A novel polypeptide ligand, p30, or LIGHT, for herpes virus entry mediator, HVEM, is provided. LIGHT is useful for modulating immune responses and in inhibiting infection and/or subsequent proliferation by herpesvirus. HVEM fusion proteins are also provided. Methods for treating subjects with lymphoid cell disorders, tumors, autoimmune diseases, inflammatory disorders or those having or suspected of having a herpesvirus infection, utilizing p30 and the fusion proteins of the invention, are also provided.

SUMMARY OF THE INVENTION

The present invention is based on the identification of an endogenous polypeptide that functions as a ligand for HVEM, which previously was known only to bind HSV gD. This HVEM-binding ligand, also referred to as p30, or LIGHT, is provided, as well as nucleic acid sequences encoding p30 and antibodies which bind to p30. The invention also includes methods for identifying compounds that modulate viral infection, e.g., herpesvirus (e.g., CMV or HSV) infection, and methods for modulating lymphoid cell responses. Thus, the methods of the invention are useful for treating subjects with autoimmune diseases, lymphoid malignancies and viral infection associated with herpesviridae including, for example, herpesvirus and cytomegalovirus infection.

In one embodiment the invention features an assay for identifying a compound which affects an HVEM-binding agent-mediated cellular response. Also within the invention is an assay for identifying a compound which affects an LT.beta.R-p30-mediated cellular response.

The present invention provides a method for inhibiting an inflammatory disorder in a subject, by contacting the subject with an inhibiting effective amount of an agent which prevents the interaction of p30 (LIGHT) with its receptor. The subject, tissue or cell used in the methods of the present invention may be any subject, tissue or cell, including of a mammalian specie, but is preferably human. The agents can be administered in vivo, ex vivo, or in vitro depending upon the source (e.g., whole organism, tissue or cell). For in vivo administration or contacting, delivery may be by systemic methods or topical. The agent may be any agent which prevents interaction of p30 with its receptor. Examples of such agents include the antibodies (e.g., anti-p30 antibodies), peptidomimetics, polypeptides, and fragments of HVEM of the invention.

In another embodiment, the present invention provides a fusion polypeptide comprising an HVEM polypeptide or fragment thereof operatively linked to a polypeptide of interest. The HVEM polypeptide can be from amino acid 1 to SER205 of HVEM and the polypeptide of interest an Fc region of an antibody.

In yet another embodiment, the present invention provides a polynucleotide sequence encoding the fusion polypeptide comprising an HVEM polypeptide or fragment thereof operatively linked to a polypeptide of interest.

In another embodiment, the present invention provides a vector containing the polynucleotide sequence encoding the HVEM fusion polypeptide.

In yet a further embodiment, the present invention provides pharmaceutical composition comprising the fusion polypeptide and/or polynucleotides and a pharmaceutically acceptable carrier.

The invention provides an isolated or recombinant homotrimeric p30 polypeptide comprising a monomer polypeptide having an apparent molecular weight (MW) of about 30 kilodaltons (kDa), wherein the homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR) polypeptide under physiologic conditions. In alternative embodiments, the isolated or recombinant homotrimeric p30 polypeptide comprises isomers having a pI from about 7 to about 8.5.

The invention provides a soluble isolated or recombinant homotrimeric p30 polypeptide lacking a transmembrane domain, wherein the homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions.

The invention provides a fusion protein comprising a p30 polypeptide of the invention and a heterologous sequence. In one embodiment, the heterologous sequence is a tag or another detectable moiety.

The invention provides a liposome comprising a p30 polypeptide of the invention, including a homotrimeric p30 polypeptide of the invention, a soluble homotrimeric p30 polypeptide lacking a transmembrane domain, a fusion protein of the invention, or a combination thereof.

The invention provides a pharmaceutical composition comprising a p30 polypeptide of the invention, including a homotrimeric p30 polypeptide of the invention, a soluble homotrimeric p30 polypeptide lacking a transmembrane domain, a fusion protein of the invention, or a liposome of the invention, or a combination thereof, and a pharmaceutically acceptable excipient.

