204, 1988; Fl.
ag®), an IgG ₁ , an Fc domain, a linker sequence (described in US Pat. No. 5,073,627), and the extracellular region of human CD40L. Also described is a soluble CD40L fusion protein called trimer CD40L, which contains a 33 amino acid sequence called "leucine zipper", an 8 amino acid hydrophilic sequence described by Hopp et al. It subsequently contains the human CD40L extracellular region. Both oligomeric forms of CD40L trigger the expansion of human B cells in the absence of any costimulation, resulting in the production of IgG, IgE, IgA and IgM (with appropriate cytokines). These CD40L
Are useful in the methods of the invention, as are other forms of CD40L that can be prepared using known methods for preparing recombinant proteins.

Further CD40 binding proteins Binding proteins can also be constructed using recombinant DNA technology to incorporate the variable region of a gene encoding an antibody to CD40 (James W. Larri).
ck et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human M
onoclonal Antibody Variable Region Genes From Single Hybridoma Cells, "B
iotechnology 7: 934-938, September 1989; Reichmann et al., "Reshaping Human An
tibodies 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: 1534-1
536, 1988; Chaudhary et al., "A Recombinant Immunotoxin Consisting of Two Ant
ibody Variable Domains Fused to Pseudomonas Exotoxin, "Nature 339: 394-39
7, 1989).

Briefly, DNA encoding the antigen binding site (or CD40 binding domain; variable region) of the CD40 mAb is isolated, amplified, and ligated to another protein, such as DNA encoding human IgG ( Verhoeyen et al., Supra; see also Reichmann et al., Supra). Alternatively, the antigen binding site (variable region) can be linked to or inserted into another completely different protein (see Chaudhary et al., Supra), so that, in addition to the antigen binding site of the antibody, Novel proteins are produced with completely different protein functional activities.

In addition, DNA sequences encoding smaller portions of an antibody or variable region that specifically bind to mammalian CD40 can be utilized within the context of the present invention. Similarly, using the CD40 binding region (extracellular domain) of the CD40 ligand
Forty binding proteins can be prepared. DNA sequences encoding proteins or peptides that form oligomers are particularly useful in preparing CD40 binding proteins that include the antigen binding domain of a CD40 antibody, or the extracellular domain of a CD40 ligand. Certain of such oligomer-forming proteins are described in US
Further useful oligomer-forming proteins are also disclosed in US Ser. No. 08 / 107,353, filed Aug. 13, 1993, and Sep. 2, 1993.
It is disclosed in USSN 08 / 145,830 filed on the 9th.

Once suitable antibodies or binding proteins are obtained, they can be isolated or purified by a number of techniques known to those skilled in the art (Antibodies: A Laborato
ry Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,
1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, protein A or protein G.
Includes column purification, or any combination of these techniques. Recombinant C
D40 binding proteins can be prepared according to standard methods, eg, ELISA
Can be tested for binding specificity to CD40 using assays known in the art, including, but not limited to, the ABC, or dot blot assays, as well as by bioactivity assays such as those described for the 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) describes several immune systems including acquired immunodeficiency syndrome (AIDS), toxoplasmosis, leishmaniasis, trypanosomiasis, and shistosomiasis. It discusses murine models of different human diseases. Nathan (Mechanisms of Host Resistance to Infections Agents, Tu
mors, and Allografts, RM Steinman and RJ North, eds., Rockefeller Un
iversity Press, New York, pp. 165-184, 1986) also outlines the use of mice in the study of various human diseases, and was also performed in humans, first confirming the results observed in murine models. Presents the results of the studies conducted.

[0027] Other species also provide useful animal models involved in HIV infection. For example, Wyand (AIDS Res. And Human Retroviruses 8: 349; 1992) discusses the use of SIV-infected rhesus monkeys for preclinical evaluation of AIDS drugs and vaccines. Monkey and cat models (Gardner, Antiviral Res. 15: 267; 1991; Stahl-Hennig et al., AIDS 4: 611;
1990) and a murine model (Ruprecht et al., Cancer Res. 50: 5618s; 1990) have been proposed for the evaluation of antiretroviral therapy.

