WO2007024668A2 - Targeting of herpes simplex virus to specific receptors - Google Patents

Abstract

The invention relates to engineered Herpes simplex virus (HSV) particles exhibiting a binding pair member that specifically binds to, or targets, one or more specific binding partners, such as receptors, including binding partners lacking at least one of a cytoplasmic domain or a transmembrane domain. Also, recombinant vectors for producing such HSV particles are provided. By reducing the affinity of HSV for its natural receptor(s) and increasing the affinity for a selected receptor, the HSV particles of the invention are useful for targeting cells that express the selected, receptor, which itself may be a product of genetic engineering. The ability to selectively target cells renders the HSV particles particularly useful in selectively diagnosing, treating, and imaging cells bearing the selected binding pair member, such as a receptor. The invention also provides for polynucleotide-based therapy to cells bearing the selected binding pair member such as a receptor.

Description

TARGETING OF HERPES SIMPLEX VIRUS TO SPECIFIC RECEPTORS

Government Interests

The U.S. Government owns rights in the invention pursuant to National Cancer Institute grant number CA71933.

Background of the Invention

A steady rate of healthcare advances has led to continuing improvement in the health and quality of life for humans and animals. Nevertheless, a variety of diseases, disorders, and conditions have largely eluded the best efforts at prevention or treatment. Chief among these maladies is the loss of cell-cycle control that frequently results in the undesirable cell proliferation characteristic of cancer in its many forms, such as malignant glioma. Malignant gliomas are devastating brain tumors that afflict animals such as humans. The average life span after diagnosis is less than one year and few patients have been reported to survive five years. Furthermore, none of the conventional anti-cancer therapies has been successful in significantly prolonging the lifespan of patients with this disease. In recent years there have been numerous attempts to use genetically engineered herpes simplex viruses (HSV) as oncolytic agents to treat malignant gliomas. Because wild-type viruses are highly virulent, the viruses used in preclinical evaluations and in phase- 1 . clinical studies have been thoroughly attenuated. While several deletion mutants have been tested, the mutants that reached clinical trials lacked the γi34.5 gene encoding infected cell protein 34.5 (ICP34.5) and, optionally, the U_L.39 gene encoding the large subunit of ribonucleotide reductase.

These attenuated HSV viruses, however, have been imperfectly engineered as oncolytic agents. One advantage of these mutant viruses is that they have a significantly reduced capacity to replicate in normal, non-dividing cells in vivo. Viral ribonucleotide reductase is an essential gene for viral replication in resting cells and, hence, the U_L39 mutant virus is dysfunctional in the normal environment of the central nervous system (Simard et al 1995). The major function of ICP34.5 is to preclude the shutoff of protein synthesis caused by activation of protein kinase R in infected cells. Once activated, this enzyme phosphorylates the α subunit of translation initiation factor 2 (eIF2α), resulting in complete cessation of translation. Mutants lacking the γ]34.5 genes are highly attenuated because the lytic life cycle is completely blocked in an interferon^"1" cellular background. In contrast, γ]34.5 mutants are nearly as virulent as wild-type virus in mice lacking interferon receptor. Although mutants deleted in both γj34.5 and UL39 are not significantly more attenuated than those lacking the γi34.5 genes, such mutants do provide added insurance in the form of a reduced risk of reversion.

A significant disadvantage of these mutant HSV viruses is their poor replication, even in dividing cells. In experimental animal systems, the mutant viruses do not exhibit sustained lytic life cycles, with the loss of a potentially amplified response to a given therapeutic dose of the virus that would be expected upon re-infection of tumor cells by the multiplied viral progeny. Consequently, maximum killing of tumors cells requires high doses of virus. Given the poor growth of these mutant HSV viruses, even in dividing Cells, production of virus pools large enough to yield efficacious inocula of >10⁹ plaque forming units (PFU) has remained a major obstacle. Moreover, indiscriminate binding of virus to non-tumor cells further diminishes the effectiveness of HSV virus dosages because mis-targeted viral particles do not contribute to the desired beneficial therapeutic effect of tumor cell destruction. One approach to overcoming these obstacles is to achieve a more thorough understanding of the HSV lytic life cycle and thereby facilitate the development of HSV mutants tailored for use as targeted therapeutic agents, such as targeted oncolytic agents.

HSV enters host cells using a two-step mechanism. The first step of entry is HSV attachment to the cell surface. This step is initiated by glycoproteins B and C (gB and gC), which project from the viral envelope, attaching to heparan sulfate proteoglycans on host cell surfaces. The gB and gC domains interacting with heparan sulfate have been mapped at the sequence level (Laquerre et al. 1998). Following this initial attachment, viral glycoprotein D (gD) interacts with one of several receptors. Of these gD receptors, two are particularly important for entry (Spear et al, 2000). One receptor, designated HveA, is a member of the family of receptors for tumor necrosis proteins. A second receptor, designated HveC, is a member of the nectin family of proteins, structurally related to the immunoglobulin superfamily, which serve as intercellular connectors (Campadelli-Fiume et al. 2000). The second step of HSV entry into a cell is fusion of the viral envelope with the plasma membrane of the cell. To effect fusion, gD, when bound to its receptor, recruits glycoproteins B, H and L, which results in fusion of the envelope with the plasma membrane.

Additional understanding of HSV infection has come from recent studies that have lent significance to an old observation that gD interacts with the 5 cation-independent mannose 6 phosphate receptor, contributing to the accumulation of HSV in endosomes. Endocytosis of viral particles results in particle degradation by lysosomal enzymes, but the cells succumb as a consequence of the degradation of cellular DNA by lysosomal DNase. HSV gD blocks this apoptotic pathway to cell death through its interaction with the mannose 6 phosphate receptor. Thus, gD 10 interacts with HveA, nectins, the mannose 6 phosphate receptor, and at least one member of the complex of viral glycoproteins involved in the fusion of HSV with the plasma membrane.

In an attempt to target HSV-I infection to specific cells, a recombinant HSV having a chimeric protein comprising gC and erythropoietin (EPO) on its i5- —surface was ^constructedr-Although the recombinant virus bound to cells expressing EPO receptor and endocytosis of the virus occurred, successful infection of these EPO-receptor expressing cells did not occur.

Previous studies have disclosed the construction of a recombinant virus (R5111) capable of infecting cells via the ILl 3α2 receptor (IL13Rα2) (Zhou G, et al.,

20 2002; and WO 2004/033639 A3). This receptor differs from the common ILl 3 receptor in that it does not signal, it is monomelic, it contains a short cytoplasmic domain, it does not interact with EL4, and in nature it is present in high-grade malignant gliomas or astrocytomas and in human testes. In R5111, the polylysine tract responsible for the binding of gB to heparan sulfate was deleted, the amino

25 terminal domain of gC was replaced with the corresponding domain of ILl 3 to abolish the interaction of gC with heparan sulfate, and the same domain of IL 13 was inserted between amino acids 24 and 25 of gD. The studies have established that the R5111 recombinant HSV enters cells via the IL13Rcc2 receptor and that it dos not depend on endocytosis as its primary mechanism of entry into cells.

30 Urokinase plasminogen activation receptor (uPAR) is a 335-residue protein attached to the cell surface via a glycosylphosphatidylinositol anchor. It lacks transmembrane and cytosolic domains. uPAR binds and localizes the urokinase plasminogen activator (uPA) at the cell surface (Ploug M, et al., 1991). Increased uPA activity has been reported in malignant astrocytomas in vitro and in malignant brain tumors in vivo. In malignant brain tumors, uPA activity was correlated with poor prognosis. Increased uPA-uPAR interactions at the surface of a cell facilitate cellular movement via extracellular matrix (ECM) degradation, which is necessary for tumor cell invasion, chemotaxis, and cellular adhesion (Moller JV, et al., 1993; Cantero D, et al., 1997). Notwithstanding these observations, there has been no disclosure or suggestion in the art to use the uPA-uPAR system as a target for therapeutics (e.g., cancer therapeutics), which is unsurprising in view of the absence of transmembrane and cytosolic domains in uPAR.

A need continues to exist in the art for viral therapeutic agents exhibiting improved targeting capacities while retaining sufficient capacity to infect to be therapeutically useful. Ideally, suitable viruses would be therapeutic agents, such as oncolytic agents, themselves, as well as providing a targeting vehicle or vector for the controlled delivery of polynucleotide coding regions ultimately useful as therapeutic agents. Another need in the art is for targeted agents useful in diagnostic applications as, e.g., imaging agents or targeted vehicles for imaging agents.

