WO1990013316A1 - Dissemination of hiv-1 infected cells - Google Patents

Dissemination of hiv-1 infected cells Download PDF

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WO1990013316A1
WO1990013316A1 PCT/US1990/002318 US9002318W WO9013316A1 WO 1990013316 A1 WO1990013316 A1 WO 1990013316A1 US 9002318 W US9002318 W US 9002318W WO 9013316 A1 WO9013316 A1 WO 9013316A1
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cells
cd18
icam
hiv
infected
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PCT/US1990/002318
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French (fr)
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Roger D. Rossen
Donald C. Anderson
C. Wayne Smith
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Baylor College Of Medicine
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546Integrin superfamily
    • C07K14/70553Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/70525ICAM molecules, e.g. CD50, CD54, CD102
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [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
    • C07K16/2845Immunoglobulins [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 against integrin beta2-subunit-containing molecules, e.g. CD11, CD18

Abstract

A therapeutic method for suppressing or preventing the migration of HIV-1 infected cells from the circulatory system by administering an effective amount of an anti-migration agent capable of impairing the ability of the HIV-infected leukocyte to bind the ICAM-1 or a member of the CD11/CD18 family of receptor molecules. Migration of HIV-infected leukocytes often results in the formation of extravascular foci of mononuclear cells. The invention may be used in the treatment of AIDS which is caused by the HIV-1 virus.

Description

- 1 -

TITLE OF THE INVENTION

DISSEMINATION OF HIV-1 INFECTED CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Patent Application Serial No. 07/344,925 (filed April 28, 1989).

FIELD OF THE INVENTION

The invention concerns a therapeutic method for suppressing the migration of HIV-1 infected cells from the circulatory system. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV-1 virus. This invention was supported in part by the U.S. Government. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

It has been determined that in order to properly defend a host against foreign invaders such as bacteria or viruses, leukocytes must be able to migrate from circulation to sites of infection and inflammation. Leukocytes must also be able to attach to antigen- presenting cells so that a normal specific immune response can occur, and finally, they must attach to appropriate target cells so that lysis of virally-infected or tumor cells can occur. Each of these migration processes requires that leukocytes have the ability to adhere to other cells, especially endothelial cells. An excellent review of the properties and characteristics of leukocytes is provided by Eisen, H. ., (In; Microbiology. 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418). -2-

Recently, a family of leukocyte surface molecules involved in mediating such adhesion have been identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T- cells (Davignon, D. et al.. Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells (Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attachment related functions described above (Springer, T. et al.. Fed. Proc. 44.2660-2663 (1985)).

The family of receptor molecules identified by the above- described method has been termed the "CD11/CD18 family of receptor molecules." The receptor molecules of the CD11/CD18 family are heterodimers containing an alpha subunit (CD11) and a beta subunit (CD18) (Sanchez-Madrid, F. et al.. J. Exper. Med. 158:1785-1803 (1983); Keizer, G.D. et al.. Eur. J. Immunol. 15:1142-1147 (1985)).

Three different alpha subunits have been characterized: CDlla (equivalently referred to as the LFA-1 alpha subunit), CDllb (equivalently referred to as the Mac-1 alpha subunit) and CDllc (equivalently referred to as the pl50,95 alpha subunit). The CDlla/CD18 heterodimer is found on most lymphocytes (Springer, T.A., et al. Immunol. Rev. 68:111-135 (1982)). The CDllb/CD18 and CDllc/CD18 heterodimers are found on macrophages, granulocytes and large granular lymphocytes. These three molecules play a role in cellular adhesion (Keizer, G. et al.. Eur. J. Immunol. 15:1142-1147 (1985)). The natural binding ligand for the CD11/CD18 receptor molecules is ICAM-l (Rothlein et al.. J. Immunol. 137:1270 (1986)), European Patent Application Publication No. 289,949, which references are incorporated herein by reference).

The beta chains of the heterodimers share extensive homology. The CD18 molecules were found to have a molecular weight of 95 kd whereas the molecular weights of the alpha chains were found to vary from 150 kd to 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)). Although the alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, close analysis of the alpha subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al.. (J . Exper. Med. 158:586-602 (1983); J. Exoer. Med. 158:1785-1803 (1983)).

A group of individuals has been identified who are unable to express normal amounts of any member of this adhesion protein family on their leukocyte cell surface (Anderson, D.C., et al.. Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et al.. J. Infect. Pis. 152:668- 689 (1985)). Leukocytes from these patients displayed in vitro defects similar to normal counterparts whose LFA-1 family of molecules had been antagonized by antibodies. Furthermore, these individuals were unable to mount a normal immune response due to an inability of their cells to adhere to cellular substrates (Anderson, D.C., et al.. Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et al.. J. Infect. Pis. 152:668-689 (1985)). These data show that immune reactions are mitigated when leukocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the LFA-1 family.

Thus, in summary, the ability of leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells). This adherence has been found to require cell-cell contacts which involve specific receptor molecules present on the cell surface of the leukocytes. These receptors enable a leukocyte to adhere to other leukocytes or to endothelial, and other non-vascular cells. The cell surface receptor molecules have been found to be highly related to one another. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms including defective antibody responses.

The migration and dissemination of leukocytes is important in protecting an individual from the consequences of infection. These processes, however, are also responsible for the migration and dissemination of viral-infected leukocytes. Of particular concern is -4-

the migration and dissemination of leukocytes infected with HIV (human immunodeficiency virus (also known as HTLV-III and LAV)). HIV is a double stranded RNA retrovirus. HIV is the causal agent of AIDS (Acquired Immunodeficiency Syndrome). The virus is believed to cause AIDS by binding to, and infecting, the T cells of a susceptible host. The migration of such cells results in the formation of extravascular foci, and may cause tumors and other abnormalities.

Since leukocyte adhesion is involved in the process through which cellular migration and dissemination occur, an understanding of this process is of significant value in the treatment of viral disease.

SUMMARY OF THE INVENTION

In detail, the invention comprises a method for suppressing the extravascular migration of a virally infected leukocyte in a patient having such a leukocyte, which comprises administering to the patient an effective amount of an anti-migration agent, the agent being capable of impairing the ability of said leukocyte to bind to ICAM-l or to a member of the CD11/CD18 family of receptor molecules.

The invention further comprises the embodiment of the above- described method wherein the virally infected leukocytes are infected with HIV.

The invention further comprises the embodiment of the above- described method wherein the agent is an immunoglobulin, or an antigen binding fragment of an immunoglobulin.

The invention further comprises the embodiment of the above- described method wherein the immunoglobulin is a human, chimeric, or humanized antibody.

