CA2231804A1 - Method for inhibiting cell-mediated killing of target cells - Google Patents

Abstract

Disclosed is a method for inhibiting cell-mediated (e.g., CTL-mediated) killing of a mammalian target cell by expressing in the target cell a poxvirus serpin proteinase inhibitor (SPI)-1. If desired, SPI-2 can be expressed in the cell in addition to SPI-1. Cells expressing these proteinase inhibitors are resistant to cell-mediated killing and apoptosis.

Description

W O 97/10~06 PCT~US96114571 ~ 1 METHOD FOR INHIBITING CELL-MEDIATED
KILLING OF TARGET CELLS

1. Cross Reference To Related Applications ,, This application claims priority under 35 U.S.C. 119 from U.S. Provisional Application Serial No.60/003,665, filed September 11,1995.

2. Statement as to Federally Sponsored Research This invention was made at least in part with funds from the Federal Government under National Institutes of Health Grant No. 5 RO1 Al 15722. The Government therefore has certain rights in the invention.

3. Background of the Invention Cell-mediated killing of target cells can be undesirable in vivo. For example, cell-mediated killing of grafts is thought to be responsible for rejection of a graft by a host.
Similarly, autoimmune ~ise~ses, such as diabetes, are thought to be arise from the undesirable induction of cell-mediated killing of an individual's cells (e.g., pancreatic islet cells). Cell-mediated killing has also hampered progress in the field of gene therapy. Many gene therapy methods entail the use of a viral vector to express atherapeutic gene in a target cell. Generally, such viral vectors also encode viral antigens that induce the pdliet,l':j immune system to effectuate cell-mediated killing of target cells that are transfected with the virus and therapeutic gene. Thus, these examples illustrate that the induction of an immune response can be undesirable. -Cell-mediated killing by cytotoxic T Iymphocytes (CTL) is thought to occur by secretion of cytoplasmic granules containing perforin and granzymes, or by signaling via the Fas pathway (Berke,1994, Ann. Rev. Immun.12:735-773 and Nagata et al., 1995, Science 267: 1449-1456) .

Summary of the Invention Applicants have discovered that cell-mediated killing of a target cell can be inhibited by expressing in the target cell a proteinase inhibitor of an orthopoxvirus (e.g., SPI-1).
Accordingly, the invention features a method for inhibiting cell-mediated cell death of a mammalian target cell by expressing in the target cell an orthopoxvirus proteinase inhibitor, thereby inhibiting cell-mediated cell death. In particular, the invention can be used to inhibit CTL-mediated cell death and apoptosis. The CA 02231804 1998-03-ll WO 97J10006 PCT~US96/14~71 invention is thus useful for suppressing the immune system of a patient, e.g., for treating an autoimmune disease, inhibiting graft rejection, or inhibiting the death of cells that are targeted in gene therapy methods.