Summary

The invention satisfies at least on of the aforementioned needs in the art by providing viral forms suitable for use as therapeutic and diagnostic agents themselves, as well as by providing a ready vehicle for the delivery of therapeutic or diagnostic polynucleotide coding regions to cells. These viral forms are modified viruses of the Herpesviridae family of viruses, and are preferably derived from herpes simplex virus type 1 or type 2. The invention provides a method of making virus particles with a novel ligand (or binding pair member), and making said particles totally dependent on a receptor of the ligand (or binding pair member) for entry into targeted cells.

Disclosed herein are methods to modify the surface of, e.g., an HSV virus particle in a manner that targets the virus to a specific receptor present on the surface of a cell of choice, typically a cell in need of therapy or a cell whose presence provides information of diagnostic value. The invention provides viral particles, e.g., HSV particles, having a reduced affinity for their natural cell-surface receptor(s), and ^methods for producing and using such particles, which minimizes or eliminates the problem of reduced efficiency associated with the mis-targeting of therapeutic and diagnostic viruses. Additionally, the invention provides viral particles, e.g., HSV particles, that exhibit specific affinity for a cell-surface component that is not a natural viral receptor and that is present solely or predominantly on a given target cell, as well as methods for producing and using such viruses. Modified viral particles (e.g., HSV). having increased affinity for a cell-surface component associated with one or more target cells exhibit improved targeting capabilities relative to known viral particles. The modified HSV particles have reduced indiscriminate binding, thereby minimizing sequestration of viral dosages away from the target cells. Other benefits of the modified viruses are described herein and will be apparent to those of skill in the art upon review of this disclosure.

One aspect of the invention provides a recombinant herpes simplex virus (HSV) particle having at least one protein on its surface, comprising: (a) an altered viral surface protein, wherein the alteration reduces binding of the viral surface protein to a sulfated proteoglycan; and (b) an altered gD, wherein the alteration reduces binding of gD to one or more of its cellular receptors, the alteration comprising a heterologous peptide binding pair member on the surface of the recombinant HSV particle forming a fusion protein with the altered gD; wherein the recombinant HSV particle preferentially binds to a cell expressing a binding partner to the heterologous peptide binding pair member, the binding partner lacking a ' cytoplasmic domain or a transmembrane domain. In some embodiments, the binding . partner is a fragment of a protein, the fragment comprising a domain that specifically binds the binding pair member. Analogously, the binding pair member may be a fragment of a protein, the fragment comprising a domain that specifically binds the binding partner. This aspect of the invention also comprehends embodiments in which the binding partner is a cell-surface receptor and/or the binding pair member is a ligand for a cell-surface receptor, a single-chain antibody, or any peptide molecule capable of specifically interacting (i.e., binding) to a binding partner on the surface of a cell. The binding partner may lack both a cytoplasmic domain and a transmembrane domain. An exemplary binding partner is urokinase plasminogen activator receptor (uPAR), and an exemplary binding pair member is urokinase plasminogen activator, a ligand of uPAR. In some embodiments, the recombinant HSV particles comprise an altered viral surface protein selected from the group consisting of gB and gC. The recombinant HSV particles of the invention include particles wherein the alteration of gD reduces binding to at least one protein selected from the group consisting of HveA 5 and HveC.

This aspect of the invention also includes embodiments in which the binding pair member forms a second fusion protein with a viral surface protein selected from the group consisting of gB and gC. Suitable target cells exhibiting a binding partner on their cell surfaces include a cancer cell, such as a malignant 10 gliomal cell.

In another aspect, the invention provides a pharmaceutical composition comprising the recombinant HSV particle described above and a pharmaceutically acceptable carrier, diluent, or excipient. In yet another aspect, the invention is drawn to a kit comprising the pharmaceutical composition and a set of instructions for -I-5-— axjmini steringihexomposition to a-subject hraeed, suclras-a human.

The invention further provides methods of using the recombinant HSV particles, including a method of targeting a recombinant HSV particle to a cell comprising (a) identifying a binding pair member specifically recognizing a binding partner exhibited on the surface of a target cell; and (b) creating an HSV particle as

20 described above, wherein the binding pair member binds to the binding partner exhibited on the surface of the target cell. In embodiments of the method, the altered viral surface protein is selected from the group consisting of gB and gC. Embodiments of the method extend to methods wherein the alteration to gD reduces binding of gD to at least one cellular receptor for gD selected from the group

25 consisting of HveA and HveC. The method comprehends a binding pair member that is a ligand for the binding partner, a single-chain antibody, or any peptide capable of specifically binding to a peptide (a binding partner) found on the surface of a cell; the binding pair member may further form a second fusion protein with gC. Suitable cells for use in the method include a cancer cell such as a malignant glioma cell.

30 Another aspect of the invention is a method of imaging a cell comprising: (a) contacting the cell with a recombinant HSV particle as described herein, the recombinant HSV particle further comprising a coding region for a marker - protein; and (b) detecting-the presence of the marker protein. -Any marker protein known in the art may be used in the method, including, e.g., thymidine kinase, green fluorescent protein, luciferase, and β-galactosidase. In this method as for the other methods of the invention, a suitable cell includes a cancer cell such as a malignant gliomal cell. This aspect of the invention further comprehends a situation in which the binding partner is present at a higher number on a cancer cell as compared to a non-cancerous cell of the same type.

Yet another method according to the invention is a method of treating a cell-based disease comprising delivering a therapeutically effective amount of a recombinant HSV particle as described above to a subject in need, such as a human patient or an animal such as commercially valuable stock or companion animals. A therapeutically effective amount will depend on a variety of factors particular to a given situation, as would be known in the art, and that therapeutically effective amount, sufficient to produce a detectably desired effect, would be determinable by those in the art using routine skill. This method is useful in treating any cell-based disease known in the art, including hyperproliferative cell disorders such as cancer, e.g., malignant glioma, and disorders arising from known genetic defects (e.g., Lesch- Nyhan syndrome, thalassemias).

Another method according to the invention is a method of ameliorating a symptom associated with a disease comprising administering a therapeutically effective amount of a recombinant HSV particle according to claim 1 to a subject in need. Any detectable disease symptom is suitable for amelioration, particularly those symptoms that negatively affect the quality of life of an afflicted human or animal, e.g., those symptoms causing pain, swelling, deformity, limited motion, and the like. Exemplary symptoms include the pain, swelling, loss of energy, shortness of breath and the like that are associated with a hyperproliferative cell disease, such as cancer.

In another aspect, the invention provides a method of delivering a therapeutically useful peptide to a cell comprising: (a) inserting a coding region for a therapeutically useful peptide into the DNA of a recombinant HSV particle as described above, thereby producing a recombinant HSV clone; and (b) delivering a therapeutically effective amount of the recombinant HSV clone to the cell. The recombinant HSV clone is delivered in vivo, ex vivo, or in vitro. Still another aspect of the invention is a method-of killing a target cell, comprising contacting the target cell with a recombinant HSV particle as described herein. In some embodiments of this aspect of the invention, the recombinant HSV particle itself effects cell death by completing the lytic life cycle of HSV and/or by promoting apoptosis; in other embodiments, the recombinant HSV particle delivers a heterologous coding region encoding a gene product that is lethal to a cell in and of itself or that contributes to the development of a lethal physiological state upon interaction with other components, such as a prodrug.

Other features and advantages of the invention will be better understood by reference to the brief description of the drawing and the description of the detailed description of the invention that follow.

Brief Description of the Drawing

Fig. 1. Schematic representation of the construction of the ATF-uPA- gD and BD-uPA-gD recombinant viruses. (Fig. IA) Schematic representation of the uPA constructs. (Line 1) Full length of uPA. (Line 2) ATF-uPA. (Line 3) BD-uPA. (Fig. IB) Schematic representation of recombinant HSV 5181 and 5182. (Line 1) Sequence arrangement of HSV-I genome where rectangular boxes represent the inverted repeat sequences ab and b'a' flanking the unique long (UL) sequence and inverted repeat c'a' and ca flanking the unique short (US) sequence. (Line 2) Schematic representation of recombinant HSV-I(F) genome, in which the N-terminal domain of gC was replaced with IL-13, and the polylysine domain (codons 68-77) of gB was deleted. The domain of gD was replaced with the immediately early promoter of cytomegalovirus to enable the expression of gl. (Line 3) Sequence arrangements of the glycoprotein D are highlighted. ILl 3 was replaced with ATF-uPA (Line 4) or BD-uPA (Line 5).