Macrophages / Monocytes and dendritic cell- activated macrophages take up microorganisms (phagocytosis), produce and release highly reactive intracellular oxygen species, and treat mammals against the microorganism (s). Secretes a variety of cytokines that up-regulate the immune and inflammatory response of the cell. Activation of macrophages is confirmed in vitro by a variety of means, including measuring one or more of these activities.

One of the primary functions of peripheral blood monocytes regulates immune and inflammatory responses by synthesizing and secreting a range of biologically active molecules, including enzymes, plasma proteins, cytokines and chemokines. That is. Activated macrophages include interleukin-6 (IL-6), interleukin-1α and β (IL-1α, IL-1
β), tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), macrophage inhibitory peptide-1α (MIP-1α), macrophage inhibitory peptide-1β (MIP-1β), interleukin-12 ( Produces and secretes various cytokines and chemokines, including IL-12) and growth regulatory proteins (GRO). These molecules have a wide range of immunomodulatory properties and are useful in modulating immune or inflammatory responses.

[0030] Monocytes / macrophages are believed to be a major target of HIV-1 in vivo and play an important role in sustaining infection and are thought to serve as viral reservoirs. Immature monocytes in the blood differentiate and infiltrate tissues as more differentiated macrophages. In culture, monocytes derived from blood mimic this differentiation and augment and express various enzymes and cell surface antigens characteristic of macrophages (Kaplan and Gaudernack, J. Exp. Med. 156: 1101; 1982). ). Correlated with monocyte / macrophage differentiation is the differential expression of chemokine receptors. Freshly isolated monocytes express low levels of CCR5 mRNA, which increases after in vitro differentiation. Increased expression of CCR5 correlates with susceptibility to infection by a macrophage chemotactic (M chemotaxis) strain of HIV-1.

[0031] Dendritic cells (DCs) are often specialized antigen-presenting cells (DCs).
As such, they play a crucial role in the development of the immune response. DCs express CD40 and prepare CDs for antigen presentation
It is known to activate by binding to 40 ligand or agonist CD40 antibodies. These cells are also believed to play a role in HIV infection and the development of anti-HIV immune responses. Like monocytes / macrophages, DC may also serve as a reservoir for the virus and allow infection of T cells by HIV. Various methods for isolating DC are known in the art, including purification from peripheral blood by elutriation or affinity purification using monoclonal antibodies, umbilical cord blood or other DC-rich organs. For example, isolation from spleen followed by growth under appropriate culture conditions.

Administration of CD40 Binding Protein The present invention provides a method of using a therapeutic composition comprising an effective amount of a CD40 binding protein and a suitable diluent and carrier, and to reduce susceptibility to infection by a retrovirus, including HIV. Provide a way. The use of CD40 binding proteins with soluble cytokine receptors or cytokines, or other immunomodulatory molecules, is also contemplated. For example, CD40 binding proteins are known to activate monocytes / macrophages, such as granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-γ), GM-CSF
Fusion proteins including, for example, those described in US Pat. No. 5,073,627, and interleukins 2 and 15. The CD40 binding protein and the factor (s) can be combined in a suitable solution or administered separately.

For therapeutic use, the purified CD40 binding protein is administered to a patient, preferably a human, for treatment in a manner appropriate for the indication. Thus, for example, a CD40 binding protein composition that is administered to reduce susceptibility to retroviral infection can be administered by bolus injection, continuous infusion, delayed release from an implant, or other suitable technique. . Typically, the therapeutic will be administered in the form of a composition comprising the purified CD40 binding protein together with a physiologically acceptable carrier, excipient or diluent. Such carriers are non-toxic to recipients at the dosages and concentrations employed.

Usually, the preparation of such a CD40 binding protein composition involves adding the CD40 binding protein to a buffer, an antioxidant (eg, ascorbic acid), a low molecular weight (less than about 10 residues) polypeptide, protein, amino acid, It involves combining with carbohydrates (including glucose, sucrose or dextrin), chelating agents (eg, EDTA), glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with allogeneic serum albumin are exemplary, suitable diluents.
Preferably, the product is formulated as a lyophilizate using a suitable excipient solution (eg, sucrose) as a dilution station.