Brief Descri~Jlion of the Drawings Figs. 1A and 1B are l~i~loy,~ s representing the inhibition of granule-meditatedcytolysis of cells infected with CPV or RPV. L1210 (Fig. 1A) or EL4 (Fig. 1B) cells were either mock-infected or infected with CPV or RPV, and cytolysis was assayedby incubation with stimulated CTL21.9 effector cells in a standard 4 hour chromium release assay at an effectorto target ratio of 5:1. To facilitate comparison of multiple experiments, the values for % specific Iysis for mock-infected cells were set to 100%, and other data are shown relative to mock-infected cells. Each bar represents the mean and standard deviation c~lcl~ted from 3-5 independent experiments, each performed in triplicate.
Effector cells (CTL21.9 - Havele, et al., 1986, Journal of Immunology 137: i448-1454) were stimulated for 24 hours prior to assay at 37~C with a 1/250 dilution of 2C11 hyl~ridoma supernatant (anti-CD3 monoclonal antibody - Leo, et al., 1g87, Proceedings of the National Academy of Sciences USA 84:13744274). L1210 cells (a murine T Iymphoma derived from DBA/2 mice; obtained from Dr. Pierre Goldstein, CNRS, Marseille, France or EL4 cells (a murine T Iymphoma cell line) were infected at a multiplicity of 10 plaque forming units per cell with either CPV (Brighton Red strain) or RPV (Utrecht strain), or were mock-infected in RPMI (RPMI 1640 conlai" ,g 5% fetal bovine serum (Gibco~ and 10~ M ~-mercaptoethanol) for 12 hours at 37~C.
Cells were labeled with 5'Cr (1 OO,uCi per 2 x 1 o6 cells; Dupont NEN) at 37~C for 90 minutes, washed three times, and added to V-bottomed 96-well plates (1 x 104 cells per well) with stimulated CTL21.9 effector cells (5 x 104 cells per wall). Plates were centrifuged at 500 rpm for 5 minutes to promote cell contact, and then incl Ihated for 4 hours at 37~C in RPMI. Supernatants were then removed and counted in a gamma counter. Each assay was performed in triplicate. Specific Iysis and relative % Specific Lysis were calculated as follows: % Specific Lysis = (sample -spontaneous release)/(total - spontaneous release)x100 Relative % Specific Lysis =
% Specific Lysis (virus infected cells)/% Specific Lysis (mock infected cells).
Figs. 2A - 2D are a series of hislo~l dn~s representing i"hil~i~ion of Fas-mediated cytolysis of virus-infected L1210-Fas (Fig. 2A), ~L4 (Fig. 2B) and YAC-1 (Fig. 2C) W O 97/1~006 PCT~US96/14571 -- 3 --cells. L1210-Fas, EL4 and YAC-1 ceils were either mock-infected or infected with the indicated virus as described in the legend to figure 1. Cytoiysis of target cells was assayed by incubation with stimulated PMM-1 effector cells in a standard 4 hour chromium release assay at an effector to target ratio of 5: 1. To facilitate comparison of multiple experiments, the values for % specific Iysis for mock-infected cells were set to 100%, and other data are shown relative to mock infected cells. Each bar represents the mean and standard deviation c~lculated from 3-5 independent experiments, each performed in triplicate. Fig. 2D is the histogram obtained after L1210-FAS and L1210 cells were either mock-infected or infected with CPV or RPV,and incl Ih~ted with stimulated PMM-1 effector cells at an effector to target ratio of 5: 1 and the % specific chromium release was determined. Representative data from a single experiment performed in triplicate are shown. Virus infections, chromium release assays, and c~'~ tions were performed as described in the legend to Figs.
1 A and 1 B PMM-1 effector cells (Kaufmann, et al.,1981, Proceedings of the National Academy of Sciences USA 78:2502) (a BALB/c derived peritoneal exudate Iymphocyte CTL hybridoma; obtained from Dr. G. Berke, Weizmann Institute of Science, Rehovot, Israel, were stimulated prior at assay by incubating with PMA (10 ng/ml; Sigma) and ionomycin (3~,ug/ml; Sigma) at 37~C for three hours. L1210-FASis an L1210 cell transfectant ex,~,r~ssi"g the murine Fas antigen (Rouvier, et al.,1993, Joumal of Experimental Medicine 177:195-200) (obtained from Dr. Pierre Goldstein, CNRS, Marseille, France), and YAC-1 is murine Lymphoma cell line.
Figs. 3A-3F are a series of histograms representing Fas-mediated cytolysis of target celis infected with virus mutants in the SPI-1 or SPI-2 genes. L1210-FAS (Figs.
3A and 3D), EL4 (Figs. 3B and 3E), or YAC-1 (Figs. 3C and 3F) were either mock-infected or infected with either wild type CPV, a CPV mutant in the SPI-1 gene (Thompson, et al.,1993, Virology 197:328-338) (CPV~SPI-1), a CPV mutant in the SPI-2 gene (Ali, et al., 1994, Virology 2~2:305-314) (CPV/~SPI-2), wild type RPV, RPV mutants in the SPI-1, SPI-2 (Ali, et al.,1994, Virology 202:305-314), or both SPI-1 and SPI-2 genes (RPV~SPI-1, RPV~SPI-2, RPV~SPI-1/2, respectively). Data are shown as the % specific Iysis relative to mock infected cells and are the mean and standard deviations of three independent experiments. Virus infections, chromiumrelease assays, and c~lcl ll~tions were performed as described in the legends to Figs.
1A, 1B, and 2A-2D. RPV mutant in both SPI-1 and SPI-2 was constructed from RPV~SPI-1 by homologous recombination leading to replacement of a 447 bp region of the SPI-2 open reading frame with the Ecoli lacZ gene driven by the vaccinia virus p11 promoter.