The invention further comprises the embodiment of the above- described method wherein the immunoglobulin is a monoclonal antibody.

The invention further comprises the embodiments of the above- described method wherein the immunoglobulin is an antibody to a CPU molecule, a CD18 molecule, ICAM-l or ICAM-2. -5-

The invention further comprises the embodiment of the above- described method wherein the agent is a soluble derivative of ICAM-l, CDlla, CDllb, CDllc, or CD11/CD18.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows cell surface expression of CD18, CDlla and ICAM-l on H9 CD4+ T cells, as determined by flow cytometer analysis. Solid line - non-immune ascites. Dotted line » uninfected H9 cells. Dashed line = HIV-1 infected cells. Note bimodal distribution of CP18, CDlla and ICAM-l on HIV-1 infected cells. 80% of infected cells showed increased cell surface expression of these markers. Fluorescence histograms of uninfected cells exposed to anti-ICAM-1 and non-immune ascites were totally superimposable.

Figure 2 shows flow cytometric analysis of THPl and U937 cells, incubated with monoclonal anti-CP18, CDlla and CDllb, followed by fluorescein conjugated goat anti-mouse IgG. The solid line shows background fluorescence of non-immune ascites. The coarsely dotted line shows fluorescence of uninfected monocytoid cells, the dashed line, fluorescence of cultured cells, >85% of which contained HIV-1, as demonstrated by in situ hybridization. One of four experiments shown, p <.02, analysis of variance, comparing mean channel fluorescence of HIV-1 infected versus uninfected cells in all experiments.

Figure 3 shows cell surface expression of CD18 (left), CDlla, (middle) and CDllb (right) of uninfected THPl cells (open bars) and HIV-1 infected THPl cella (slashed bars) at weekly intervals following inoculation or sham inoculation with HIV1, as determined by glow cytometry.

Figure 4 shows (left panel) that THPl cell surface expression of CD18 (dashed line) is increased on HIV-1 infected as compared to uninfected (dotted line) cells, as shown by flow cytometric analysis. Solid lines indicate baseline fluorescence of cells exposed to non- -6-

immuπe mouse ascites. Expression of CD4 is decreased on HIV-1 infected (middle panel) and ICAM-l is not expressed on either the HIV- 1 infected or the uninfected THPl cells (right panel). F(ab')2 fragments of the anti-ICAM- monoclonal antibody R6-5-D6 were used in these experiments to prevent non-specific binding of this IgG2a monoclonal by high affinity Fc receptors present on THPl cells.

Figure 5 shows the percent adherence of THPl and U937 cells to monolayers of human umbilical vein endotheliu . Slashed bars - percent adherence of HIV-1 infected cells; open bars » percent adherence of uninfected cells of the same line. In each of the experiments shown, infected cells were more adherent (p <.01, paired t test). Preincubation with monoclonal anti-CD18 (TS 1/18) and with anti-CDlla (TS 1/22) significantly suppressed adherence of both HIV- infected and uninfected U937 cells; similarly, preincubation of the endothelial monolayer with the R 6.5-P6 monoclonal anti-ICAM-1, also suppressed adherence of the monocytoid cells.

Figure 6 shows the ho otypic aggregation of THPl cells, cultured in the presence of 50 ng/ml phorbol yristate acetate (PMA). This figure shows results of one of three experiments with equivalent outcomes. Slashed bars show percent of HIV infected cells, open bars, the percent of uninfected THPl cells incorporated in aggregates containing more than 50 cells. Addition of saturating concentrations of the W6/32 monoclonal antibody directed against the framework of class I major histocompatibility antigens (an irrelevant monoclonal antibody (Mab)) had no effect on aggregate formation. Addition of equivalent concentrations of anti-CD18 suppressed aggregation. Anti- CDlla had a similar effect on PMA induced aggregation of uninfected cells, but was less effective in suppressing aggregation of the HIV-1 infected THPl cells. Anti-ICAM-1 had little if any effect on homotypic aggregation of this cell line. -7-

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The CD18 Family of Receptor Molecules

As discussed above, the CD11/CD18 family is comprised of three heterodimers which share a common beta subunit, (CD18) and a distinct α chain: CDlla, (LFA-1 alpha or αL, a 177,000 dalton molecule), CDllb, (Mac-1 alpha, αM, 165,000 daltons) and CDllc (αX or P150,95 alpha, a 150,000 dalton molecule) (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)).

As used herein, the term "CD11/CD18" is intended to generally refer to any and all members of the family of receptor molecules which contain CD18. The term "CDH" is intended to refer to any alpha subunit of the CD11/CD18 family of receptor molecules. In contrast, the terms "CDlla," "CDllb," and "CDllc" are intended to refer to the specific alpha subunit molecule designated (i.e. the LFA-1, Mac-1 and pl50,95 alpha subunits, respectively). The term "CD18" is intended to refer to the beta subunit of the CD11/CD18 family. The terms "CDlla/CD18," "CDllb/CD18," and "CDllc/CD18" are intended to refer to the specific receptor molecule designated (i.e. the LFA-1, Mac-1 and pl50,95 receptor molecules, respectively).

CDllb/CD18 and CDllc/CD18 are found, in various quantities on monocytes, macrophages, and granulocytes (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)); CDlla/CD18 is expressed on lymphocytes, monocytes, granulocytes and large granular lymphocytes (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)).

The importance of the CDlla/CD18 complex in host defense has been illuminated by identification of an autosomal recessive trait characterized by recurrent, severe bacterial infections in which affected individuals are unable to synthesize normal CD18 molecules (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 28:175 (1987)). Leukocytes from such individuals are -8-

unresponsive to stimuli which induce leukocytes to adhere to and move across vascular endothelial cells (C. . Smith et al.. J. Clin. Invest. 82:1746 (1988)).

The CD11/CD18 complex is also involved in other cell-cell interactions involved in host defence against infection, including binding and phagocytosis of iC3b-opsonized particles, a property of CDllb/CD18 on granulocytes and monocytoid cells, and Mg^+-dependent adhesion and killing of target cells by T cells and NK cells, a property of the CDlla/CD18 heteroduplex (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)).

ICAM-l

ICAM-l is a cell surface glycoprotein expressed on non- hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal centered B cells and dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-l is highly expressed on vascular endothelial cells in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-l is expressed in low amounts on peripheral blood lymphocytes. Phorbol ester-stimulated differentiation of some yelomonocytic cell lines greatly increases ICAM-l expression. Thus, ICAM-l is preferentially expressed at sites of inflammation, and is not generally expressed by quiescent cells. ICAM-l expression on dermal fibroblasts is increased threefold to fivefold by either inter!eukin 1 or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible.