The appropriate dosage can be determined initially in animal models and then in trials with the species to be treated. The amount and frequency of administration will, of course, depend on factors such as the nature and severity of the condition being treated, the desired response, the condition of the individual being treated.
Typically, a therapeutically effective dose of a CD40 binding protein will range from about 0.01 to about 1 mg / kg of body weight.

The relevant disclosures of all references cited herein are expressly incorporated by reference. The following examples are intended to illustrate certain aspects of the present invention, but not to limit its scope. Example 1 In this example, a soluble oligomeric CD40 ligand (CD
40L) describes the preparation of cells used to determine the effect. Monocytes were isolated from fresh peripheral blood mononuclear cells (PMBC) by attaching to plastic at 37 ° C. After culturing for 1 hour, the adherent cells were thoroughly washed with medium and then removed from the flask by gentle scraping. T and B remaining
Lymphocytes were further depleted using anti-CD2 and anti-CD19 coated beads (Dynal Inc.). These monocytes were typically analyzed by flow cytometry at 9%.
It was 5% CD14 positive. Purified monocytes were treated with 10% fetal bovine serum (FC
Mature macrophages were induced by culturing in RPMI 1640 containing S) in a 5% CO ₂ incubator at 37 ° C. with or without GM-CSF. At this point, 95% of the cells had expanded or formed spindles with extended protrusions.

For RNA extraction and RT-PCR of chemokine receptors on newly isolated monocytes, immunomagnetic microbeads conjugated with monoclonal anti-CD14 antibody (MACS Magneti
c The cells were further purified using the Cell Sorting Kit, Miltenyi Biotec Inc.).
Monocyte purity was assessed by staining with anti-CD14 and by forward and side scatter on flow cytometry. By this analysis, these cell populations were> 9
It appeared to contain 9% 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 converted to PBS / 0.5 mM E
Plates were removed by treatment with DTA and incubated for 15 minutes at room temperature with PBS / 4% human AB serum to block Fc receptors. The cells were then incubated for 15 minutes on ice in the presence of appropriate dilutions of the following monoclonal antibodies (mAbs) in PBS with 2% human AB serum: anti-CD4 (Leu-3A, Becton) Dickinson); anti-CD14 (LeuM3
Becton Dickinson); anti-CXCR4 (12G5, Dr. J. of the University of Pennsylvania).
Courtesy supply from Hoxie); anti-CCR5 (2D7 and 2F9, LeukoSite,
Courtesy of Dr. CR MacKay of Inc.). Reactivity was compared to an isotype matched control mAb (Zymed).

After washing three times with cold PBS, phycoerythrin (PE) (Boehringer Mannhei
The cells were incubated for 15 minutes on ice with 0.1 ml of a 1:50 dilution of goat anti-mouse IgG (H + L) F (ab ') 2 labeled with m). 3 more with PBS
After washing twice, the cells were suspended in PBS containing 4% formaldehyde,
Analyzed by FACS using ecton Dickinson FACSsort. 10,000 cells were analyzed for each determination. CellQuest software (Becton Dic
kinson) was used to analyze the fluorescence. To assess the level of non-specific binding of goat anti-mouse IgG to monocyte-macrophages, cells were incubated with an isotype-matched IgG control mAb before adding a secondary PE-conjugated antibody,
Processed as indicated above.