Figs. 4A4D are a series of histograms representing Granule-mediated cytolysis of target cells infected with virus mutants in the SPI-1 or SPI-2 genes. L1210 (Figs.
4A and 4C) or EL4 {Figs. 4B and 4D) cells were infected with wild type viruses or virus mutants in either the SPI-1 or SPI-2 genes, or with RPV Gor,ldi~ ,i, Ig mutations in both the SPI-1 and the SPI-2 genes (RPV~SPI-1/2; described above). Data are shown as the % specific Iysis relative to mock infected cells and represent the mean and standard deviations of three independent experiments. Virus infections, chromiumrelease assays, and c~ic~ tions were performed as described in the legends to Figs.
1A, 1B, and 2A-2D.
Fig. 5 is a listing of the DNA and amino acid sequences for SPI-1 (SEQ ID Nos:
1 and 2, respectively).
Fig. 6 is a listing of the DNA and amino acid sequences for SPI-2 (SEQ ID NOs:
3 and 4, respectively).

nePile~l D~scri~.lion of the Invention The invention provides a method for inhibiting cell-mediated killing of a target cell.
"Cell-mediated" killing refers to ability of a cell (e.g., a cytotoxic T Iymphocyte (CTL)) to effectuate the death of a target cell, e.g., by cytolysis or ~poptnsic. In practicing the invention, cell death is inhibited by expressing in the target cell a proteinase inhibitor of a pox virus, such as an orthopoxvirus, thereby inhibiting cell-mediated (e.g., CTL-mediated) killing of the target cell.
If desired, the method can include identifying the target cell as a target of cell-mediated killing. Numerous cells are known to be targets of cell-medi~tPd cell death.
P, ~ft:rdbly, the cell is a human cell. For example, cells of heterologous or autologous grafts are targets for cell-me-~iat~d killing, and such cells can be used in the invention.
Bone marrow cells are particularly suitable for use in the invention, as such cells can readily be obtained, maniru'~ d in vitro, and then introduced into a patient. Other suitable cells include cells that are assoc;dled as tissues (e.g., liver tissue) or organs (e.g., hearts) that can be grafted in methods of transplantation. Likewise, cells that are destroyed due to autoimmune diseases, e.g., pancreatic islet cells of diabetes patients, also are targets for cell-mediated killing. In addition, CD4' Iymphocytes of patients infected with I luman Immunodeficiency Virus (HIV) are considered target cells in the invention. Such CD4+ Iymphocytes are thought to be killed by apoptosis that is induced by stimulation of the CD4 molecule on the Iymphocytes by a complex of gp120 and antibody. The Fas antigen/Fas ligand system is thought to mediate the W O 97/10006 PCT~US96/14571 --5-- _ death of CD4+ cells even though the cells are not thought to be infected with HIV.
Thus, cell-mediated killing of these cells results in the massive depletion of CD4 cells that is observed in AIDS patients. When the invention is used in a method of treating a patient, the cell can be autologous or heterologous to the patient.
In a preferred embodiment, the invention can be used to inhibit cell-mediated killing of cells that are the targets of conventional gene therapy methods. Manyconventional gene therapy methods employ viral vectors to express a therapeutic gene in a cell of a mammal. A "therapeutic" gene is any gene that, when expressed, confers a beneficial effect on a cell. In vivo, such a gene is one that ameliorates a sign or symptom of a disorder, or confers a desired phenotype on a cell or another cell of the patient. Such a therapeutic gene can be, for example, a gene that corrects a deficiency in gene expression (e.g., an insulin gene for correcting a deficiency in insulin expression). Traditional gene therapy methods are hindered because viralvectors carrying therapeutic genes also express viral antigens that induce cell-mediated killing of target cells that are ll~nsf~ ed with the viral vectors. Theinvention thus provides a method for inhibiting cell-mediated death of these target cells, with inhibition being accomplished by expressing a poxvirus proteinase inhibitor in the target cell. If desired, the proteinase inhibitor can be expressed from the same vector, or a different vector, as the vector used to express the therapeutic gene.
Additional cells that are targeted for cell-mediated cell killing can readily be identified in conventional in vitro CTL cytotoxicity assays, such as chromium release assays.