Fig. 2. Amino acid sequence alignment of ATF-uPA-gD junction and BD-uPA-gD junction. (Fig. 2A) The amino-terminal sequence of ATF-uPA-gD (SEQ ID NO: 1). The first underlined sequence identifies the gD signal peptide. ATF-uPA (bracketed by arrows) was inserted between residues 24 and 25 (underlined) of gD, between the Xhol and Kpnl restriction enzyme sites. (Fig. 2B) The amino-terminal sequence of BD-uPA-gD (SEQ ID NO:2). BD-uPA(bracketed by arrows) was inserted between residues 24 and 25 (underlined) of gD, between the Xhol and Kpnl restriction enzyme sites.

Fig. 3. Photograph of electrophoretically separated proteins from lysates of cells infected with ATF-uPA-gD or BD-uPA-gD virus reacted with antibody to ICPO, gD, USl 1 or ATF-uPA. Vero cells grown in 25-cm² flasks were exposed to 10 pfu of HSV-I , ATF-uPA-gD or BD-uPA-gD virus per cell. The cells were harvested 24 hours after infection, solubilized, subjected to electrophoresis in 10% denaturing polyacrylamide gels, electrically transferred onto a nitrocellulose sheet, and reacted with a monoclonal antibody against ICPO, gD, USl 1 or ATF-uPA, respectively. The protein bands corresponding to the ICPO, gD, ATF-uPA-gD fusion protein, BD-uPA-gD fusion protein and USl 1 are indicated.

Fig. 4. Human uPAR expression from the individual clones of stable transfectants of the Jl.1 cell line. The individual clones were amplified as below. The cells were harvested from 25-cm². flasks, solubilized, and subjected to electrophoresis in 10% denaturing polyacrylamide gels, electrically transferred onto a nitrocellulose sheet, and reacted with a monoclonal antibody to human uPAR.

Fig. 5. Replication of R5182 and R5182 virus and HSV-I(F) in J- uPAR, Jl.1, J-HveA and J-Nectin cells. Cells grown in 25-cm² flasks were exposed to 0.1 pfu of the recombinant virus or wild-type HSV per cell and harvested 24 hours after infection. Progeny virus was titered on Vero cells.

Fig. 6. Photographs of DNA bands derived by reverse transcription and PCR amplification of RNAs extracted from Jl.1 or J-uPAR cells using primers as described below. The PCR was performed with primers specific for the hamster (A) and human uPAR (B) ORFs indicated. Fig. 7. scuPA competition assay on R5181 virus infectivity in J-uPAR and J 1.1. J- uPAR and J 1.1 were exposed 1 hour to either 10 nM or 100 nM of scuPA, respectively. The cells were then infected with 0.1 pfu of R5181 virus per cell and harvested 24 hours post-infection. Progeny virus was titrated on Vero cells.

Fig. 8. The Effect OfNH₄Cl on R5181 virus infectivity in J-uPAR and Jl .1. Cells grown in 25-cm² flasks were exposed to increasing concentrations of

NH₄Cl for 30 minutes, infected with R5181 virus at 0.1 pfu/cell for 120 minutes in the same medium, and harvested 24 hours after infection. Progeny_virus was titered on Vero cells.

Detailed Description

The invention provides benefits that will improve the health and well- being of animals such as man by providing a targeted approach to the treatment of a variety of conditions and diseases that currently impair health, resulting in significant economic burdens using conventional treatments. In providing modified viral particles having controllable targeting capacities, the diagnostic and therapeutic benefit of the viruses themselves can be delivered with greater precision to particular cells. Additionally, these viral particles can be used as targeting vehicles for the delivery of a wide variety of therapeutic and diagnostic biomolecules, such as polynucleotides encoding therapeutic or diagnostic peptides.

Beyond the modified viral particles, the invention provides methods for making such therapeutic and diagnostic agents as well as methods for using the agents to diagnose or treat a variety of diseases and conditions, or to ameliorate a symptom associated with such a disease or condition, such as tumorigenic disease (e.g., gliomas). To facilitate an understanding of the invention and all of its aspects, illustrative embodiments are described below. The descriptions of these illustrative embodiments are not meant to limit the invention to the embodiments disclosed herein. In light of the description, one of skill in the art will understand that many changes and modifications can be made to the illustrative embodiments and still remain within the scope of the invention. The illustrative embodiments are disclosed using as an exemplary virus a member of the Herpesviridae family of viruses, herpes simplex virus (HSV). As noted above, HSV-I and HSV-2 are members of the family of viruses known as the Herpesviridae, whose structures are well known in the art. The targeting methods of the invention are applicable to any member of the Herpesviridae and are not limited to the exemplary embodiments described in the examples. Furthermore, a large number of recombinant HSV viruses are known in the art. Such viruses may contain one or more heterologous genes. Also, such viruses may contain one or more mutated HSV genes, for example, mutations that render the virus replication-deficient or affect the virulence of the virus in one or more cell types. Examples-of recombinant HSV- containing-a heterologous gene and methods of making and using such viruses are described in U.S. Patent No. 5,599,691 (incorporated herein by reference in its entirety). Preferred heterologous genes include genes encoding marker proteins. Marker proteins, such as green fluorescent protein, luciferase, and beta-galactosidase, allow detection of cells expressing the protein. In other embodiments, the heterologous gene encodes an enzyme that activates a prodrug, thereby killing adjacent uninfected cells. In yet other embodiments, the heterologous gene encodes a protein that affects the immune response, such as interleukin 12 (IL-12). Such proteins that activate the immune response against a tumor are particularly useful.

In one aspect, the invention relates to altering the surface of an HSV particle to target the virus to a specific receptor. By creating a fusion protein comprising a portion of gD and a ligand (or binding pair member), the virus is targeted to a cell having the other member of the binding pair, such as a cell surface receptor that binds the ligand (or binding pair member). In preferred embodiments, one or more HSV surface proteins, such as gB (SEQ ID NOs.: 3 and 4), gC (SEQ ID NOs.: 5 and 6), or gD (SEQ ID NOs.: 7 and 8), are altered to reduce binding to natural HSV receptors.

"Alterations" of the surface of an HSV particle or HSV surface protein include insertions, deletions, and/or substitutions of one or more amino acid residues. One type of alteration is an insertion, which involves the incorporation of one or more amino acids into a known peptide, polypeptide or protein structure. For ease of exposition, alterations will be further described using a protein exemplar. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of known proteins, which yield proteins such as fusion proteins and proteins having amino acid tags or labels. Although described in terms of proteins, polypeptides, and/or peptides, corresponding alterations to encoding nucleic acids are also comprehended by the invention.

Another type of alteration is a deletion, wherein one or more amino acid residues in a protein are removed. Deletions can be effected at one or both termini of the protein, or with removal of one or more residues within the amino acid sequence. Deletion alterations, therefore, include all fragments of a protein described herein. Yet another type of alteration is a substitution, which includes proteins wherein one or more amino acid residues are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables A or B₅ below.

Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A as described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc.New York (1975), pp.71-77] and set out immediately below.

Table A Conservative Substitutions I

SIDE CHAIN CHARACTERISTIC AMINO ACID

Non-polar (hydrophobic):

A. Aliphatic A L I V P

B. Aromatic F W

C. Sulfur-containing ] M

D. Borderline G Uncharged-polar:

A. Hydroxyl S TY

B. Amides N Q

C. Sulfhydryl C

D. Borderline G Positively charged (basic) KR H Negatively charged (acidic) D E

Alternative, exemplary conservative substitutions are set out in Table

B, immediately below. Table B - Conservative Substitutions II

ORIGINAL RESIDUE EXEMPLARY SUBSTITUTION

Ala (A) VaI, Leu, He

Arg (R) Lys, GIn, Asn

Asn (N) GIn, His, Lys, Arg

Asp (D) GIu

Cys (C) Ser

GIn (Q) Asn

GIu (E) Asp

His (H) Asn, GIn, Lys, Arg

He (I) Leu, VaI, Met, Ala, Phe,

Leu (L) lie, VaI, Met, Ala, Phe

Lys (K) Arg, GIn, Asn

Met (M) Leu, Phe, He

Phe (F) Leu, VaI, He, Ala

Pro (P) GIy

Ser (S) Thr

Thr (T) Ser

Tip (W) Tyr

Tyr (Y) Trp, Phe, Thr, Ser

VaI (V) He, Leu, Met, Phe, Ala

The binding site of HveA has been reported to be at the amino terminal domain of gD (Carfi A., et al, 2001) The precise binding sites of gD for Nectin 1 are not known, although it has previously been reported to involve gD amino acids 38 and 221 (Manoj S., et al, 2004; Zago A., et al, 2004; Connolly SA., 2005). Accordingly, one aspect the invention relates to amino acid alterations in the N-terminal region of gD such that the ability of gD to bind HveA or Nectinl is reduced or eliminated A "natural receptor" as used herein is a cell surface molecule that interacts with wild- - -

type HSV in the absence of human intervention. For example, gB and gC of HSV-I interact with heparan sulfate proteoglycans in a natural infection. In preferred embodiments, gB and/or gC are altered to reduce or eliminate binding to heparan sulfate proteoglycans. As another example, gD is known to bind to several receptors, including HveA and HveC, in a natural infection. In preferred embodiments, gD is altered to reduce or eliminate binding to HveA and/or HveC.