ICAM-l displays molecular weight heterogeneity in different cell types with a molecular weight of 97 kd on fibroblasts, 114 kd on the myelomonocytic cell line U937, and 90 kd on the B lymphoblastoid cell JY. ICAM-l biosynthesis has been found to involve an approximately 73 kd intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment (which inhibits glycosylation) has a molecular weight of 55 kd.

ICAM-l isolated from phorbol ester stimulated U937 cells or from fibroblast cells yields an identical major product having a molecular weight of 60 kd after chemical deglycosylation. ICAM-l monoclonal antibodies interfere with the adhesion of phytohe agglutinin blasts to LFA-1 deficient cell lines. Pretreatment of fibroblasts, but not leukocytes, with monoclonal antibodies capable of binding ICAM-l inhibits leukocyte-fibroblast adhesion. Pretreatment of leukocytes, but not fibroblasts, with antibodies against LFA-1 has also been found to inhibit leukocyte-fibroblast adhesion.

ICAM-l is, thus, a binding ligand of the CD 18 complex on leukocytes. It is inducible on fibroblasts and endothelial cells in vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor in a time frame consistent with the infiltration of lymphocytes into inflammatory lesions in vivo (Dustin, M.L., et^. al.. J. Immunol 137:245-254. (1986); Pober, J.S., et. al.. J. Immunol 137:1893-1896, (1986)). Further ICAM-l is expressed on non- hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, other epithelial cells, and fibroblasts and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center B-cells and dendritic cells in tonsils, lymph nodes and Peyer's patches (Dustin, M.L., et. al.. J__ Immunol 137:245-254. (1986)). ICAM-l is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen pianus, exanthema, urticaria and bullous diseases. Allergic skin reactions provoked by the application of a hapten on the skin to which the patient is allergic also revealed a heavy ICAM-l expression on the keratinocytes. On the other hand toxic patches on the skin did not reveal ICAM-l expression on the keratinocytes. ICAM-l is present on keratinocytes from biopsies of skin lesions from various dermatological disorders and ICAM-l expression is induced on lesions -10-

from allergic patch tests while keratinocytes from toxic patch test lesions failed to express ICAM-l.

ICAM-l is, therefore, a cellular substrate to which lymphocytes can attach, so that the lymphocytes may migrate to sites of infection or inflammation.

ICAM-2

A second LFA-1 ligand, distinct from ICAM-l, has been postulated (Rothlein, R. et al.. J. Immunol. 137:1270-1274 (1986); Makgoba, M.W. et al.. Eur. J. Immunol. 18:637-640 (1988); Dustin, M.L. et al.. cY_ Cell. Biol. 107:321-331 (1988)). This second ligand has been designated ICAM-2 (Staunton, D.M. et al.. FASEB J. 3:a446 (1989)).

Characteristics of HIV Infection

HIV infection is believed to occur via the binding of a viral protein (termed "gpl20") to a receptor molecule (termed "CD4") present on the surface of T4 lymphocytes. The virus then enters the cell and proceeds to replicate, in a process which ultimately results in the death of the T cell. The destruction of the T cells results in an impairment in the ability of the infected patient to combat opportunistic infections. Although individuals afflicted with AIDS often develop cancers, the relationship between these cancers and HIV infection is, in most cases, uncertain.

The first symptom of AIDS is typically chronically swollen lymph nodes. This stage of the disease.may last from 3-5 years. During this period the HIV virus replicates and gradually reduces the number of T4 cells which are available to prevent disease. Eventually, the number of T4 cells falls to less than 400 cells/cc.

Generally, 18 months after an individuals T cells have fallen below the 400 cells/cc level, the disease can be shown to have impaired the patient's ability to mount and sustain an immune response -11-

against specific proteins which are injected under the skin (i.e. impairment of the patient's ability to mount a delayed hypersensitivity response). This conditions rapidly worsens until the patient is completely unable to mount a systemic delayed hypersensitivity response. At this stage, patients frequently develop opportunistic infections, such as oral thrush (candidiasis), herpes simplex, cytomegalovirus and molluscum contagiosum infections. AIDS patients generally succumb to the disease within 10 years from infection. The disease and its treatment are reviewed in Scientific American 259:40-134 (1988); Clumeck, N. et al.. Eur. J. Clin. Microbiol. Infect. Pis. 7:2-10 (1988); Waldmann, T.A. et al.. Blood 72:1805-1816 (1988); Sarin, P.S., Ann. Rev. Pharmacol. 28:411-428 (1988); which references are incorporated herein by reference).

Although immunodeficiency associated with CD4+ T cell depletion remains the most obvious consequence of HIV-1 infection, it is more and more evident that organs outside the hematopoietic and immune systems are affected by this virus in man, and by viruses with similar characteristics in other primates (R.T. Johnson, et al . FASEB J. 1:2970 (1988); M.H. Stoler et al.. J. Amer. Med. Assn. 256:2360 (1986); S. Gartner et al.. J. Amer. Med. Assn. 156:2365 (1986); S. Gartner et al.. Science 233:215 (1986); S.P. Raffanti et al.. Chest 93:592, (1988); M.C. Dalakas et al.. Neurology 36:569 (1986); M.C.Dalakas et al.. J. Amer. Med. Assn. 156:2381 (1986); R.D. Bailey et al.. Human Pathology 18:749 (1987); C. Cammarosano, et al .. J__ Amer. Coll. Cardiol. 5:703 (1985); B.K. Valle et al.. Heart-Lung 16:584 (1987); T.K. Rao et al.. N. Enol . J. Med. 110:669 (1984); N.W. King et al.. Amer. J. Pathol. 113:382 (1983); P. Solal-Celigy et al.. Amer. Rev. Resoir. Pis. 131:956 (1985); C.C. Tsai et al.. Amer. J. Pathol. 120:30 (1985); L.J. Couderc et al.. Arch Intern. Med. 147:898 (1987)).