Conventional gene delivery and gene expression methods can be used to express a proteinase inhibitor in a cell. For example, vectors derived from ma",n,-' -n viruses, such as retrovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors, are well known in the art and can be used in the invention. Other art-known gene delivery and expression techniques can also be used in the invention. For example, the proteinase inhibitor can be expressed from a genetic construct (i.e., any nucleic acid, such as a plasmid or cosmid, engineered to express a gene). If desired, the vector can be engineered to contain a "suicide' gene. Such genes are known in the art for their ability to encode a factor that renders the cell sensitive to a substance that can be administered to the cell in the event that subsequent killing of the target cell should be desired.
The various genetic constructs can be introduced into a cell by conventional methods, such as liposome-based methods, direct uptake, electroporation, CaCI-based methods, and the like. When liposome-based methods are used, the WO 97/10006 PCT~US96/14571 -6-liposomes can be engineered to have on their surface desired markers, e.g., receptors, antibodies, lectins, or carbohydrates.
Typically, the nucleic acid encoding the proteinase inhibitor is operably linked to a promoter that is active in a mammaiian cell (e.g., a promoter that naturally drives expression of a gene in a mammalian cell or a promoter of a virus that infects amammalian cell). Preferably, the promoter is a promoter of an poxvirus, such as a promoter that naturally drives expression of a proteinase inhibitor. If desired, the promoter can be a cell-specific promoter, a tissue-specific promoter, or a stage-specific promoter. Such promoters are known in the art and can be used to conferspecificity in the practicing the invention. When tissues or organs are used as the target cells in the invention, conventional organ perfusion methods are well suited for delivering the nucleic acid encoding the prot~i. ,ase inhibitor to the cells of the tissue or organ P, ~r~r~bly, the p, uLei"ase inhibitor is a serpin Droteinase Inhibitor. such as SPI-1 (SEQ ID N0: 2; GenBankAccession No. U07766; Ali et al., supra), SPI-2 (SEQ ID
N0: 4; GenBank Acces~ion No. U07763; Ali et al., supra (SPI-2 is also known as crmA in CPV), or SPI-3 (Turner et al., 1995, Viroceptors, Virokines, and RelatedImmune IModulators Encoded by DNA Viruses, Mcradden ed, R.G. Landes Company, Austin, TX). The p,~ ase inhibitor can be derived from any pox virus; preferably, the virus is an orthopox virus, such as a cowpox virus, rabbitpox virus, smallpox virus, or vaccinia virus. Such virus can be obtained from ATCC. Each of these viruses naturally encodes members of the serpin family of proteinase i"hibilor:j, which can be used in the assay. Mutant strains of these viruses that have decreased pathogenicity while nonetheiess ek~,ressi~lg a proteinase inhibitor can used. In addition, variants (e.g., conservative variants) and mutants of these proteinase inhibitors can be used, provided that the resulting polypeptide retains a detect~h'Q ability to inhibit CTL-mediated cell death (e.g., as determined in a CTL cytotoxicity assay as described herein).
By "conservative variant" is meant a polypeptide having an amino acid 3~ substitution where the native amino acid and the substituted amino acid are of approximately the same charge and polarity. Such sl Ihstitutions typically include, e.g., sl Ihstitutions within the following groups: glycine, alanine; valine, isoleucine, leucine, methionine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;
Iysine, arginine; and phenylalanine, tyrosine. In general, such conservative amino acid sl Ihstit~ Itions do not significantly affect the function of the polypeptide. If desired, the ability of a variant or mutant to function as a proteinase inhibitor can be measured W O 97/10006 PCT~US96/14571 in a CTL cytotoxicity assay as described herein, using the wild-type proteinase inhibitor as a standard.
The invention also presumes the use of nucleic acids encoding a protease inhibitor where the nucleic acid is a degenerate variant of the nucleic acid sequences ~ 5 expressly described herein. By "degenerate variants" of a nucleotide sequence is meant nucleotide sequences that encode the same amino acid sequence as a given nucleotide sequence, but in which at least one codon in the nucleotide sequence is different. Degenerate variants occur due to the degeneracy of the genetic code, whereby two or more different codons can encode the same amino acid. Applicants have discovered that the presence of nucleotide encoding SP-1 polypeptide and nucleotide encoding SP-2 polypeptide provide enhanced inhibition of cell-mediated killing.
Included within the invention is an isol~ted target cell that is resistant to cell-mediated killing. Such cells contain a nucleic acid that consists essentially of a nucleic acid that expresses a poxvirus SPI-1 polypeptide, as described herein. Such cells can also include a nucleic acid that consists essentially of a nucleic acid that expresses an SPI-2 polypeptide. If desired, these cells can also express a therapeutic gene. The term "isolated" target cell means that a gene is delivered to a cell and expressed non-systemically in a population of cells (e.g., in liver cells or pancreatic islet cells). Such cells can be in the form of a cell suspension (e.g., bone marrow cells) or they can be in the form of a tissue or organ (e.g., a liver) for use in organ ll~nspla. ,l~Lion methods. Enco" ,p~ssed by this term are cell populations of a mammal that are targeted for SPI-1 expression, without systemic expression of SPI-1 in the mammal. For example, liver cells of a mammal that are targeted for SPI-1 expression are considered "isolated," even when the cells are contained within the mammal.
The invention also provides a method for determining whether a nucleic acid (i.e., a "test" nucleic acid) encodes a polypeptide that inhibits cell-mefJi-terl killing of a target cell. Thus, this aspect of the invention provides a method for identifying additional proteinase inhibitors that can be used in the methods described above.
One method entails introducing into a target cell a mutant poxvirus that does not express a functional SPI-2 polypeptide; expressing in the target cell the "test" nucleic acid; and detecting inhibition of cell-me~i~ted killing of the target cell as an indication that the test nucleic acid encodes a polypeptide that inhibits cell-mediated killing. In this sense, the "test" nucleic acid is tested for its ability to complement the SPI-2 deletion. Mutant viruses that fail to encode functional SPI-2 are known in the art (Ali CA 02231804 1998-03-ll W O 97/10006 PCT~US96/14~71 --8-- .
et al., infra), and additional viruses can be produced using conventionai mutagenesis methods. It is expected that the majority of such polypeptides will be proteinase inhibitors that, like SPI-2, belong to the serpin family of proteinase inhibitors.
Typicaliy, the polypeptide encoded by the "test" nucleic acid is further characterized by testing its ability to confer resistance to cell-mediated killing in the presence of a 4 poxvirus that does not encode a functional SPI-1 polypeptide. Such an SPI-1 mutant has been described (Thompson et al., infra), and additional mutant viruses can be produced using conventional gene manipulation techniques. A "test" nucleic acid encoding a polypeptide that confers resistance to cell-mediated killing in both of the aforementioned genetic settings is considered a new inhibitor of cell-mediated killing.
The nucleic acid encoding such a polypeptide can then be used in the invention to confer on a target cell resistance to cell-mediated killing.