[0039] A significant decrease in the expression of the chemokine receptors CXCR4 and CD4 was observed after 24 hours of culture, with no detectable CXCR4 and CD4 on day 7 of culture.
This finding has been replicated in cells from at least five different donors. Some donor cells maintained CXCR4 expression along with CCR5 and CD4, probably due to monocyte / macrophage culture conditions, cell type or differentiation stage. Example 3 In this example, chemokine receptor mRNA from cells isolated as described above
RNA-PCR is described. Total cellular RNA was prepared using Triazol (Gibco / BRL), treated with RNase-free DNase (Boehringer-Mannheim), templated and used in RT-PCR. 1 μg total RNA, 1 μg oligo-d
T, 200U Superscript reverse transcriptase (Gibco / BRL) and dATP, dCTP
RNA was reverse transcribed in a 20 μl reaction containing 0.5 mM each of dGTP, dGTP, and dTTP. After incubation at 37 ° C. for 1 hour, 1:10 cDNA product is
0.1 μg each of the primers that hybridize to the 5 ′ and 3 ′ ends of the chemokine receptors (CCR1-5 and CXCR4) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 1 U of a Taq / Pwo polymerase mixture (Boehringer Amplification in a reaction solution containing Mannheim).

After denaturing the CCR at 94 ° C., 30 cycles (94 ° C., 40 seconds;
(0 second; 72 ° C., 1 minute). Cellular RNA in genomic DNA
A control cDNA reaction omitting the reverse transcriptase was prepared in parallel to control contamination of E. coli. These were uniformly negative. PCR products were separated by electrophoresis through 1% agarose. To test the linearity of the amplification, a 10-fold dilution series starting from 1 pg of the chemokine receptor plasmid DNA was amplified under the same conditions as described above.

The sequences of the primers used are as follows: GAPDH 5 ′ CCA TGG AGA AGG CTC
GGG, 3 'CAA AGT TGT CAT GGA TGA CC; CCR1 5' GCC CAG AAA CAA AGA CTT CAC
GG, 3 'GGA ATC CTG GGG AAC ATA GAA C; CCR2A 5' ACG CAT TTC CCC AGT ACA
TC, 3 'TGT GAT TCA AAC GAC ATC AT; CCR2B 5' TGA GAC AAG CCA CAA GCT G, 3
'CTG AAT GCG TGA GCC CTT TCG TC; CCR3 5' TTC TAT CAC AGG GAG AAG TG, 3 '
ACT GGA AGT TTG AAG GAC TGT T; CCR4 5 'GCA AGC TGC TTC TGG TTG GGC CCA
G, 3 'GAA TGT GGA AAA GTT CAT TGA C; CCR5 5' GGT GGA ACA AGA TGG ATT AT, 3 'ATG TGC ACA ACT CTG ACT G; CXCR4 5' AGC GAG GTG GAC ATT CAT C, 3 'A
CG TGA TTC ACT ACA GCT C.

CXCR4 mRNA levels were reduced during in vitro differentiation, although still high in cells after 24 hours of culture. In contrast, levels of CCR5 mRNA, barely detectable in freshly isolated monocytes, increase after in vitro differentiation, which is a factor in the M-taxis HIV-1 isolate of these cells. Consistent with a higher likelihood of infection. CCR2A, CCR2B and CCR3 were up-regulated 24 hours after adhesion to plastic; mRNA levels of these chemokine receptors remained high until day 3, at which time they dropped dramatically (CC
R3 mRNA was still detectable). In contrast, CCR1 is constitutively expressed in both freshly isolated and cultured monocytes, whereas CCR1
CR4 is not expressed. These results indicate that CXCR4 during macrophage differentiation
And that there is a disparity in the regulation of the CCR5 receptor. Therefore, CCR5
mRNA and protein levels are increased during culture, which is due to the HI of the M
Only V-1 allows entry into these cells. Example 4 This example describes the preparation of a luciferase reporter virus and an infectivity assay designed to determine the ability of these viruses to infect cells.
The NL4-3-Luc-RE-virus stocks pseudotyped with various Envs were
HIV-1 JRFL, ADA, HXB2 or eutrophic MLV (A-MLV) E
10 μg of each of the NL4-3-Luc-RE- and pcDNA-I / AMP-based expression vectors (Invitrogen) encoding nv were added as previously described (Co
nnor et al., Virology 206: 936; 1995) by transfecting 293 T cells. Forty-eight hours after transfection, virus-containing supernatants were collected and frozen at -70 ° C. Virus was quantified by p24 ^gag ELISA.