Dysfunctions of the central and peripheral nervous systems in HIV infected individuals are particularly striking (R.T. Johnson, et a . FASEB J. 2:2970 (1988); M.H. Stoler et al.. J. Amer. Med. Assn. -12-

256:2360 (1986); S. Gartner et al. J. Amer. Med. Assn. 256:2365 (1986); S. Gartner et al. Science 133:215 (1986)). The heart (S.P. Raffanti et al . Chest 93:592. (1988); C. Cammarosano, et al.. J. Amer. Coll. Cardiol. 5:703 (1985); B.K. Valle et al.. Heart-Lung 16:584 (1987)) and skeletal muscle (M.C. Da akas et al.. Neurology 36:569 (1986); M.C.Dalakas et al.. J. Amer. Med. Assn. 256:2381 (1986); R.D. Bailey et al.. Human Pathology 18:749 (1987); N.W. King et al.. Amer. J. Pathol. 113:382 (1983), the kidney (T.K. Rao, et al.. N. Engl . J. Med. 310:669 (1984)), the lung (P. Solal-Celigy et al.. Amer. Rev. Respir. Pis.131:956 (1985)) and other organs (N.W. King et al.. Amer. J. Pathol. 113:382 (1983); C.C. Tsai et al.. Amer. J. Pathol. 120:30 (1985); L.J. Couderc et al.. Arch Intern. Med. 147:898 (1987)) also may be involved.

The Relationship Between HIV Infection and the Expression of the CP18 Family of Molecules and Their Binding Ligands

Histologic examination of affected organs reveals focal extravascular mononuclear cell infiltrates. Attempts to identify virus-infected cells in such infiltrates in the central nervous system have revealed the presence of HIV-1 infected cells. These studies have shown that HIV-1 resides primarily in monocytes and macrophages, and other cells of this lineage (R.T. Johnson, et al. FASEB J. 2:2970 (1988); M.H. Stoler et al.. J. Amer. Med. Assn. 256:2360 (1986); S. Gartner et al. J. Amer. Med. Assn. 156:2365 (1986); S. Gartner et al . Science 233:215 (1986)).

The mechanisms which stimulate formation of extravascular infiltrates of HIV-1-infected monocytoid cells have not previously been well defined. The mechanisms may involve either the transport of cell-free virus or the transport of virus across endothelial barriers within the cytoplasm of infected mononuclear cells. One aspect of the present invention concerns the discovery that infection with HIV-1 stimulates cell surface expression of molecules which facilitate -13-

adherence of leukocytes to vascular endothelial cells and the translocation of leukocytes from the blood to extravascular tissue sites (C.W. Smith et al.. J. Clin. Invest. 82:1746 (1988), herein incorporated by reference).

Thus, the effect of HIV-1 infection on T lymphocyte and monocytoid cell expression of molecules involved in adherence and translocation of leukocytes across endothelial cell barriers was studied in order to identify mechanisms which might initiate emigration of HIV-1 infected cells from the circulation.

Following infection with Human Immunodeficiency Virus-1 (HIV-1), both the alpha, and beta subunits of the LFA-1 receptor molecule (CDlla and CD18, respectively), and ICAM-l, the cell surface ligand for LFA-1, were found to be present on approximately 80% of HIV-1 infected T cells. These molecules were not evident on uninfected T cells. Increased cell surface expressions of both the alpha and beta subunits of LFA-1, but not of ICAM-l, was also observed on HIV- infected monocytoid U937 and THPl cells.

HIV-1 infection also increased homotypic aggregation in the presence of phorbol myristate acetate. Adherence of monocytoid cells to cultured human umbilical vein endothelial cells was also increased by infection with HIV-1. Preincubating T cells with anti-LFA-1 alpha subunit or anti-LFA-1 beta subunit antibodies suppressed the ability of such cells to adhere to endothelial cells. Similarly, cellular adhesion could be impaired by preincubating HUVEC with anti-ICAM-1 antibody.

Thus, HIV-1 infection stimulates expression of molecules involved in leukocyte adherence and transendothelial migration in vitro. n vivo, these effects can facilitate extravascular migration of HIV-1 infected cells and their localization in the central nervous system, the lung and other tissues. Treatment with either anti-ICAM-l antibodies, or antibodies to either the LFA-1 alpha or beta subunit can suppress the cell to cell interactions, and thereby prevent such adherence and transendothelial migration. -14-

The Anti-Migration Agents of the Present Invention

The present invention thus derives, in part, from the discovery that HIV infection results in increased expression of the CDlla/CD18 heterodimer, and/or its binding ligand, ICAM-l. This increased expression is significant in that it enhances the ability of HIV- infected T cells to migrate from the circulation. Such migration may result in the formation of extravascular foci and have diverse and serious consequences.

The present invention provides a method for suppressing such migration of HIV-infected cells which comprises administering an effective amount of an anti-migration agent to an HIV-infected individual.

The anti-migration agents of the present invention include any agent capable of impairing the ability of an HIV-infected T cell to bind to either CDlla, CD18, CDlla/CD18 or their ligand ICAM-l and ICAM-2. Molecules which bind to CDlla, CD18, or CDlla/CD18 will suppress migration by impairing the ability of the CDlla/CD18 expressed by HIV-infected T cells to bind to endothelial cells. Molecules which bind to ICAM-l will suppress migration by impairing the ability of the ICAM-l expressed by HIV-infected T cells to bind to cells expressing a CD11/CD18 receptor. In order to impair the ability of a cell to bind to the CDlla/CD18 receptor, or to the ICAM-l ligand molecule, it is possible to employ either immunoglobulin or non- immunoglobulin antagonists of these molecules.

Examples of immunoglobulin antagonists include monoclonal or polyclonal antibodies which are capable of binding to either the CDIla/CD18 molecule, or either of its subunits, or ICAM-l or ICAM-2. Suitable antagonists also include the antigen-binding fragments of such antibody molecules (for example F(ab) or F(ab)2 fragments. Such antibodies can be derived from mouse, or other mammalian cells (including human). -15-

As indicated above, both polyclonal and monoclonal antibodies may be employed in accordance with the present invention. Of special interest to the present invention are antibodies to ICAM-l (or their functional derivatives), or to members of the CD18 family (or their functional derivatives), which are produced in humans, or are "humanized" (i.e. non-immunogenic in a human) by recombinant or other technology. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies disclosed herein, but are less immunogenic, and are better tolerated by the patient.

Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al .. International Patent Publication PCT/US86/02269; Akira, K. et al.. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S.L. et al.. European Patent Application 173,494; Neuberger, M.S. et al.. PCT Application WO 86/01533; Cabilly, S. et al.. European Patent Application 125,023; Better, M. et al.. Science 140:1041-1043 (1988); Liu, A.Y. et al..