Exal... les The following exalllFlEs and meant to illustrate, not limit, the invention, the metes and bounds of which are determined by the claims. While the methods described inthese examples are typical of those that can be used, other procedures known to those skilled in the art may alternatively be used.

Example l: Cowpox Virus and Rabbitpox Virus Inhibit CTL-Mer~ 1 Cytolysis This example shows that cells infected with an orthopoxvirus are resistant to CTL-mediated cytolysis. In this example, target cells were Infected with wild-type cowpox virus (CPV (Brighton Red strain) or rabbbitpox virus (RPV (Utrecht strain)), (American Type Culture Collection, Rockville, Mr~.), at a m~ ''i,1i~;ily of infection (moi3 of ten piaque forming units (pfu)/cell for twelve hours. Each of these viruses expresses a serpin proteinase inhibitor in the infected cells. The target cells were then assayed for cytolysis by CTL21.9 effector cells in a conventional chromium release assay. In this example, the target cells were L1210 (ATCC) and EL4 cells(thymoma cells). These target cells are known to be er~i~;erilly Iysed by CTL21.9, and iysis occurs in a caicium-dependent reaction that involves the secreti3n of cytoplasl lliC
granules (Garner et al., 1 994, J. Immun. 153:5413-5421).
Infection of L1210 cells with CPV resulted in a dramatic and reproducible inhibition of cytolysis by CTL21.0 (Fig. 1A), as compared with mock-infected cells.
Infection with RPV also resulted in significant inhibition of CTL-mediated cytolysis W O 97/10006 PCT~US96/14S71 _9_ (Fig. 1A). Infection of E~4 cells with either CPV or RPV resulted in an approximately 50~/0 reduction in the level of cytolysis by CTL21.g (Fig.1 B). The calcium-dependent nature of cytolysis of these cells, was confirmed by carrying out the assays in the presence of EGTA (data not shown). In sum, this example illustrates that infection , 5 by CPV and RPV each can inhibit the ability of CTL to Iyse target cells.