The invention provides a pharmaceutical composition comprising a LT.beta.R or TNFR1 agonist. In one embodiment, the agonist comprises a ligand such as a polypeptide p30 (LIGHT), LT.alpha., TNF or LT.alpha.1.beta.2, or an antibody, such as a fully human or humanized form, or a fusion protein or functional fragment thereof. In a particular aspect, the antibody is a monoclonal antibody denoted 3C8, 3H4 and 4H8. Pharmacuetical compositions additionally ionclude those having one or more antiviral agents (e.g. an agent for treatment of herpesvirus, such as CMV).

The invention provides antibodies having a LT.beta.R or TNFR1 agonist activity. In one embodiment, an antibody has the binding specificity of a monoclonal antibody denoted 3C8, 3H4 or 4H8. In particular aspects, the antibody having the binding specificity of a monoclonal antibody denoted 3C8, 3H4 or 4H8 is humanized or fully human.

The invention provides a kit comprising a pharmaceutical composition and printed matter, wherein the pharmaceutical composition comprises a p30 polypeptide, wherein the p30 polypeptide comprises a homotrimeric p30 polypeptide comprising a monomer polypeptide having an apparent molecular weight of about 30 kDa, wherein the bomotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR) polypeptide under physiologic conditions, or, a soluble homotrimeric p30 polypeptide lacking a transmembrane domain, wherein the soluble homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions, and a pharmaceutically acceptable excipient, wherein the printed matter comprises instructions for a use of the pharmaceutical composition, wherein a use comprises inhibiting virus entry into a cell or virus proliferation in a cell. The instructions can include use of the pharmaceutical composition for inhibiting virus entry into a cell or inhibiting virus proliferation in a cell in vivo. In alternative embodiments, the inhibited virus is a herpesvirus, a herpes simplex virus (HSV), a cytomegalovirus (CMV), a .gamma.-herpesvirus or an Epstein Barr virus (EBV). The inhibition of virus entry or virus proliferation in the cell can be in a mammal, including a human.

The invention provides a kit comprising a pharmaceutical composition and printed matter, wherein the pharmaceutical composition comprises a p30 polypeptide, wherein the p30 polypeptide comprises a homotrimeric p30 polypeptide comprising a monomer polypeptide having an apparent molecular weight of about 30 kDa, wherein the homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR) polypeptide under physiologic conditions, or, a soluble homotrimeric p30 polypeptide lacking a transmembrane domain, wherein the soluble homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions, and a pharmaceutically acceptable excipient, wherein the printed matter comprises instructions for a use of the pharmaceutical composition, wherein a use comprises modulating diseases with unwanted lymphocyte proliferation. In alternative embodiments, the instructions comprise use of the pharmaceutical composition to modulate a T or a B lymphoma or leukemia, or an autoimmune disease. The autoimmune disease can be rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus or myasthenia gravis.

The invention provides a pharmaceutical composition comprising an expression vector encoding a p30 polypeptide having an apparent molecular weight of about 30 kDa or a p30 polypeptide lacking a transmembrane domain, wherein the p30 polypeptide forms a homotrimeric polypeptide that binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions.

The invention provides a kit comprising a pharmaceutical composition and printed matter, wherein the pharmaceutical composition comprises an expression vector encoding a p30 polypeptide having an apparent molecular weight of about 30 kDa or a p30 polypeptide lacking a transmembrane domain, wherein the p30 polypeptide forms a homotrimeric polypeptide that binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions, and a pharmaceutically acceptable excipient, and, wherein the printed matter comprises instructions for a use of the pharmaceutical composition, wherein a use comprises targeting of tumor cells or activated lymphocytes. In one embodiment, the use comprises treatment of a tumor by direct injection of the pharmaceutical composition into the tumor.

The invention provides a kit comprising an LT.beta.R or TNFR1 agonist, and instructions for use in treating a subject having or at risk of having a herpesvirus infection. The invention provides a kit comprising a pharmaceutical composition comprising an LT.beta.R or TNFR1 agonist and instructions for use in treating a subject having or at risk of having a herpesvirus infection. The invention provides a kit comprising an LT.beta.R or TNFR1 agonist, an antiviral agent, and instructions for use in treating a subject having or at risk of having a herpesvirus infection. The invention provides a kit comprising a pharmaceutical composition comprising an LT.beta.R or TNFR1 agonist, an antiviral agent, and instructions for use in treating a subject having or at risk of having a herpesvirus infection.