Receptor-ligands

As used herein, "receptor" and "ligand" refer to two members of a specific binding pair and, hence, are binding partners. Moreover, a "ligand" and a "receptor" as used herein refer to intact molecules or portions of such molecules that retain the capacity to specifically bind to the other member of the binding pair.

One advantage of the invention is the ability to tailor HSV to target a specific receptor while maintaining infectivity of the virus, hi an exemplary embodiment, an HSV particle contains a fusion protein comprising a portion of gD and at least the receptor-binding domain portion of uPA. Such a virus is able to infect cells expressing the cell-surface receptor uPAR. HSV containing the gD/uPA fusion protein are effectively targeted to such cells. Ligands that bind to receptors which are overexpressed or differentially expressed on either tumor cells or cells associated with tumor growth (e.g., neo vasculature) are particularly preferred. Examples include the α_vβ₃-α_vβ₅ integrins, which are overexpressed in tumor neovasculature; epidermal growth factor receptor (EGFR), which is overexpressed in head, neck, lung, colon, breast, and brain cancer cells; HER-2/Neu, which is overexpressed in breast cancer cells; MUC-I, which is overexpressed in breast, lung, and pancreas cancer cells; and prostate-specific membrane antigen, which is overexpressed in prostate cancer cells. hi certain embodiments, the ligand is a single-chain antibody that binds to its cognate specific binding pair member, herein referred to as a receptor. Single-chain antibodies have been shown to be effective in targeting applications, as evidenced by their ability to target retroviruses to specific receptors. More generally, the invention includes recombinant HSV comprising a gD fusion to, at least, the binding domain of a binding pair member.

Essentially any two binding pair members or partners may be used as receptor-ligand binding pairs in the invention. It is contemplated that certain factors, such as the distance from the binding site on the receptor to the membrane, or the conformation of the ligand when fused to gD, may affect the efficiency of recombinant HSV fusion to the cell membrane. Therefore, screens for effective receptor-ligand pairs are contemplated, using no more than routine procedures known in the art. Additional screens, conventional in nature, may be used to optimize constructs. One routine method of screening is to follow the protocol provided in the example for candidate receptor/ligand pairs, using uPAR/uPA as a control receptor/ligand pair.

Alternatively, one may use a membrane fusion assay as described in Turner et ah, 1998, incorporated herein by reference in its entirety. In the Turner assay, cells transfected with construct(s). encoding gB, gH, gL, and the gD/ligand fusion protein, and cells expressing the receptor, are co-cultured and the cells are examined for membrane fusion. Membrane fusion between gD/ligand-expressing cells and receptor-expressing cells indicates that the candidate receptor-ligand pair (the ligand being a gD/ligand fusion protein) is functional. Constructs encoding functional gD/ligand proteins can then be used to create recombinant HSV that are targeted to cells expressing the receptor.

Cell Targeting

Evident from the preceding discussion, another aspect of the invention is the targeting of a recombinant HSV to a cell having a specific binding pair member or receptor on its surface. In preferred embodiments, a recombinant HSV is designed to comprise a binding pair member or ligand that interacts with a receptor known to be expressed on a cell of interest. The cell of interest is then infected with recombinant HSV. Such targeting methods may be used for a variety of purposes. In one aspect, a recombinant HSV is used to introduce a heterologous gene into a cell that expresses the receptor. In preferred embodiments, the cell is not infected by, or is poorly infected by, wild-type HSV. Thus, in certain embodiments, the invention provides a vector for transforming a cell of interest with a heterologous gene. Further, a cell can be rendered a target of a recombinant HSV of the invention. The cell can be rendered a target by transforming the cell to express one member of a binding pair, e.g., a receptor capable of specifically binding a ligand - -

expressed on a recombinant HSV. For example, as described in Example 2* the Jl.1 cell line, which was resistant to infection by a recombinant HSV expressing an uPA ligand, was rendered susceptible to infection by transforming the cell line with a vector encoding uPAR to produce the cell line J-uPAR. Generally, the targeted HSV according to the invention exhibit one member of a binding pair, with the other member of that pair found on the surface of a target cell. In some embodiments of the invention, targeting is achieved with a ligand-receptor binding pair, with the ligand exhibited on the targeted HSV and the cognate receptor found on the surface of the target cell, as described above. Although the invention comprehends embodiments involving binding pairs that do not exhibit a ligand-receptor relationship (e.g., biotin-avidin) and embodiments in which the receptor is exhibited by the targeted HSV, embodiments in which the targeted HSV exhibits a ligand and the target cell presents the cognate receptor on its surface is used as an illustrative embodiment to reveal the versatility of the invention and to disclose the full scope thereof. For example, several ligands have been used for receptor- mediated polynucleotide transfer. Some ligands that have been characterized are asialoorosomucoid (ASOR) and transferrin (Wagner et al., Proc. Natl. Acad Sci. USA, 87(9):3410-3414, 1990). A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has also been used in a polynucleotide delivery vehicle (Ferkol et al, FASEB J., 7:1081-1091, 1993; Perales et al, Proc. Natl. Acad. Sci., USA 91 :4086-4090, 1994) and epidermal growth factor (EGF) has further been used to deliver polynucleotides to squamous carcinoma cells (Myers, EPO 0273085). Each of these specific approaches, and other approaches known in the art to achieve some selectivity in DNA delivery, or targeting, are amenable to use in the compositions and methods of the invention and are contemplated as embodiments of the invention.

For embodiments in which a targeted HSV harboring a coding region, e.g., a therapeutic coding region or gene, is delivered to a target cell, the nucleic acid encoding the therapeutic gene product may ultimately be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the therapeutic polynucleotide may be stably integrated into the genome of the cell. This integration may place the gene in its native location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation), hi yet further embodiments, the nucleic acid maybe - -

stably maintained in the-cell-as a separate, episomal segment of DNA. Suitable episomes encode functions sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is 5 dependent on the type of expression construct employed, as would be understood in the art.

It is envisioned that promoters subject to cell cycle regulation will be useful in the present invention. For example, in a bicistronic HSV vector designed to treat a disease, disorder or condition by killing a target cell, use of a strong CMV

10 promoter to drive expression of a first gene, such as pi 6, that arrests a cell in the Gl phase is accompanied by expression of a second gene, such as p53, under the control of a promoter that is active in the Gl phase of the cell cycle, thus providing a dual- gene approach to ensure that the target cell undergoes apoptosis. Other promoters, such as those of various cyclins, PCNA, galectin-3, E2F1, p53, BRCAl, and, indeed,

J 5_ _.,__ any suitable promoter_or_expression element known jn.the art, could be used.

In embodiments of the invention designed to treat diseases, disorders, or conditions associated with unwanted or excessive cell proliferation, such as cancer or restenosis, HSV is targeted to proliferating cells, thereby killing the cells. Because HSV is typically lethal to infected cells, expression of a heterologous gene is not 0 required. However, in embodiments wherein the lethality of HSV is attenuated, an

HSV harboring a gene that is lethal to the infected cell or that prevents proliferation of the infected cell may be used to target a cell.

Alternatively, HSV targeted to specific surface markers can be used to visualize the distribution of tumor cells in tissues. This diagnostic tool had been 5 unavailable because of the potentially indiscriminate binding of HSV to cells.

Modification of HSV by eliminating (ablating) or reducing the indiscriminate binding of HSV to heparan sulfate proteoglycans without deleteriously affecting the capacity of such HSV to replicate in both dividing and non-dividing cells makes possible the use of these modified viral forms to visualize the distribution of tumor cells. 0 In one preferred method for visualizing the distribution of tumor cells, radioactive visualization is achieved by viral thymidine kinase (TK)-dependent incorporation of a radioactive precursor. Methods of molecular imaging of gene expression are well known in the art. Methods often use highly sensitive detection techniques such as positron emission tomography (PET) or single-photon emission- computed tomography (SPECT). In one embodiment, TK expression is measured

1 S using a gancyclovir analog, such as 9-(3-[ F]fluoro-l-hydroxy-2- propoxy)methylguanine, as the tracer or marker (Vries et ah, 2002). For a review of imaging TK gene expression using PET or SPECT, see Sharma et al., 2002 or Vries et al., 2002.