For one cycle infection with NL-Luc virus pseudotyped with different Envs, 1 × in 24-well plates stimulated as described for individual experiments
10 ⁶ cells are infected with 100 ng of the virus p24 ^gag for 4 hours. This amount of virus is equivalent to 5-7 × 10 ⁴ TCID ₅₀ (determined by limiting dilution analysis of virus stocks using activated PBMC), which corresponds to an MOI of about 0.4. After this 4 hour period, the cells are washed again and cultured for a further 8 days. At the indicated time points, cells are lysed in 200 μl lysis buffer (Promega) and stored at −70 ° C. Luciferase activity was determined using 20 μl of each lysate with a commercially available reagent (Pr
omega) using a Lumat LB 9501 luminometer.

In one such experiment, cells were cultured with CD40L (300 ng / ml), C
D40L + GM-CSF (300 ng / ml and 1,000 U / ml, respectively)
) Or SDF-1α (10 nM) for 7 days. Luciferase activity is C
In the absence of D40L, it was higher for both the M-taxis and broad host HIV-1 reporter viruses. However, in the presence of CD40L, luciferase activity was low in the HIV-1 reporter virus _pseudotyped with HIV-1 _JFRL , which indicates that CD40L downregulates CCR5 mRNA and protein levels, thereby invading HTV-1 entry. Suggests that In addition, luciferase activity in the eutrophic reporter virus was also reduced, suggesting that CD40L treatment of macrophages / monocytes could also result in post-entry blockade of retroviral replication.

In another experiment, monocytes were treated with GM-CSF (100 U / ml or 10 U / ml).
ml; Immunex Corporation, Seattle, WA), CD40L (100 n
g / ml) or a combination of GM-CSF and CD40L. The cells were stimulated with GM-CSF for 72 hours, then CD40L for 48 hours, analyzed by FACS for chemokine receptor expression, and subjected to infectivity assays. In addition, supernatants were collected and analyzed for the presence of chemokines. The results of the infectivity assay are shown in Table 1 below.

Table 1: Effect of cytokines on HIV-1 luciferase pseudotyped virus entry and replication GM-CSF, 100 U / ml JRFL 4,451 ^* A-MLV 11,818 NoENV 40 GM-CSF, 10 U / ml JRFL 6,495 A-MLV 7,497 NoENV 15 CD40L JRFL 4,273 A-MLV 11,051 NoENV 21 GM-CSF, 100U + CD40L JRFL 267 A-MLV 1474 NoENV 16 GM-CSF, 10U + CD40L , 334 NoENV 15 ^* per second counts in luciferase activity Furthermore, as shown in FIG. 1, the combination of CD40L and GM-CF is a large amount of than either cytokine alone several chemokine To induce the production of. In addition, FACS analysis showed that GM-CS
The chemokine receptor CCR5 was shown to be down-regulated in cells treated with F. Example 5 Monocytes were obtained and neutralizing monoclonal antibodies against CD40L (M90; Immunex Co.)
(rporation, Seattle, WA) or in the absence of two different preparations of CD40L for 7 days. These cells were then infected as described previously and the effect of CD40L on infectivity was assessed by luciferase assay. The results are shown in FIG. Both preparations of D40L inhibited the replication of JRFL pseudotyped virus; this inhibition was reversed by adding a neutralizing antibody against CD40L.

In addition, cells were analyzed for expression of various surface markers, including the chemokine receptor CCR5. Both preparations resulted in reduced expression of CCR5. These results indicate that CD40L can reduce the replication of retroviruses that use CCR5 as a coreceptor to infect macrophages / monocytes in several ways: increased production of chemokines Because the required co-receptors are not available for virus binding, this results in a blockade of virus entry; down-regulation of the CCR5 chemokine receptor further reduces the number of available co-receptor molecules. in addition,
CD40L, alone or in combination with GM-CSF, can trigger a post-entry block of replication, as indicated by the reduced infectivity of the eutrophic murine retrovirus in the previous example. Example 6 Peripheral blood mononuclear cells were obtained and stimulated with phytohemagglutinin (PHA) for 72 hours.
Next, these cells are washed and interleukin-2, CD40L, or IL-
Medium containing 2 + CD40L was added and cells were incubated for 24 or 48 hours. At this point, these cells are transformed as described above with JRFL or A-MLV or n
Infected with HIV-1 luciferase pseudotyped with oENV. Five days later, these cells were collected and subjected to a luciferase assay. The results at 48 hours are shown in Table 2 below; similar results were observed at 24 hours.