The invention provides a method for inducing a proliferation-inducing signal to a lymphocyte comprising (a) providing a composition that binds to cell surface expressed HVEM, and (b) contacting the lymphocyte with a proliferation-inducing amount of the composition. In alternative embodiments, the composition comprises an anti-HVEM antibody or a polypeptide comprising an anti-HVEM antibody binding site. In alternative embodiments, providing a composition that binds to cell surface expressed HVEM comprises providing a composition comprising a p30 polypeptide, a soluble p30 polypeptide, a liposome-associated p30 polypeptide, or, a vector encoding a p30 polypeptide or a cell expressing a recombinant p30 as a cell-associated p30 polypeptide. In this method, the lymphocyte can be a T cell or a B cell. The lymphocyte can be contacted in vivo.

The invention provides a method for inhibiting a p30 polypeptide-mediated cellular response comprising (a) providing an composition that inhibits binding of a cell surface expressed p30 polypeptide to a cell surface expressed HVEM or LT.beta.R, and, (b) contacting the cell expressing the cell surface expressed p30 polypeptide or the cell surface expressed HVEM or LT.beta.R with an amount of the composition sufficient to inhibit a p30 polypeptide-mediated cellular response. In this method, the cell can be contacted with the composition in vivo. In alternative embodiments, the inhibited p30 polypeptide-mediated cellular response comprises inhibition of a lymphocyte cellular response, the inhibited lymphocyte response is lymphocyte proliferation, and the inhibited lymphocyte is a pathogenic effector cell. The inhibited lymphocyte response can comprise modulation of a T or a B lymphoma or leukemia or an autoimmune disease. The autoimmune disease can be rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus or myasthenia gravis. The inhibited lymphocyte response can comprise modulation of a reaction to a transplant.

In one embodiment, the contacted cell expresses HVEM and the composition is a soluble p30 polypeptide. In an alternative embodiment, the contacted cell expresses LT.beta.R and the composition is a soluble p30 polypeptide. In other embodiments, the contacted cell expresses p30 polypeptide on its cell surface and the composition is a soluble HVEM polypeptide; and, the contacted cell expresses p30 polypeptide on its cell surface and the composition is an anti-p30 antibody.

The invention provides a method for treating tumors comprising (a) providing a pharmaceutical composition comprising an expression vector encoding a p30 polypeptide having an apparent molecular weight of about 30 kDa or a p30 polypeptide lacking a transmembrane domain, wherein the p30 polypeptide forms a homotrimeric polypeptide that binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions, and (b) directly injecting the pharmaceutical composition into the tumor.

The invention provides a method of modulating a lymphotoxin beta receptor (LT.beta.R)-mediated cellular response, the method comprising: (a) providing a composition that inhibits binding of an LT.beta.R to a p30 polypeptide; and (b) contacting a cell expressing the LT.beta.R or the p30 polypeptide with an amount of the composition sufficient to modulate the lymphotoxin beta receptor (LT.beta.R)-mediated cellular response. In one embodiment, the cell expresses LT.beta.R and the composition comprises a pharmaceutical composition comprising a p30 polypeptide comprising a homotrimeric p30 polypeptide comprising a monomer polypeptide having an apparent molecular weight of about 30 kDa, wherein the homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR) polypeptide under physiologic conditions, or, a soluble homotrimeric p30 polypeptide lacking a transmembrane domain, wherein the soluble homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions, and a pharmaceutically acceptable excipient. In one embodiment, the cell expresses a p30 polypeptide and the composition comprises an anti-p30 antibody. In another embodiment, the lymphotoxin beta receptor (LT.beta.R)-mediated cellular response comprises binding of a herpesvirus to a cell. In one embodiment, the herpesvirus is blocked from entry into the cell. In another embodiment, the herpesvirus is inhibited from proliferating in the cell. In alternative embodiments, the herpesvirus is a herpes simplex virus (HSV), a cytomegalovirus (CMV), a .gamma.-herpesvirus or an Epstein Barr virus (EBV).