A second preferred imaging method is to fuse a non-critical tegument protein (e.g., UsI 1, which is present in nearly 2000 copies per virus particle) to a marker protein, such as green fluorescent protein, which is capable of being visualized in vivo. Alternatively, a non-critical protein can be fused to a luciferase and the presence of the luciferase visualized with a luminescent or chromatic luciferase substrate. Although a marker protein can be fused to essentially any viral structural protein, preferred viral proteins include <gC, gE, gl, gG, gJ, gK, gN, ULI 1, ULI 3, U_L14, U_L21, U_L41, U_L35, U_L45, U_L46, U_L47, U_L51, U_L55, U_L56, U_s10, and U_sl 1. The marker protein also may be fused to thymidine kinase (Soling et al., 2002).

Library screening

As noted above, HSV comprising, e.g., a gD/ligand fusion protein can bind and infect cells expressing a receptor to the ligand. In one embodiment, a cell line expressing a receptor is used in screening for ligands of the receptor. cDNA from a cDNA library is cloned into a vector encoding a portion of gD to produce a gD/cDNA-encoded fusion protein. The resulting vectors are then screened for membrane fusion using the assay of Turner et al. described above or by creating recombinant HSV expressing the gD/cDNA-encoded fusion protein and screening the viruses for the ability to infect receptor-expressing cells. Such methods may be used, e.g., to identify a ligand to an orphan receptor.

In other embodiments, mutations in, or variants of, the members of binding pair are screened to determine whether the mutants or variants maintain the ability to interact with the respective partner. Such methods may be useful in determining the specific residues important in receptor-ligand interaction. Therapeutic methods

Another aspect of the invention is the use of the targeted HSV in therapeutic methods. By altering the cell-binding and infectivity properties of the virus, many routes and methods of administration become viable. For example, non- targeted HSV has the potential to bind indiscriminately to a variety of cells. Because of this property, large virus numbers are used and intravenous administration is generally not effective. However, by targeting the virus, one may lower the viral load (i.e., quantity of virus), yet maintain or increase efficacy. Furthermore, the targeted HSV can be administered intravenously and produce therapeutic effects. Therapeutic methods of the invention include those methods wherein an HSV is targeted to a binding pair member, such as a cell receptor, that contributes to, or is the basis of, a disease or disorder. These targeted HSV can either exploit the

- therapeutic properties of HSV itself (e.g., the lethality of HSV to infected cells) or the

- targeted HSV can serve as a vector for the targeted delivery of at least one therapeutic - — polynucleotide, such^~as"an-τexpressϊbie polynucleotide comprising a coding region.

For example, in methods wherein the targeted HSV contains one or more gene products that render the virus toxic to the cell or that prevent or inhibit cell proliferation, a preferred receptor is overexpressed or selectively expressed on harmful or undesirable cells, such as cancer cells. In other embodiments, the targeted HSV encodes a gene product that provides a desired function or activity in the targeted cell, e.g., when a cell has one or more genetic defects preventing the cell from functioning properly.

Additionally, it is contemplated that a therapeutic polynucleotide (e.g., gene or coding region) of a targeted HSV may be engineered to be under the expression control of a cell- or tissue-specific expression control element, e.g., a promoter. In such embodiments, the targeted HSV provide a further enhancement to the selective treatment of a suitable disorder, disease or condition. The targeted HSV is specific for a binding partner located on the surface of those cells for which treatment is intended, and expression of the therapeutic coding region or gene borne by the targeted HSV is limited to particular cells or tissues.

As HSV has been engineered to overcome the barriers to vector-based therapies, the choice of recombinant polynucleotide to be inserted into the vector has widened to the point where a wide- variety of diseases, disorders and conditions are amenable to treatment with targeted HSV. A number of diseases are amenable to polynucleotide-based therapy using HSV (see, e.g., Kennedy, et al.. Brain.120, 1245- 1259, 1997, incorporated by reference herein in its entirety). Though most attention has focused on cancers, there has been success in treating Parkinson's disease by expressing tyrosine hydroxylase in striatal cells, thus restoring L-dopa-induced nerve repair following axotomy of the superior cervical ganglion. Injection of a vector expressing nerve growth factor resulted in restored levels of tyrosine hydroxylase. More generally, HSV can now be used in polynucleotide-based therapy to replace missing or defective coding regions in the target cells. In the event of an inherited single-gene disorder (such as Lesch-Nyhan syndrome) where the complete DNA sequence, cause, and effect of the disorder are known, a single polynucleotide replacement mediated by targeted HSV is appropriate and contemplated. Another strategy amenable to the use of targeted HSV is the enhancement of endogenous expression levels of a eene product, e.e.. a growth factor or enzvme. Yet another strategy for using targeted HSV is HSV-directed enzyme pro-drug therapy. The delivery of a drug-sensitivity gene would be beneficial in the treatment of, e.g., a malignant brain tumor, making the tumor more susceptible to conventional anticancer agents. In other embodiments, the targeted HSV of the invention provide for vector-mediated delivery of anti-sense oligodeoxyribonucleotides (oligonucleotides). An oligonucleotide, or short segment of DNA(e.g., 2-100 nucleotides in length), is delivered to a target cell and therein binds to complementary mRNA, thus blocking the expression of a specific gene within the target cell. The encoded protein fails to be synthesized, as the mRNA is not be recognized by the translational components of the cell, hi preferred embodiments, a deleterious gene is targeted.

In yet other embodiments, targeted HSV are used to deliver polynucleotides, e.g., DNAs encoding gene products, that can recruit or enhance an immune system response, thereby bringing a subject's or patient's own immune system Io bear in the treatment of a disease, disorder or condition known in the art to be affected by to immune system activity. For example, an increase in cellular antigen expression of tumor cells, mediated by delivery of an expressible coding region for the antigen by a targeted HSV, would enhance the immune response and increase the susceptibility of such tumor cells to host cytotoxic immunity.

In some embodiments, a targeted HSV composition of the invention is delivered to a patient at or around the site of a tumor, which is a very efficient method for counteracting clinical disease. Alternatively, systemic delivery of targeted HSV compositions may be appropriate in other circumstances, for example, where extensive metastasis has occurred, or where inaccessible tumors are encountered.

It is contemplated that in certain embodiments of the invention a protein that acts as an angiogenesis inhibitor is targeted to a tumor. Also, an angiogenesis inhibitor agent may be administered in combination with a targeted HSV of the invention. These agents include, for example, Marimastat (British Biotech, Annapolis MD; indicated for non-small-cell lung, small-cell lung and breast cancers); AG3340 (Agouron, LaJolla, CA; for glioblastoma multiforme); COL-3 (Collagenex, Newtown PA; for brain tumors); Neovastat (Aeterna, Quebec, Canada; for kidney and non-small-cell lung cancer) BMS-275291 (Bristol-Myers Squibb, Wallingford CT; for metastatic non-small-cell lung cancer); Thalidomide (Celgen; for melanoma, head and neck cancer, ovarian, and metastatic prostate cancers; Kaposi's sarcoma; recurrent or metastatic colorectal cancer (with adjuvants); gynecologic sarcomas, liver cancer; multiple myeloma; CLL, recurrent or progressive brain cancer, multiple myeloma, and non-small-cell lung, nonmetastatic prostate, refractory multiple myeloma, and renal cancer); Squalamine (Magainin Pharmaceuticals Plymouth Meeting, PA; non-small-cell lung cancer and ovarian cancer); Endostatin (EntreMEd, Rockville, MD; for solid tumors); SU5416 (Sugen, San Francisco, CA; recurrent head and neck, advanced solid tumors, stage IIIB or IV breast cancer; recurrent or progressive brain (pediatric) cancer; ovarian cancer, AML (acute myeloid leukemia); glioma, advanced malignancies, advanced colorectal cancer, von-Hippel Lindau disease, advanced soft tissue cancer; prostate cancer, colorectal cancer, metastatic melanoma, multiple myeloma, malignant mesothelioma: metastatic renal, advanced or recurrent head and neck cancer, metastatic colorectal cancer); SU6668 (Sugen San Francisco, CA; advanced tumors); interferon-α; anti-VEGF antibody (National Cancer Institute,

Bethesda MD; Genentech, San Franscisco, CA, for refractory solid tumors; metastatic renal cell cancer, in untreated advanced colorectal cancer; EMDl 21974 (Merck KGaA, Darmstadt, Germany, for HIV-related Kaposi's sarcoma, and progressive or recurrent anaplastic.glipm_.a);_Interleukin 12_. (Genetics Institute, Cambridge, MA, for Kaposi's sarcoma) and IM862 (Cytran, Kirkland, WA, for ovarian cancer, untreated metastatic cancers of colon and rectal origin, and Kaposi's sarcoma). The parenthetical information following the agents indicates the cancers against which the 5 agents are being used in these trials. It is contemplated that any of these disorders may be treated with the targeted HSV compositions of the invention, either alone or in combination with the agents listed.