Figure imgf000017_0001
84:3439-3443 (1987); Liu, A.Y. et al.. jh Immunol . 139:3521-3526 (1987); Sun, L.K. et al .. Proc. Natl. Acad. Sci . USA 84:214-218 (1987); Nishimura, Y. et al.. Cane. Res. 4Z.-999-1005 (1987); Wood, C.R. et al .. Nature 314:446-449 (1985)); Shaw et al.. J. Natl.Cancer Inst. 80:1553-1559 (1988); all of which references are incorporated herein by reference). General reviews of "humanized" chimeric antibodies are provided by Morrison, S.L. (Science. 119:1202-1207 (1985)) and by Oi, V.T. et al.. BioTechnioues 4:214 (1986); which references are incorporated herein by reference).

Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones, P.T. et al.. Nature 321:552-525 (1986); Verhoeyan et al.. Science 139:1534 (1988); Beidler, C.B. et al.. J. Immunol. 141:4053-4060 (1988); all of which references are incorporated herein by reference). -16-

Examples of preferred immunoglobulin antagonists of the CDlla/CD18 receptor include monoclonal antibody TS 1/18 (anti-CD18) or monoclonal antibody TS 1/22 (anti-CDlla). Examples of immunoglobulin antagonists of the ICAM-l ligand include RR 1/1 (Rothlein et al.. J__ Immunol. 137:1270 (1986)), and R6-5-D6 (disclosed in European Patent Application Publication No.289,949, which application is incorporated herein by reference).

The non-immunoglobulin antagonists which may be employed in accordance with the present invention as anti-migration agents include soluble derivatives of the CDlla/CD18, CDllb/CD18, or CDllc/CD18 receptor molecules, as well as soluble derivatives of each of the subunits of the receptor molecule (i.e. CDlla, CDllb, CDllc or CD18). Such molecules will suppress migration by binding to the ICAM-l expressed by HIV-infected T cells. Alternatively, soluble derivatives of ICAM-l may be employed. Such molecules will suppress migration by binding to the CDlla/CD18 receptors expressed by HIV- infected T cells.

The soluble derivatives referred to above are derivatives which are not bound to a membrane of a cell. Such derivatives may comprise truncated molecules which lack a transmembrane domain. Alternatively, they may comprise mutant forms of the natural molecules which lack the capacity to be bound (or stably bound) to the membrane of a cell even though they contain a transmembrane domain. Soluble derivatives of ICAM-l and their preparation are disclosed by Marlin, S.D. et al.. Nature 344:70-72 (1990), which reference is incorporated herein by reference).

The therapeutic effects of the present invention may be obtained by providing to a patient any of the above-described anti-migration agents. Such agents may be obtained either synthetically, through the use of recombinant DNA technology, or by proteolysis, or by a combination of such methods.

Both the antibodies of the present invention and the ICAM-l molecule discussed herein are said to be "substantially free of -17-

natural contaminants" if preparations which contain them are substantially free of materials with which these products are normally and naturally found.

Administration of the Agents of the Present Invention

In providing a patient with antibodies, or fragments thereof, capable of binding to any of the CD11, CD18 or ICAM-l molecules, or when providing CD11, CD18, CD11/CD18, ICAM-l or ICAM-2 (or a fragment, variant, or derivative thereof) to a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient), although a lower or higher dosage may be administered. When providing CD11, CD18, CD11/CD18, ICAM-l or ICAM-2 molecules or their functional derivatives to a patient, it is preferable to administer such molecules in a dosage which also ranges from about 1 pg/kg to 10 mg/kg (body weight of patient) although a lower or higher dosage may also be administered. As discussed below, the therapeutically effective dose can be lowered if anti-CDlla or anti-CD18 antibody is additionally administered with a soluble form of CD11, CD18, CD11/CD18, ICAM-l or ICAM-2. As used herein, one compound is said to be additionally administered with a second compound when the administration of the two compounds is in such proximity of time that both compounds can be detected at the same time in the patient's serum.

The agents of the present invention may be administered to patients intravenously, intranasally, intramuscularly, subcutaneously, enterally, or parenterally. When administering such agents by injection, the administration may be by continuous infusion, or by single or multiple boluses. -18-

The agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to suppress the migration of HIV (or other virally) infected T cells. An amount is said to be sufficient to "suppress" migration of T cells if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent such migration.

Soluble CD11, CD18, CD11/CD18, ICAM-l, or ICAM-2 or a fragment or derivative thereof, may be administered either alone or in combination with one or more additional agents (such as antibody to CDlla, CD18, ICAM-l, or ICAM-2). The administration of such compound(s) may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compound(s) are provided in advance of any symptom of viral infection (for example, prior to, at, or shortly after) the time of such infection, but in advance of any symptoms of such infection). The prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent migration of virally infected T cells. When provided therapeutically, the compound(s) is provided at (or shortly after) the detection of virally infected T cells. The therapeutic administration of the compound(s) serves to attenuate any additional migration of such T cells.

The agents of the present invention may, thus, be provided either prior to the onset of viral infection (so as to suppress the anticipated migration of infected T cells) or after the actual detection of such virally infected cells.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

The agents of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined -19-

in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the agents of the present invention, or their functional derivatives, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the agents of the present invention, or their functional derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxy ethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the agents of the present invention, or their functional derivatives, into particles of a poly¬ meric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copoly ers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, πanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980). -20-

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 FLOW CYTOMETRIC ANALYSIS OF T CELLS

Flow cytometric analysis of H9 infected T cells was performed as described by D.C. Anderson et al. (J. Clin. Invest. 74:536 (1984)). In brief, H9 T cells were exposed to monoclonal antibodies (Mabs) specific for CD18, CDlla and ICAM-l. The cells were then incubated, in the presence of fluorescein conjugated anti-murine IgG antibodies, under conditions sufficient to permit the anti-murine IgG antibodies to bind to any of the Mabs which may have bound to the H9 T cells. The results of one of three replicate experiments, shown in Figure 1, revealed that up to 80% of HIV-1 infected cells, expressed the CD18, CDlla and ICAM-l molecules. The figure also shows that CDlla, CD18 and ICAM-l were not detected on uninfected H9 cells. Binding of the Mabs to the infected H9 T cells was not due to stimulated expression of Fc receptors for IgG, since equivalent results were obtained with F(ab')2 fragments of these antibodies. In contrast, surface expression of CD4 was not different in infected and uninfected H9 cells.