The invention provides a method for inhibiting virus production in a cell, the method comprising (a) providing a p30 polypeptide; and, (b) contacting a cell infected with a herpesvirus or a cell susceptible to infection by a herpesvirus with an effective amount of a p30 polypeptide, thereby inhibiting herpesvirus production in the cell. In one embodiment, the entry of the herpesvirus into the cell is inhibited. In another embodiment, the contacting is in vivo and the p30 composition is provided as a pharmaceutical composition, wherein the pharmaceutical composition comprises a homotrimeric p30 polypeptide comprising a monomer polypeptide having an apparent molecular weight of about 30 kDa, wherein the homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR) polypeptide under physiologic conditions, or, a soluble homotrimeric p30 polypeptide lacking a transmembrane domain, wherein the soluble homotrimeric polypeptide binds to a herpes virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R) polypeptide under physiologic conditions, and a pharmaceutically acceptable excipient.

The virus can be a herpes simplex virus (HSV), a cytomegalovirus (CMV), a .gamma.-herpesvirus or an Epstein Barr virus (EBV). In one embodiment, the contacting is in a mammal, such as a human.

In another embodiment, a herpesvirus infection (e.g., CMV, a .beta.-herpesvirus or a .gamma.-herpesvirus) or a disorder associated with herpesvirus infection is treated. In one aspect, a method includes contacting a subject having or at risk of having a herpesvirus infection or a disorder associated with herpesvirus infection with an amount of a LT.beta.R or TNFR1 agonist sufficient to treat herpesvirus infection. Agonists include ligands, such as polypeptides (e.g., p30 (LIGHT), LT.alpha., TNF or LT.alpha.1.beta.2) and antibodies, such as fully human or humanized forms, as well as agonists that do not produce substantial apoptosis in cells infected with the virus. In additional aspects, virus proliferation, nucleic acid replication or protein expression, or virus reactivation from latency is reduced in the subject following treatment. Target subjects include subjects that are or are at risk of being immunosuppressed, subjects having an HIV infection or a tumor, subjects having or is at risk of having a blood or bone marrow, organ, or tissue transplant. Target subjects further include neonates. Disorders include pneumonia, arteriosclerosis, CMV hepatitis, CMV retinitis, CMV pneumonitis, CMV nephritis or CMV mononucleosis, child febrile illness, cytomegalaic inclusion disease or a demyelinating disease (e.g., multiple sclerosis), Kaposis sarcoma, Hodgkins leukemia or non-Hodgkins leukemia or lymphoma. Various embodiments include additionally contacting the subject with an antiviral agent or other form of herpesvirus treatment.

The invention provides a method for identifying a compound that inhibits CMV infection, including (a) contacting LT.beta.R or TNFR1 with a test compound under conditions allowing binding; (b) measuring LT.beta.R or TNFR1 activity in the presence of the test compound; and (c) comparing activity in the presence of the test compound to the absence of the test compound, wherein an increase in LT.beta.R or TNFR1 activity in the presence of the test compound identifies the test compound as a compound that inhibits CMV infection. In one aspect, the test compound is a library of compounds, for example, a peptide or small molecule library. In other aspects, the activity comprises activation of an NFkB target gene (e.g., ICAM1, VCAM1, interleukin-8 (IL-8) or secondary lymphoid organ chemokine (SLC), for example), or activation of IFNgamma or IFNbeta gene expression. In yet other aspects, the identified compound is tested for inhibiting CMV infection in cells or animals.

The invention provides a method for identifying a compound that inhibits CMV infection, including (a) contacting a cell that expresses LT.beta.R or TNFR1 in the presence of CMV with a test compound under conditions allowing binding between LT.beta.R or TNFR1 and the test compound; (b) measuring CMV proliferation, replication, protein expression or cytopathicity in the presence of the test compound; and (c) comparing CMV proliferation, replication, protein expression or cytopathicity in the presence of the test compound to the absence of the test compound, wherein a decrease in CMV proliferation, replication, protein expression or cytopathicity in the presence of the test compound identifies the test compound as a compound that inhibits CMV infection. In one aspect, the test compound is a library of compounds, for example, a peptide or small molecule library.

 

Claim 1 of 31 Claims

1. A method for treating a subject having a CMV infection comprising contacting the subject with an amount of an agonist antibody or antigen binding fragment thereof that binds to lymphotoxin beta receptor (LT.beta.R) sufficient to treat the CMV infection.

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