In order to prepare a therapeutic composition for clinical use, it will be necessary to prepare the therapeutic composition as a pharmaceutical composition, 10 i.e., in a form appropriate for in vivo application. Generally, this will entail preparing a composition that is essentially free of pyrogens, as well as other impurities that could be harmful to humans or other vertebrates. .

Generally, appropriate salts and buffers are included to render delivery vectors stable and to allow for uptake by target cells. Aqueous compositions of the

15 invention comprise an effective amount of the targeted HSV, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an

20. animal or a human. As used herein, "pharmaceutically acceptable carriers" includes any and all solvents, dispersion media,, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless a conventional medium or agent is incompatible with either the vectors of the invention

25 or the intended subject receiving treatment, its use in therapeutic compositions is contemplated. Supplementary active or inert ingredients also can be incorporated into the compositions.

The active compositions of the invention include standard pharmaceutical preparations. Administration of these compositions according to the 30 invention is by any known route, provided that the target tissue is accessible via that route. The pharmaceutical compositions may be introduced into the subject by any conventional method, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intravesicular, intrapulmonary (e.g., term release); sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site. The treatment may consist of a singlέ dose or a plurality of doses over a period of time.

Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

Appropriate dosages may be ascertained through the use of established routine assays. As studies are conducted, further information will emerge regarding optimal dosage levels and duration of treatment for specific diseases, disorders, and conditions.

In preferred embodiments, the unit dose may be calculated in terms of the dose of viral particles being administered. Viral doses are defined as a particular number of virus particles or plaque forming units (pfu). Particular unit doses include Iθ3, Iθ4, 10⁵, 106, io?, 10«, \Φ, K)IO, ₁₀H, 10¹², 10 ¹³ Or IO¹^ PfU. Particle doses may be somewhat higher (10- to 100-fold) due to the presence of infection- defective particles, which is deterniinable by routine assays known in the art. The pharmaceutical compositions and treatment methods of the invention are useful in the fields of human medicine and veterinary medicine. Thus, the subject to be treated may be a vertebrate, e.g., a mammal, preferably human. For veterinary purposes, subjects include, for example, farm animals such as cows, sheep, pigs, horses and goats, companion animals such as dogs and cats, exotic and/or zoo animals, laboratory animals including mice, rats, rabbits, guinea pigs and hamsters; and poultry such as chickens, turkeys, ducks and geese.

In some embodiments of the invention, it is contemplated that the targeted HSV is administered in conjunction with cbemo- or radiotherapeutic intervention, immunotherapy, or with any other therapy conventionally employed in the treatment of cancer.

To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce malignant phenotypes using the methods and compositions of the invention, one contacts a "target" cell, a tumor, or its vasculature with a targeted HSV composition and at least one other agent. The components of these compositions are provided in a combined amount effective to kill or inhibit proliferation of cancer cells. This process may involve contacting the cells with the targeted HSV composition and the agent(s) or factor(s) at the same time. -This may be achieved by contacting the subject organism, or cell of interest, with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same or different times, wherein one composition includes a targeted HSV composition of the invention and the other composition includes the second agent.

Another aspect of the invention provides diagnostic methods that involve imaging a tumor or diseased tissue using a targeted HSV. Such methods are useful in diagnosing a patient with a disease, disorder, or condition that is indicated by the presence of a binding pair member, e.g., a receptor, on the surface of a cell. Diagnostic imaging methods are discussed above.

Kits

Kits according to the invention may include recombinant viruses of the invention or may include vectors for producing such recombinant viruses. A vector for producing a recombinant virus of the invention may encode the gD/ligand fusion protein or may be designed to facilitate cloning of a ligand to produce, a gD/ligand fusion protein (e.g., a vector containing a multiple cloning site within the gD coding region that facilitates in-frame insertions).

Other components that can be included in a kit of the invention include a receptor-expressing cell line (useful as a control), a nucleic acid molecule for expressing the receptor in a particular cell type, and instructions for using the kit to effect diagnostic analyses or therapeutic treatments. In certain embodiments, a therapeutic kit will further contain a component for bringing about a therapeutic effect, such as a prodrug or a toxic compound. In other embodiments, a diagnostic kit will contain a compound useful in imaging methods, such as a chromophore or fluorophore, or an antibody for detecting infected cells.

Having provided a general description of the various aspects of the invention, the following disclosure provides examples illustrative of the invention, wherein Example 1 describes construction of a targeted HSV, Example 2 illustrates the construction of a cell line expressing a targeted HSV receptor target, and Example 3 describes the selective infection of a desired cell by a targeted HSV. Example 1

Construction of HSV recombinant viruses R5181 (ATF-uPA-gD) and

R5182 (BD-uPA-gD)

I. Construction of the ATF-uPA-gD plasmids. The complete cDNA of amino-terminal fragment (ATF) of uPA without the N-terminal signal peptide was amplified from pULscuPA plasmid using the following primers:

ATF-uPA-XhoI: 5 '-CCGCTCGAAGCAATGAACTTCATCAAGT- TCCATC-3' (SEQ ID NO: 9) ATF-uPA-Kpnl: 5 '-GGGGTACCTTTTCCATCTGCGCAGTCAT-

GCACC-3' (SEQ ID NO: 10)

The PCR product was gel purified and ligated into pGEM-T Easy Vector (Promega, Madison, WI). The sequence of ATF-uPA was verified by sequencing the entire ATF-uPA. Plasmid pGG5112 carries a 3648 bp fragment containing gD, mutant IL- 13(El 3Y) and flanking sequences in the EcoRI/Xbal sites of pBR322 (Reuning U, et al., 1998). To construct the ATF-uPA-gD chimeric plasmid, pGG5112 was digested with Xhol and Kpnl to release two fragments, 6.2 and 0.4 kb, respectively. Plasmid ATF-uPA was digested with Xhol and Kpnl and then inserted into the Xhol and Kpnl sites of the 6.2-kb fragment to generate the ATF- uPA-gD chimeric transfer plasmid.

II. Construction of the R5181 (ATF-uPA-gD) and R5182 (BD- uPA-gD) recombinant viruses.

The R5181 and R5182 viruses were generated by co-transfection of transfer plasmid ATF-uPA-gD or BD-uPA-gD and the R5110 viral DNA into rabbit skin cells by Lipofectamine reagent (Life Technologies, Grand Island, NY). The R5110 vector was described in WO 2004/033639 A3, incorporated by reference herein; R5110 contains a deletion of gD, a deletion of the heparan sulfate binding domain of gB, and a substitution of the amino terminal domain of gC with IL13 (Fig. IB). The progeny of the transfection was plated at a high dilution on Vero cell cultures so as to yield individual, well-spaced plaques. From each of the infected cell cultures, four single plaques were picked, frozen-thawed, sonicated, and then re- plated on fresh cultures of Vero cells for preparation of virus stocks and viral DNA for sequencing. Vero cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modification of Eagles minimal essential medium (DMEM) supplemented with 5% newborn calf serum (NBCS). Rabbit skin cells were maintained in DMEM supplemented with 5% NBCS.

III. Viral DNA Extraction.

Infected cells were removed from each of the 25-cm² flasks exposed to the individual plaque isolates, rinsed, and resuspended in 500 μl of Lyse-O-Lot (150 niM NaCl/10 mM Tris/1.5 mM MgCl₂) in the presence of 0.1 % of Nonidet P-40 (Zhou et al., 2002). The nuclei were removed by low-speed centrifugation. SDS to 0.2%, EDTA to 5 mM, and β-ME to 50 mM were added to the supernatant fluid, which was then extracted twice with phenol/chloroform. Viral DNA was finally precipitated by ethanol and resuspended, and the ATF-uPA-gD or BD-uPA-gD chimeric reading frames were amplified by PCR with the following primers:

5 '-CGGAATTCGATGGGGGGGGCTGCCGCCAG-S' (SEQ ID NO: 11)

5'-AACTGCAGCTAGTAAAACAAGGGCTGGTGCG-S' (SEQ ID NO: 12) The gD PCR products were sequenced to determine whether the gD and ATF-uPA, or BD-uPA sequences contained deletions or substitutions. The R5181 virus encodes an uPA peptide insert of 135 residues in length (residues 20-155 of uPA of SEQ ID NO: 14) between residues 24 and 25 of the HSV-I gD of SEQ ID NO: 8. The complete polynucleotide and amino acid sequences of ATF-uPA-gD are set out in SEQ BD NOs: 15 and 16. R5182 contains a much smaller peptide insert of 23 residues (residues 34-57 of uPA of SEQ ID NO: 14) and consisting of the binding site of uP A for uPAR. The complete polynucleotide and amino acid sequences of BD-uPA-gD are set out in SEQ ID NOs: 17 and 18. The structures of R5181 and R5182 viruses were verified as follows: (i) The presence of a chimeric ATF-uPA-gD gene in the R5181 vims and of a BD-uPA-gD gene in R5182 were verified by sequencing of the entire open reading frames amplified by PCR (Fig. 2A and Fig. 2B, respectively). (ii) As. expected, anti-gD antibody reacted with the ATFruPA-gD band in lysates of cells infected with the R5181 mutant virus but not with the gD band in lysates of wild-type virus (Fig. 3). Moreover, as expected, the chimeric ATF-uPA- gD protein of the R5181 virus (Fig. 3, lanes 3-5), migrated more slowly than wild- type gD (Fig. 3, lane 1).