HIV-1 infection was verified by indirect immunofluorescence (B. Yoffe et al.. Proc. Natl. Acad. Sci. fU.S.A.) 84:1429 (1987), which reference is incorporated herein by reference), using pooled high titer polyclonal patient antisera as a source of HIV-1 specific antibodies, and by in situ hybridization (M.E. Harper et al.. Proc. Natl. Acad. Sci. fU.S.A.l 83:772 (1986) which reference is incorporated herein by reference) using an

Figure imgf000022_0001
probe specific for the long terminal repeat ("LTR") of HIV-1 (NEP 200, DuPont Co. Biotechnology systems, Wilmington, DE 19898). -21-

In summary, to investigate mechanisms which might facilitate migration of HIV-1 infected cells from the circulation, the effect of HIV-1 on expression of CD11, CD18 and ICAM-l on T lymphocytes and monocytoid cells was expressed. CDlla, CD18 and ICAM-l were readily demonstrated by flow cytometry on up to 80% of HIV-1 infected H9 cells, but were not evident on uninfected H9. Within 2 weeks after inoculation at a time when > 50% of the cells contained HIV-1, (as shown by in situ hybridization ), surface expression of CDlla and CD18, but not ICAM-l, was significantly increased in THPl and U937 cells. 36.9 ± 2.3% (SE) of HIV-1 infected U937 cells adhered to human umbilical vein endothelial cells (HUVEC), cultured on glass cover slips, as compare to 20.7 ± 1.8 % (SE) of uninfected U937 cella (p <.01). Adherence was decreased 3 fold by preincubation of U937 with monoclonal anti-CD18 or anti-CDlla and 2 fold by preincubation of HUVEC wit monoclonal anti-ICAM-l. Homotypic aggregation of infected THP-1 in the presence of 50 ng/ml phorbol myristate acetate was virtually abolished by incubation with anti-CD18; it was reduced b anti-CDlla and but little stimulates expression of molecules critical for leukocyte adherence and transendothelial migration in vitro. Similar mechanisms may influence leukocyte trafficking, in vivo, and may play a role in the localization of HIV-1 infected cells in the central nervous system and other tissues.

EXAMPLE 2

EFFECT OF HIV INFECTION ON THE

EXPRESSION OF CD18, CDlla, CDllb AND ICAM-l

Continuous monocytoid cell lines, U937 (ATCC CRL 1593; L.J. Miller et al.. J. Immunol. 137:2891 (1986), which reference is incorporated herein by reference), and THPl (ATCC TIB 202; S. Tsuchiya et al.. Int. J. Cancer.16:171 (1980) which reference is incorporated herein by reference) were obtained from the American Type Culture -22-

Collection. Cells from the cell lines were cultured in RPMI 1640 culture medium (supplemented with 10% fetal calf serum). As shown in Figure 2, uninfected cells were found to constitutively express CD18, CDlla and CDllb.

The cells were inoculated with 1 x 103 TCID50 (Tissue Culture Infectious Dose 50) of the HTLV-IIIB prototype strain (multiplicity of infection equal to 0.001. After three weeks (when greater than 90% of the cells were found to be infected with the virus), the expression of CD18, CDlla, CDllb and ICAM-l were determined by indirect immunofluorescence (B. Yoffe et al.. Proc. Natl. Acad. Sci. (U.S.A.) 84:1429 (1987), which reference is incorporated herein by reference) and by in situ hybridization (M.E. Harper et al.. Proc. Natl. Acad. Sci. (U.S.A.) 83:772 (1986), which reference is incorporated herein by reference). As shown in Figure 2, expression of these integrins was coordinately increased.

To determine whether increased expression of these molecules might be stimulated simply by exposure to the virus, THPl cells were studied at intervals following inoculation. Uninoculated THPl cells from the same passage, cultured under the same conditions, were used as a control. At one week post-infection, no virally infected cells could be detected by indirect immunofluorescence or in situ hybridization. But, by 14 days post-infection, 50% of the cells were HIV-1 positive by both of these tests. As shown in Figure 3, at one week pot-infection, no difference in surface expression of the CD11/CD18 complex was detectable. In contrast, by 14 days post- infection, increased surface expression of CD18 and CD11 was observed. Expression of CD4, as measured with the Mab 0KT4 (so as not to compete with the binding site for HIV-1), was decreased on HIV-1 infected THPl cells (Figure 4). No ICAM-l was detected either on uninfected or infected THPl cells (Figure 4); ICAM-l was also not demonstrable on U937 cells. -23-

EXAMPLE 3 VISUAL ENDOTHELIAL CELL ADHERENCE ASSAY

The endothelial adherence assay described herein measured the percentage of HIV-1 infected as compared to uninfected cells which bind firmly, under standard conditions, to monolayers of cultured human umbilical vein endothelial cells ("HUVEC"). The assay is described by C.W. Smith et al. (J. Clin. Invest. 82:1746 (1988), which reference is incorporated herein by reference).

HUVEC, harvested and characterized with respect to acetylated low density lipoprotein binding and factor VIII expression (E.A. Jaffe et al.. J Clin Invest 51:2745 (1973); M.A. Gimbrone, Prog. Hemostasis Thromb. 3:1 (1975); J.C. Voyta et al.. J. Cell. Biol. 99:2034 (1984), all of which references are incorporated herein by reference), were cultured as confluent monolayers on fibronectin-coated 25 mm round glass cover slips. These cells in special chambers designed to hold two cover glasses, one of which carried the endothelial monolayer. The two cover slips, separated by an "0-ring," create a chamber of approximately 1 mm in height. Monocytes (lO^/ml) were injected into the chamber. After settling for 500 seconds, the monocytes in contact with the endothelial surface were counted, using an inverted microscope, equipped with phase contrast optics. To estimate the percent which became adherent during this interval, the chamber was inverted for an additional 500 seconds to allow non-adherent cells to fall away. The number of cells which remained adherent were then counted, divided by the number in contact with the endothelial surface before inversion of the chamber, and this fraction was multiplied by 100, to obtain the percentage of adherent cells. -24-

EXAMPLE 4

EFFECT OF HIV INFECTION ON THE ABILITY OF

MONOCYTES TO ADHERE TO ENDOTHELIUH

The ability of HIV-1 to influence the ability of virus-infected monocytes to adhere to vascular endothelium was studied using a modification of the visual adherence assay described above. A significantly higher percentage of HIV-1-iπfected monocytoid cells adhered to HUVEC, under the conditions of this assay (Figure 5). Pretreatment of both HIV-1-infected and uninfected U937 cells for 15 min at room temp (22 *C) with saturating doses of Mabs to CD18 (TS 1/18) (F. Sanchez-Madrid et al.. J. EXP. Med. 158:1785. (1983), which reference is incorporated herein by reference) or to CDlla (TS 1/22) (R. Rothlein et al.. J. Immunol. 137:1270 (1986), which reference is incorporated herein by reference) significantly reduced the percentage of adherent cells. Similarly, pretreatment of HUVEC with Mab R6-5-D6 specific for ICAM-l, the endothelial cell receptor for CDlla (R. Rothlein et al .. J. Immunol. 137:1270 (1986)), also significantly reduced the percentage of adherent U937 cells (Figure 5).