Table 2: Effect of cytokines on HIV-1 luciferase pseudotyped virus entry and replication in PBMCs 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 indicate that CD40L results in reduced JRFL invasion, IL-2 and CD
Figure 4 shows that the combination with 40L provides a powerful method of inhibiting HIV-1 entry and replication. Since IL-15 shares many activities with IL-2, similar results are expected from this combination of cytokines. Example 7 This example shows that CD40L has effects similar to those observed for monocytes on the expression of chemokines and chemokine receptors in dendritic cells (DCs). Peripheral blood mononuclear cells are obtained and cultured in GM-CSF and IL-4 for 5-7 days. Both monocytes and DC (cultured in GM-CSF and IL-4) show high levels of CCR5. When CD40L is added to the culture for the last 24-48 hours, CCR5 levels are greatly reduced. In addition, CD40L-stimulated DC also express high levels of β-chemokines MIP-1α, MIP-1β, and RANTES. CD40L also results in reduced levels of some strains of HIV (as measured by analyzing reverse transcriptase (RT) activity) in co-culture of T cells and DCs infected with HIV of the BAL strain. However, in T cell / DC cocultures infected with HIV of strain IIIB, CD40L does not result in a decrease in the level of RT activity;
Stimulation of CD40L appears to result in slightly higher RT activity in these co-cultures. Example 8 This example shows that CD40L inhibits replication of simian immunodeficiency virus in rhesus monkey cells. When peripheral blood mononuclear cells were obtained and stimulated with CD40L, proliferation was induced in a dose-dependent manner. The production of viral antigens by SIVmac239-infected PBMC was approximately 70% (2712 ± 719 pg / ml; P <0.05) in PBMC activated in the presence of CD40L (50 μg / ml).
5) decreased. In RT-PCR of total RNA derived from PBMC, SIVgag
And a 226% increase in interleukin-16 (IL-16) mRNA expression. Supernatants from CD40L-stimulated and control cultures had similar amounts of IL-16 (26.4 ± 2.5 vs. 25.3 ± 1.7 p, respectively)
g / ml), the inhibition of this virus was not due to increased secretion of IL-16. Thus, CD40L is responsible for reduced replication of SIV in rhesus monkey cells in a manner that involves expression of IL-16 mRNA.

[Brief description of the drawings]

FIG. 1 depicts the results showing that CD40L alone or in combination with GM-CSF induces monocytes to produce various chemokines.

FIG. 2 shows the results showing that CD40L inhibits the replication of JRFL pseudotyped retrovirus.

[Procedure amendment]

[Submission date] June 20, 2000 (2000.6.20)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

6. The pharmaceutical composition according to claim 1, wherein the retroviral infectivity is HIV infectivity.

[Procedure amendment]

[Submission date] March 1, 2001 (2001.3.1)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

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Claims (4)

[Claims] 1. A method for reducing infectivity of a cell expressing a CCR5 chemokine receptor to a retrovirus using the CCR5 chemokine receptor as a co-receptor, the method comprising: expressing the cell expressing the CCR5 chemokine receptor. Contacting with a CD40 binding protein in an amount sufficient to reduce the amount of the CD40 binding protein. 2. The method of claim 1, wherein the CD40 binding protein is a soluble oligomer CD40L. 3. The method of claim 1, wherein the cells are also contacted with a cytokine selected from the group consisting of granulocyte macrophage colony stimulating factor, interleukin-2, interleukin-15, and combinations thereof. 4. The method of claim 2, wherein the cells are also contacted with a cytokine selected from the group consisting of granulocyte macrophage colony stimulating factor, interleukin-2, interleukin-15, and combinations thereof.