Example 2

Construction of a cell line expressing uPAR

I. Construction of J-uPAR, a Cell Line Stably Expressing human uPAR. To determine whether R5181 and R5182 viruses were able to use the human uPAR protein as a receptor for entry, we constructed, in parallel, a cell line expressing this protein in the absence of other HSV-I entry receptors. JLl cells stably transfected with the human uPAR expression plasmids by using Lipofectamine kit (GIBCO/BRL)._were selected.on.the basis of their resistance to hygromycin B (Invitrogen, Carlsbad, CA). Hygromycin B-resistant clones were amplified and screened for uPAR expression by immunoblotting with monoclonal anti-human uPAR antibody (R&D Systems, Inc., Minneapolis, MN).

Parental and transformed cells were solubilized in SDS, electrophoretically separated in a denaturing gel (100 μg/lane), transferred to a nitrocellulose sheet, and probed with antibody against uPAR followed by the appropriate secondary antibody conjugated to alkaline phosphatase (Bio-Rad). The protein bands were visualized with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (Denville Scientific, Metuchen, NJ). As shown in Fig. 4, all clones expressed a protein reactive with the anti-human uPAR antibody but differed in the level of expression. The apparent size of the protein was consistent with the reported size of uPAR. Of 14 J-uPAR-positive clones, J-uPAR-7 (Fig. 4, lane 8) was selected for further studies because it provided the highest expression level of uP AR_¬ IL Cell surfaces immunostaining.

Hygromycin B-resistant cells (J-uPAR) in 25-cm² flasks were removed from the dish by gentle scraping and reacted with human uPAR antibodies (5 μg/ml) for 15 minutes on ice. The cells were then rinsed with cold PBS and reacted for 15 minutes-with a-l-:64-dilution of a-goat anti-mouse immunoglobulin G conjugated to fluorescein isothiocyanate (FITC) (Sigma, St, Louis, MO) in ice-cold PBS. After rinsing with cold PBS, the cells were suspended in 100 μl of ice-cold PBS for further immunofluorescence analyses. 10 μl of the surface-stained cells were plated onto four-well glass slides and mounted in 90% glycerol. Slides were analyzed with the aid of a Zeiss confocal microscope. A total of 200 cells in adjacent fields were examined for surface fluorescence.

As noted above, uPAR is anchored to the plasma membrane via glycosylphosphatidylinositol and lacks transmembrane and cytosolic domains. The expression of uPAR was investigated in J-uPAR-7 cells using immunofluorescence as the detection method for uPAR. in a first series of experiments, 70% of the J-uPAR-7 cells stained positive for uPAR. (Fig. 4, lane 8) in cultures passaged by gentle scraping of the cells. The number of positive cells after single detachment of cells with versene and totally disappeared after detachment with trypsin. These results — indie-ated that-treatment-with-trypsin or versene-ir-reversibly-altered the properties of uPAR..

In a second series of experiments, the effect of fetal bovine serum (FBS) on the expression of uPAR was investigated because FBS could internalize the cell surface receptor. J-uPAR cells were grown in serum-free and specialty medium (AIM-V Medium, Invitrogen, Carlsbad, CA) containing different concentrations of FBS. FBS affected the signal intensity but not the percentage of positive cells. Cells grown in ATM-V medium in the absence of serum grew poorly. Moreover, the cells could not be passaged. The results indicated that although FBS negatively affected the presence of uPAR on the cell surface, at least a low concentration of FBS was needed to maintain J-uPAR cells. It is expected that culturing J-uPAR cells, or equivalent cells, in one of the relatively new media formulations known in the art to reduce cell dependence on sera for growth will allow greater expression and cell- surface presentation of uPAR.

Example 3 Infection by the HSV Targeting Vector R5181 and R5182

I. Virus titration. Replicate cultures of J-uPAR, Jl .1, J-HveA (expresses HveA alone) and J-Nectinl (expressed Nectinl alone) were exposed to 0.1 pfu of ATF-uPA-gD., BD-uPA-gD, or HSV-I (F) virus per cell. After 24 hours of incubation, the cells were harvested, sonicated and titrated on Vero cells. For controls, the indicated cells were mock-infected or exposed to 10 pfu of recombinant or wild-type HSV-I (F) per cell. The cells were harvested 24 hours after infection, disrupted in SDS disruption buffer, boiled, cleared by centrifugation, electrophoretically separated on a 10% denaturing polyacrylamide gel, transferred onto a nitrocellulose membrane, and exposed to appropriate antibodies under standard conditions. The results shown in Fig. 5 are as follows:

(i) HSV- 1 (F) replicated poorly in J-uPAR or J 1.1. The amounts recovered from infected cells may represent, in large part, attached unpenetrated virus.

(ii) The titer obtained from cells infected with R5181 virus from infected J-uPAR cells was about 10² to 10³-fold higher than that obtained from HSV- 1(F)- infected J-uPAR cells.

(iii) The concentration of FBS did not affect the replication of R5181 virus in J-uPAR cells.

(iv) The titer obtained from infected J-uP AR cells infected with R5181 mutant virus was 10- to 100-fold lower than that obtained from J-uPAR cells infected with R5182 mutant virus.

(v) Unexpectedly, the titer obtained from ATF-uPA-gD virus infected J 1.1 cells was of the same order as that obtained from ATF-uPA-gD virus-infected J- uP AR cells. II. Isolation and reverse transcription of RNA extracted from

Jl .1 and J-uPAR cells.

The experiments described above indicated that R5181 virus replicated in Jl .1 cells. One possible explanation for these results is that Jl-I cells express an endogenous hamster uPAR. Consistent with this hypothesis is the evidence reported elsewhere that human uPA binds to hamster uPAR with high affinity (Fowler, B., et al., 1998). To test this hypothesis, two series of experiments were performed. In the first, J 1.1 cells were tested to determine if they express hamster uPAR mRNA.

Total RNA was extracted with the aid of TRIzol reagent according to the manufacturer's instructions (Life Technologies, Rockville, MD). DNase I treatment (Life Technologies), phenol/chloroform extraction, and ethanol precipitation (Fisher Scientific, Houston, TX) were carried out to remove possible DNA contamination. Total RNA (3 μl) was reverse-transcribed to yield single- stranded cDNA with 20 U of AMV reverse transcriptase (Promega) in a total reaction volume of 30 μl. The reverse transcription was primed with the following primers: Human uPAR reverse: 5 ' -GGC AGTC ATTAGC AGGGTG ATGGTG-

3' (SEQ ID NO: 19)

Hamster uPAR reverse 5 ' -GTTGCCCTCGC AGCTGTAAC ACTGG- 3' (SEQ ID NO: 20)

JReverse. transcription .was performed-usmg_a pool of nucleotides consisting of 10 mM concentrations (each) of dGTP, dATP, dTTP, and dCTP

(Promega). Forty units of RNasin (Promega) were added to each reaction mixture. The mixture containing only the RNA template, and the primer was first heated at 70⁰C for 10 minutes, chilled on ice, and after the addition of the other components, incubated at 42°C for 30 minutes, shifted to 52°C for 30 minutes, and then heat- inactivated at 95⁰C for 5 minutes. cDNAs obtained from reverse transcription of RNA extracted from Jl .1 and J-uPAR cells were amplified by PCR under the following conditions: 1 minute at 94°C, 1 minute at 60⁰C, and 75 seconds at 72°C. The following primers were used for PCR: uPAR-start forward: 5'-ATGGGTCACCCGCCGCTGCTGCCGC-S' (SEQ ID NO: 21)

Human uPAR reverse primer or hamster uPAR reverse primer used in PCR were as described above. As shown in Figure 6, the J 1.1 cell line contained detectable levels of hamster uPAR mRNA but not human uPAR mRNA.