EXAMPLE 5

EFFECT OF A TI-CD18 AND CDlla Mabs

ON THE ABILITY OF T CELLS TO ADHERE TO ENDOTHELIUM

Since even uninfected THPl cells were highly adherent to endothelial monolayers, other experimental systems were used to evaluate the effects of Mabs to CD18 and CDlla on the adherence properties of HIV-1 infected THPl. HIV-infected THPl cells were cultured in flat bottom 96 well microtiter trays in the presence of 50 ng/ml phorbol myristate acetate (PMA), under conditions previously shown to promote leukocyte homotypic aggregation (R. Rothlein et al.. J. Immunol. 137:1270 (1986)). -25-

PMA induced aggregation of both infected and uninfected THPl cells, although the infected cells were strikingly affected by this treatment (Figure 6). The percentage of HIV-1-infected or uninfected THPl cells incorporated in large cell-cell aggregates (>50 cells) was significantly reduced (Figure 6) by pre-incubation with concentrations of monoclonal anti-CD18 (TS 1/18) sufficient to saturate the cell surface antigens, as determined by flow cytometry. Monoclonal antibodies to CDlla (TS 1/22) also reduced homotypic aggregation, but to a lesser extent, whereas an irrelevant monoclonal antibody directed against HLA class I framework antigens (C.J. Barnstable et al.. Cell 14:9 (1978)) or Mab specific for ICAM-l had little effect on homotypic aggregation of THPl cells (figure 6). The same culture conditions in the presence of PMA failed to induce significant aggregation of uninfected or HIV-1 infected U937 cells.

EXAMPLE 6

ROLE OF HIV IN THE EXPRESSION OF MOLECULES OF

THE CD18 FAMILY AND ICAM-l

HIV-1 infection thus increases cell surface expression on T cells and continuous monocytoid cell lines of molecules belonging to the CD11/CD18 family of integrins (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987) both of which references are incorporated herein by reference). Interactions between CDlla/CD18 heteroduplexes on the leukocyte surface and specific receptors on endothelial cell surfaces are required for leukocytes to move across vascular endothelial barriers in response to chemotactic stimuli (C.W. Smith et al.. J. Clin. Invest. §2:1746 (1988); E. Ruoslahti et al.. Science 138:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)).

The fact that monoclonal antibodies to either CDlla or CD18 suppressed endothelial adherence of both HIV-1-infected and uninfected U937 cells to the same baseline level (Figure 5) indicates that the -26-

enhancement of endothelial cell adherence associated with HIV infection was mediated by increased cell surface expression of CDlla/CD18. The involvement of ICAM-l in these adherence reactions was shown by that fact that treatment of the endothelium with anti- ICAM-1 also suppressed adherence of HIV-1 infected cells, although adherence was not reduced to the same extent as treatment of U937 cells with anti-CDlla or anti-CD18. Homotypic aggregation of HIV-1- infected, as well as uninfected THPl cells, stimulated with PMA, was found to have involved interactions of CD11/CD18 with ligands other than ICAM-l (M.L. Dustin et al.. J. Cell. Biol. 107_:321 (1988); M.P. Bevilacqua et al.. Proc. Natl. Acad. Sci. (U.S.A.) 84:9238 (1987), which references are incorporated herein by reference). While anti- CD18 monoclonal antibodies effectively suppressed homotypic aggregation of THPl cells, monoclonal anti-ICAM-1 had little effect on this cell-cell interaction. Other molecules that mediate cell to cell adhesion have been recognized (C.W. Smith et al.. J. Clin. Invest. 81:1746 (1988); M.L. Dustin et al.. J. Cell. Biol. 107:321 (1988); M.P. Bevilacqua et al.. Proc. Natl. Acad. Sci. (U.S.A.) 84:9238 (1987)); one or more of these may also be stimulated by infection with HIV-1.

EXAMPLE7 HIV-1 STIMULATES MONOCYTOID CELL ADHERENCE TO ENDOTHELIUM

HIV may stimulate mechanisms responsible for translocation of monocytoid cells across endothelial barriers. Thirty seven + 2 % (SE) of HIV-infected U937 cells, (>90% positive by in situ hybridization ), adhered to human umbilical vein endothelial cells (HUVEC) compared to 21 ± 2 % of uninfected U937 cells (p <.01). Monoclonal anti-CD18 decreased HUVEC adherence of both infected and uninfected U937 to 13 ± 1 %. Only 4 ± 0.8 % of infected and 3 ± 1.1% of uninfected cells, treated with anti-CDlla, adhered to HUVEC. Preincubation of HUVEC with monoclonal anti-ICAM-l also decreased adherence of infected and -27-

uninfected cells. Flow cytometer analysis showed increased expression of CP18 & CPlla on both U937 and THP-1 cells within 14 days after virus inoculation, at which time 50% of the cells were positive by in situ hybridization. Homotypic aggregation of infected THP-1 treated with 50 ng/ml phorbol myristate acetate (PMA) was virtually abolished by anti-CP18, reduced by anti-CPlla, and but little affected by anti- ICAM-1. U937, cultured under the same conditions, showed little homotypic aggregation. Thus, infection with HIV stimulated monocytoid cell surface expression of CP18 & CDlla

EXAMPLE 8

EFFECTS OF INFECTION WITH OTHER VIRUSES

ON THE EXPRESSION OF CD11/CD18 AND ICAM-l

Except for studies of Burkitt lymphoma cells, there is little information concerning the effects of infection with other viruses on the expression of CD11/CD18 and ICAM-l in lymphoid cells (CD. Gregory et al. J. Exoer. Med. 167:1811 (1988); M. Patarroyo et al.. Int. J. Cancer 41:901 (1988), which references are incorporated herein by reference). Epstein-Barr virus (EBV) positive cell lines, isolated from Burkitt's lymphomas, particularly those which grow in single cell suspensions in vitro, express little if any CDlla/CD18 or ICAM-l; in contrast EBV transformed lymphoblastoid cells customarily express high levels of these integrins and grow in large cell-cell aggregates, unless dispersed in saturating concentrations of monoclonal antibodies to CDlla or ICAM-l (CD. Gregory et al. J. Exoer. Med. 167:1811 (1988); M. Patarrovo et al.. Int. J. Cancer 41:901 (1988)). Recently, Petit et al (A.J.C. Petit et al.. J. Clin. Invest. 79:1883 (1987)) ' noted PMA-enhanced homotypic aggregation of HIV-1 infected U937 cells. The flow cytometric studies described herein demonstrated that cell surface expression of CD18, CDlla and CDllb was increased significantly on HIV-1 infected U937 cells. In addition, the results disclosed herein showed that the expression of these integrins was -28-

also increased on HIV-1 infected THPl monocytoid cells and H9 T cells. Thus, the stimulated expression of the CD11/CD18 complex is a general consequence of HIV-1 infection.