In the second series of experiments, human single-chain uPA was used to compete with the virus for the putative hamster uPAR receptor on Jl .1 cells. Human single chain uPA (scuPA) was purchased from American Diagnostics Inc. (Stamford, CT). Cells were exposed to increasing concentrations of the human scuPA for 60 minutes at 37⁰C and then exposed to 0.1 pfu of R5181 for 120 minutes at 37⁰C in the presence of human scuPA. After 24 hours of incubation, the cells were harvested, sonicated and titrated on Vero cells.

Previous studies showed that human scuPA bound to human uPAR with high affinity (Kd of about 1 nM; Roldan, A. L., et al., 1990; Barnathan, E. S., et al., 1990) and human scuPA binds to hamster uPAR with the same affinity as for the human receptor (Kd=Ll nM) (Fowler,.et al., 1998). The Jl.1 cells exposed to the highest concentration of scuPA produced 10-fold less virus than control untreated cells (Fig. 7). The results (Fig. 7) showed that scuPA competed with the virus more successfully in Jl-I cells than in J-uPAR cells, consistent with the expectation that J- uPAR cells would have more receptors than JLl cells.

III. Treatment with endosome inhibitors.

Cells were exposed to freshly prepared NH₄CI at the concentrations indicated in Fig. 8 for 30 minutes at 37°C and then exposed to 0.01 nfu of R5181 virus or HSV-I (F) per cell in the presence of the NH₄Cl. After 24 hours of incubation, the cells were harvested, sonicated and titrated on Vero cells; The entry of R5181 virus into J-uPAR or JLl cells was not inhibited by NH₄Cl even at the highest concentration tested (Fig. 8). The results indicated that the R5181 virus does not depend on the endocytic pathway as a primary mechanism for entry into J-uP AR cells.

The preceding Examples describe the construction and properties of recombinant viruses designed to target cells expressing uPAR, with the results summarized below.

(i) R5181 virus infected and replicated in cells exhibiting uPAR. R5182 virus was unable to infect cells via uPAR. It is unknown whether the R5182 virus failed to infect cells via uPAR because the secondary structure of the chimeric gD blocked the binding site from interacting with uPAR or whether the insert was incompatible with the predicted modification of gD following its interaction with a receptor. (ii) Two lines of evidence indicated that R5181 entered JLl cells by interacting with uPAR. Foremost, scuPA competed with R5181 virus for entry into JLl cells. The second line of evidence is based on studies that indicated that virions may undergo endocytosis in the absence of a cell-surface receptor. The results herein (e.g., Fig. 8) show that endocytosis was not the major mechanism whereby R5181 entered J 1.1 or J-uPAR cells inasmuch as it was unaffected by NH₄Cl. In contrast, NH₄Cl reduced the already low yield of HSV-I(F) by approximately 10-fold in both Jl .1 and J-uPAR cells which is consistent with the expectation that a small amount of virus may enter cells by endocytosis in the absence of a specific receptor.

(iii) Cells transduced with uPAR required special care in the maintenance of the receptor on serial passage. The stability of the receptor was dependent on cell type, serum concentration, and, as in the studies described herein, the method by which the cells were dislodged for serial passage. Although this study began with the premise that BHK TK^" cells would not exhibit an uPA receptor, BHK TK^" cells that were passaged numerous times by a variety of means still expressed the endogenous receptor, albeit at levels significantly lower than that that of transduced cells. Thus, receptors may not always be present in tumor cells passaged serially in culture or they may be unstable in transduced cells.

(iv) Ligands or other binding pair members may be inserted into the HSV-I virion to provide a mode of entry into cells. Additionally, binding pair members may be targeted to receptors that are not anchored via their own transmembrane domain.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

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Claims

Cl aims

1. A recombinant herpes simplex virus (HSV) particle having at least one protein on its surface, comprising:

(a) an altered viral surface protein, wherein the alteration reduces binding of the viral surface protein to a sulfated proteoglycan; and

(b) an altered gD, wherein the alteration reduces binding of gD to one or more of its cellular receptors, said alteration comprising a heterologous peptide binding pair member on the surface of the recombinant HSV particle forming a fusion protein with the altered gD wherein said recombinant HSV particle preferentially binds to a cell expressing a binding partner to said heterologous peptide binding pair member, said binding partner lacking a cytoplasmic domain or a transmembrane domain.

2. The recombinant HSV particle of claim 1 , wherein said binding partner is a fragment of a protein, said fragment comprising a domain that specifically binds said binding pair member.

3. The recombinant HSV particle of claim 1 , wherein said binding partner is a cell-surface receptor.

4. The recombinant HSV particle of claim 1 , wherein said binding partner lacks both a cytoplasmic domain and a transmembrane domain.

5. The recombinant HSV particle of claim 4 wherein said binding partner is urokinase plasminogen activator receptor.

6. The recombinant HSV particle of claim 1 , wherein said binding pair member is a fragment of a protein, said fragment comprising a domain that specifically binds said binding partner.

7. The recombinant HSV particle of claim 1 wherein said binding pair member is a ligand for a cell-surface receptor.

8. The recombinant HSV particle of claim 7 wherein said ligand is a urokinase plasminogen activator.

9. The recombinant HSV particle of claim 1 , wherein the viral surface protein is selected from the group consisting of gB and gC.

10. The recombinant HSV particle of claim 1 , wherein the alteration of gD reduces binding to at least one protein selected from the group consisting of HveA and HveC.

11. The recombinant HSV particle of claim 1 , wherein the binding pair member forms a second fusion protein with a viral surface protein selected from the group consisting of gB and gC.

12. The recombinant HSV particle of claim 7, wherein the binding partner is a cell surface receptor for said ligand.

13. The recombinant HSV particle of claim 12, wherein the cell is a cancer cell.

14. The recombinant HSV particle of claim 13, wherein the cancer cell is a malignant gliomal cell.

15. The recombinant HS V particle of claim 1. wherein the binding pair member is a single-chain antibody.

16. A pharmaceutical composition comprising the recombinant HSV particle of claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.

17. A kit comprising the pharmaceutical composition according to claim 16 and a set of instructions for administering the composition to a subject in need.

18. A method of targeting a recombinant HSV particle to a cell comprising

(a) identifying a binding pair member specifically recognizing a binding partner exhibited on the surface of a target cell; and

. (b) creating an HSV particle according to claim 1 , wherein the binding pair member binds to the binding partner exhibited on the surface of said target cell. .

19. The method of claim 18, wherein said altered viral surface protein is selected from the group consisting of gB and gC.

20. The method of claim 18, wherein the alteration to gD reduces binding of gD to at least one cellular receptor for gD selected from the group consisting of HveA and HveC.

21. The method of claim 18, wherein the binding pair member is a ligand for said binding partner.

22. The method of claim 18, wherein the binding pair member forms a second fusion protein with gC.

23. The method of claim 18, wherein the cell is a cancer cell.

24. The method of claim 23, wherein the cancer cell is a malignant gliomal cell.

25. The method of claim 18, wherein the binding pair member is a single-chain antibody.

26. A method of imaging a cell comprising:

(a) contacting the cell with a recombinant HSV particle according to claim 1 , said recombinant HSV particle further comprising a coding region for a marker protein; and

(b) detecting the presence of the marker protein.

27. The method of claim 26, wherein the cell is a cancer cell.

28. The method of claim 26, wherein the binding partner is present at a higher number on the cancer cell as compared to a non-cancerous cell of the same type.

29. The method of claim 26, wherein the marker protein is selected from the group consisting of thymidine kinase, green fluorescent protein, and luciferase.

30. A method of treating a cell-based disease comprising delivering a therapeutically effective amount of a recombinant HSV particle according to claim 1 to a subject in need.

31. The method according to claim 28 wherein the disease is cancer.

32. A method of ameliorating a symptom associated with a disease comprising administering a therapeutically effective amount of a recombinant HSV particle according to claim 1 to a subject in need.

33. The method according to claim 32 wherein the disease is characterized by hyperproliferative cells.

34. A method of delivering a therapeutically useful peptide to a cell comprising:

(a) inserting a coding region for a therapeutically useful peptide intoJhe DNA-θf a recombinant HSV particle according to claim 1, thereby producing a recombinant HSV clone; and

(b) delivering a therapeutically effective amount of said recombinant HSV clone to said cell.

35. The method according to claim 3 wherein the recombinant HSV clone is delivered in vivo.

36. A method of killing a target cell, comprising contacting the target cell with a recombinant HSV particle according to claim 1.