The increased expression of CDlla/CD18 on HIV-1 infected cells is particularly noteworthy because, it indicates that biochemical events associated with viral infection stimulate biosynthesis of CDlla/CD18. This heterodi er, in contrast to CDllb/CD18 and CDllc/CD18, is not stored, pre-formed, within intracellular vesicular compartments in monocytes (E. Ruoslahti et al .. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987); R.F. Todd, III et al.. J. Clin. Invest. 74:1280 (1984)).

Consistent with the hypothesis of stimulated biosynthesis is the observation that increased expression of CD18 and CD11 was not detected in THPl cells until 14 days after inoculation at which time evidence of viral infection was widespread.

The failure of the U937 cells to show homotypic aggregation even when stimulated with PMA may reflect the fact that these cells do not express ICAM-l, and presumably do not express other cell surface molecules which can serve as ligands for the CD11/CD18 heteroduplex. In contrast, HIV-1 appeared to stimulate expression of both ICAM-l and CD11/CD18 heterodimers by H9 T cells. Thus-, CD11/CD18-ICAM-l interactions enhance the tendency of these cells to grow as aggregates once they are infected with HIV-1. Aggregation mediated by CD18/CD11 - ICAM-l interactions may be distinguishable from, and can facilitate interactions of, the viral gpl20 and cellular CD4 molecules involved in binding HIV-1 to the surface of CD4+ T cells, an interaction which may result in the fusion of virus-infected and uninfected CD4+ cells and cause the formation of multinucleated giant cells in vitro (B. Yoffe et al.. Proc. Natl. Acad. Sci. (U.S.A.) £4:1429 (1987); D. Klatzmann et al.. Nature 312:767 (1985); J.S. McDougal et al.. Science 131:382 (1986); D.H. Smith et al.. Science 138:1704 (1987); J.D. Lifson et al.. Science 141:712 (1988)). -29-

Increased expression of both CD11/CD18 and ICAM-l on HIV-1- infected T cells may enhance their ability to interact with monocytoid cells which express appropriate reciprocal ligands. Such an enhancement would prolong cell surface interactions, and thereby facilitate the transfer of virions to uninfected monocytes. Similarly, CD11/CD18 - ICAM-l mediated adherence reactions could facilitate transfer of virus from infected monocytes to uninfected T cells.

In the circulation, increased expression of CDlla/CD18 HIV-1 infected leukocytes facilitates adherence of HIV-1 infected cells to vascular endothelium which expresses a high density of molecules, such ICAM-l, which provide attachment sites for these integrins. A number of proinflammatory molecules such as interleukin-1, γ-interferon, or tumor necrosis factor-α stimulate endothelial cell expression of ICAM- 1 (M.L. Dustin et al.. J. Immunol. 137:245 (1986); J.S. Pober et al .. J. Immunol. 137:1893 (1986)). Thus collections of HIV-1 infected mononuclear cells should be concentrated in tissues where inflammatory responses, perhaps to secondary opportunistic infections, have stimulated endothelial cells to express ICAM-l or other leukocyte receptors. With regard to this conclusion, it is noteworthy that extravascular accumulations of mononuclear leukocytes associated with myositis, myocarditis, hepatitis or various central nervous system syndromes in HIV-1 infected individuals are often focal, and occur, typically, in patients who are co-infected with agents likely to create microenvironments which induce endothelial cell expression of molecules that facilitate leukocyte adhesion and transmigration (5- 7,11,12, J.R. Berger et al.. Ann. Int. Med. 107:78 (1987); A.F. Suffredini et al.. Ann. Int. Med. 107:7 (1987); D.R. Johns et al.. J Eng. J. Med. 316:1569 (1987)).

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in -30-

general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential, features hereinbefore set forth as follows in the scope of the appended claims.

Claims

-31-WHAT IS CLAIMED IS:
1. A method for suppressing the extravascular migration of a virally infected leukocyte in a patient having such a leukocyte, which comprises administering to said patient an effective amount of an anti-migration agent, said agent being capable of impairing the ability of said leukocyte to bind to ICAM-l or to a member of the CD11/CD18 family of receptor molecules.
2. The method of claim 1, wherein said virally infected cells are infected with HIV.
3. The method of claim 1, wherein said agent is an immunoglobulin, a derivative of an immunoglobulin, an antigen binding fragment of an immunoglobulin or a derivative of an antigen binding fragment of an immunoglobulin.
4. The method of claim 3, wherein said immunoglobulin is a monoclonal antibody.
5. The method of claim 3, wherein said immunoglobulin is an antibody capable of binding to a CD11 molecule.
6. The method of claim 5, wherein said antibody is capable of binding to a CD11 molecule selected from the group consisting of: CDlla, CDllb, and CDllc.
7. The method of claim 3, wherein said immunoglobulin is an antibody capable of binding to a CD18 molecule.
8. The method of claim 3, wherein said immunoglobulin is an antibody capable of binding to ICAM-l or ICAM-2. -32-
9. The method of claim 8, wherein said antibody is capable of binding to ICAM-l, and wherein said antibody is the antibody R6.5.
10. The method of claim 1, wherein said agent is a soluble derivative of ICAM-l or ICAM-2.
11. The method of claim 1, wherein said agent is a soluble derivative of CD11.
12. The method of claim 11, wherein said soluble derivative of CD11 is selected from the group consisting of: a soluble derivative of CDlla; a soluble derivative of CDllb; and a soluble derivative of CDllc.
13. The method of claim 1, wherein said agent is a soluble derivative of CD18.
14. The method of claim 1, wherein said agent is a soluble derivative of CD11/CD18.
15. The method of claim 14, wherein said soluble derivative of CD11/CD18 is selected from the group consisting of: a soluble derivative of CDlla/CD18; a soluble derivative of CDllb/CD18; and a soluble derivative of CDllc/CD18.
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EP0462205A1 (en) * 1989-03-09 1991-12-27 Dana Farber Cancer Institute Leukocyte adhesion receptors
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