Example ll: CPV and RPV Each Inhibit the Fas Pd~ ay of Cytolysis This example demonstrates that cytolysis that occurs via the Fas pathway can be inhibited by infecting a target cell with CPV or RPV. In this example, PMM-1 cells were used as the cytolytic effectors. PMM-1 is a cytolytic hybridoma cell line that does not express perforin or granzymes (Kaufmann et al.,1981, PNAS 78:2502 and Kelgason et al., 1992, Eur. J. Immunol. 22:3187-3190) . These CTL iyse target cells via the Fas pathway (Garner et al., 1994, 153:5413-5421). Each of the target cells in this experiment expressed Fas. These cells were L1210-Fas (an L1210 cell transfected with sequences ex,u, ~ssi"g murine Fas (Rouvier et al.,1993, J. Exp. Med.
177:195-200)), EL4 cells, and YAC-1 cells (ATCC).
Infection of the Fas-expressing target cells with CPV or RPV inhibited the ability of PMM-1 cells to Iyse the target cells via the Fas pathway (Figs. 2A-2C). As a control, L1210 cells that do not express Fas were used in the experiment. L1210 cells, unlike L1210-Fas cells, are not Iysed efficiently, indicating that cytolysis by PMM-1 cells is dependent upon expression of Fas (Fig. 2D). In addition, the mechanism of killing is independent of calcium concer,l,~lion, as determined by the observation that killing is unaffected by the presence of EGTA. In sum, this example illustrates that expression of CPV of RPV in a target cell can inhibit CTL-mediated cell death the occurs by the Fas pathway of cytolysis.

2~ Example lll: CPV SPI-2 and SPI-1 Inhibit Fas-mediated Cytolysis This ~xdlllple demonstrates that expression of SPI-2 and SPI-1 are responsible for the ability of CPV and RPV to inhibit CTL-mediated cytolysis. Mutated versions of SPI-1 and SPI-2 were used in these experiments to demonsl~te the role of SPI-1 and SPI-2. The SPI-1 mutant, CPV~,SPI-1, has been described previously (Thompson et al., 1993, Virology 197:328-338). ~ikewise, the SPI-2 mutant, CPV~SPI-2, has been described (Ali et al., 1994, Virology 202:305-314). Cells infected with CPV containing a mutation in the SPI-2 gene were Iysed by PMM-1 W O 97/10006 PCTrUS96/14571 effectors at a level comparable to the level of Iysis obtained with mock-infected cells (Figs. 3A-3C). Cells infected with a CPV mutant having a mutation in the SPI01 gene showed comparable inhibition to wild-type CPV-infected cells. Similarly, mutation of the RPV SPI-2 gene almost completely relieved virus-mediated inhibition of cytolysis of infected L1210-FAS cells. In addition, mutation of SPI-2 partialiy relieved virus-mediated inhibition of EL4 cells and YAC-1 cells (Figs. 3D-3F). In these experiments, mutation of both the SPI-1 and SPI-2 genes completely abrogated RPV-mediated inhibition of cytolysis in EL4 and YAC-1 cells (Figs. 3E and 3F). These data indicate that both SPI-1 and SPI-2 confer resistance to CTL-mediated cytolysis that occurs via the Fas pathway.

Example IV: Use of SPI-1 and SPI-2 in Combination to l~hibit Granzyme-Me~ 1 Cytolysis In conl-~L to the results obtained with PMM-1 effector cells, mutations in either the SPI-1 or SPI-2 genes alone for both CPV and RPV did not completely relieve virus-rrlediated inhibition of granule-mediated cytolysis by CTL21.9 effector cells (Figs. 4A 4 D). However, an RPV strain that contained a mutation in both the SPI-1 and SPI-2 genes completely lacked the ability to inhibit cytolysis by CTL21.9 cells (Figs. 4C and 4D). These data indicate that, while SPI-1 and SPI-2 individually are able to inhibit cytolysis via the Fas pathway, complete inhibition of granule-mediated cytolysis is best achieved by expression of both SPI-1 and SPI-2.
The description provided herein is meant to illustrate, but not limit, the scope of the invention. Indeed, following the guidance provided herein, those of ordinary skill in the art can readily practice various additional embodiments of the invention.

~U~NU~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Bleackley et al., R. Chris (ii) TITLE OF INVENTION: METHOD FOR INHIBITING CELL-MEDIATED
KILLING OF TARGET CELLS
(iii) NUMBER OF ~Uu~N~: 4 (iv) ~uKKEs~uN~ENCE ADDRESS:
(A) Ann~c~cEE Fish & Richardson P.C
(B) STREE:T: 4225 Executive Square, Suite 1400 (C) CITY: La ~olla (D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 92037 (v) ~:u.. ~ READABLE FORM:
~A) MEDIUM TYPE: Floppy disk (B) CU.I~U~K: IBM PC compatible ~C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CL~SSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/003,665 (B) FILING DATE: ll-SEP-1995 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Wetherell, ~r., vIohn R.
(B) REGISTRATION NUMBER: 31,678 (c) REFERENCE/DOCKET NUMBER: 07254/019001 ~ix) TELEuu. .uNlCATION INFORMATION:
(A) TELEP~ONE: 619/678-5070 (B) TELEFAX: 619/678-5099 (2) INFORMATION FOR SEQ ID NO:l:
(i) ~;.2UI:;N--~; rTT~p7~TRFl T .cTICS
(A) LENGTH: 1074 base pairs (B) TYPE: nucleic acid (C) ST~NnEr'-~CS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1074 (Xi) ~:~U~NU~ DESCRIPTION: SEQ ID NO:1:

- ATG GAT ATC TTT AAA GAA CTA ATC TTA A~A CAC CCT GAT GAA AAT GTT 48 45 Met Asp Ile Phe Lys Glu Leu Ile Leu Lys His Pro Asp Glu Asn Val Leu Ile Ser Pro Val Ser Ile Leu Ser Thr Leu S~r Ile Pro Asn His Gly Ala Ala Gly Ser Thr Ala Glu Gln Leu Ser Lys Tyr Ile Glu Asn GTG AAT GAG AAT ACC CCC GAT GaT AAG AAG GAT GAC AAT AAT GAC ATG 192 Val Asn Glu Asn Thr Pro Asp Asp Lys Lys Asp Asp Asn Asn Asp Met GAC GTA GAT ATT CCG TAT TGT GCG ACA CTA GCT ACC GCA AAT A~A ATA 240 Asp Val Asp Ile Pro Tyr Cy8 Ala Thr Leu Ala Thr Ala Asn Ly3 Ile Tyr Cy5 Ser Asp Ser Ile Glu Phe His Ala Ser Phe Leu Gln Lys Ile AAA GAC GGT TTT CAA ACT GTA AAC TTT A~T AAT GCT AAC CAA ACA AAG 336 Lys Asp Gly Phe Gln Thr Val Asn Phe A3n Asn Ala Asn Gln Thr Lys GAA CTA ATC AAC GAA TGG GTT AAG ACG ATG ACA AAT GGT AA~ ATT AAT 384 Glu Leu Ile Asn Glu Trp Val Lys Thr Met Thr Asn Gly Lys Ile Asn Ser Leu Leu Thr Ser Pro Leu Ser Ile Asn Thr Arg Met Thr Val Val Ser Ala Val His Phe Lys Ala Met Trp Lys Tyr Pro Phe Ser Lys His Leu Thr Tyr Thr Asp ~y3 Phe Tyr Ile Ser Lys Asn Ile Val Thr Ser Val Asp Met Met Val Ser Thr Glu Asn A3p Leu Gln Tyr Val His Ile Asn Glu Leu Phe Gly Gly Phe Ser Ile Ile Asp Ile Pro Tyr Glu Gly Asn Ser Ser Met Val Ile Ile Leu Pro Asp Asp Ile Glu Gly Ile Tyr Asn Ile Glu Ly3 Asn Ile Thr Asp Glu Lys Phe Lys Ly3 Trp Cys Gly Met Leu Ser Thr Lys Ser Ile Asp Leu Tyr Met Pro Lys Phe Lys Val Glu Met Thr Glu Pro Tyr Asn Leu Val Pro Ile Leu Glu Asn Leu Gly Leu Thr Asn Ile Phe Gly Tyr Tyr Ala Asp Phe Ser Lys Met Cys Asn Glu Thr Ile Thr Val Glu Lys Phe Leu His Thr Thr Phe Ile Asp Val Asn Glu Glu Tyr Thr Glu Ala Ser Ala Val Thr Gly Val Phe Met Thr W O 97/10006 PCT~US96/14571 -13- -' Asn Phe Ser Met Val Tyr Arg Thr Lys Val Tyr Ile Asn His Pro Phe ATG TAC ATG ATT A~A GAC AAC ACA GGA CGT ATA CTT TTT ATA GGG A~A 1056 Met Tyr Met Ile Lys Asp Asn Thr Gly Arg Ile Leu Phe Ile Gly Lys Tyr Cys Tyr Pro Gln (2) INFORMATION FOR SEQ ID NO:2:
(~: CHARACTERISTICS:
(A) LENGTH: 358 amino acids (B) TYPE: amino ac~d (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~UU~N~ DESCRIPTION: SEQ ID NO:2:
Met Asp Ile Phe Lys Glu Leu Ile Leu Lys His Pro Asp Glu Asn Val Leu Ile Ser Pro Val Ser Ile Leu Ser Thr Leu Ser Ile Pro Asn His Gly Ala Ala Gly Ser Thr Ala Glu Gln Leu Ser Lys Tyr Ile Glu Asn Val Asn Glu Asn Thr Pro Asp Asp Lys Lys Asp Asp Asn Asn Asp Met Asp Val Asp Ile Pro Tyr Cys Ala Thr Leu Ala Thr Ala Asn Lys Ile Tyr Cys Ser Asp Ser Ile Glu Phe His Ala Ser Phe Leu Gln Lys Ile 85 90 g5 Lys Asp Gly Phe Gln Thr Val Asn Phe Asn Asn Ala Asn Gln Thr Lys loo lo5 llo Glu Leu Ile Asn Glu Trp Val Lys Thr Met Thr Asn Gly Lys Ile Asn Ser Leu Leu Thr Ser Pro Leu Ser Ile Asn Thr Arg Met Thr Val Val Ser Ala Val His Phe LYB Ala Met Trp Lys Tyr Pro Phe Ser Lys His Leu Thr Tyr Thr Asp Lys Phe Tyr Ile Ser Lys Asn Ile Val Thr Ser Val Asp Met Met Val Ser Thr Glu Asn Asp Leu Gln Tyr Val His Ile Asn Glu Leu Phe Gly Gly Phe Ser Ile Ile ASp Ile Pro Tyr Glu Gly Asn Ser Ser Met Val Ile Ile Leu Pro Asp Asp Ile Glu Gly Ile Tyr Asn Ile Glu Lys Asn Ile Thr Asp Glu Lys Phe Lys Lys Trp Cys Gly Met Leu Ser Thr Lys Ser Ile Asp Leu Tyr Met Pro Lys Phe Lys Val Glu Met Thr Glu Pro Tyr Asn Leu Val Pro Ile Leu Glu Asn Leu Gly Leu Thr Asn Ile Phe Gly Tyr Tyr Ala Asp Phe Ser Lys Met Cys Asn Glu Thr Ile Thr Val Glu Lys Phe Leu Hi3 Thr Thr Phe Ile Asp Val Asn Glu Glu Tyr Thr Glu Ala Ser Ala Val Thr Gly Val Phe Met Thr Asn Phe Ser Met Val Tyr Arg Thr Lys Val Tyr Ile Asn His Pro Phe Met Tyr Met Ile Lys Asp Asn Thr Gly Arg Ile Leu Phe Ile Gly Lys 15 Tyr Cy8 Tyr Pro Gln (2) INFORMATION FOR SEQ ID NO:3:
(i) ~-~u~ ~hRhrT~RTcTIcs:
(A) LENGTH: 1038 base pairs (B) TYPE: nucleic acid (C) sTRhr~nN~.cs: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
25 (B) LOCATION: 1.. 1038 (Xi) ~U~:NC~: DESCRIPTION: SEQ ID NO:3:

Met Asp Ile Phe Arg Glu Ile Ala Ser Ser Met Ly3 Gly Glu Asn val Phe Ile Ser Pro Ala Ser Ile Ser Ser Val Leu Thr Ile Leu Tyr Tyr GGA GCT AAT GGA TCC ACT GCT GAA CAG CTA TCG AhA TAT GTA GAA AAG 144 Gly Ala Asn Gly Ser Thr Ala Glu Gln Leu Ser Lys Tyr Val Glu Lys Glu Glu Asn Met Asp Lys Val Ser Ala Gln Asn Ile Ser Phe Lys Ser ATA AAT AAA GTA TAT GGG CGA TAT TCT GCC GTG TTT AhA GAT TCC TTT 240 Ile Asn Lys Val Tyr Gly Arg Tyr Ser Ala Val Phe Ly3 Asp Ser Phe Leu Arg Lys Ile Gly Asp Lys Phe Gln Thr Val Asp Phe Thr Asp Cy9 Arg Thr Ile Asp Ala Ile Asn Lys Cys Val Asp Ile Phe Thr Glu Gly Lys Ile Asn Pro Leu Leu Asp Glu Gln Leu Ser Pro Asp Thr Cys Leu CTA GCA ATT AGT GCC GTA TAC TTT A~A GCA AAA TGG TTG ACG CCA TTC 432 Leu Ala Ile Ser Ala Val Tyr Phe Lys Ala Lys Trp Leu Thr Pro Phe Glu Lys Glu Phe Thr Ser A3p Tyr Pro Phe Tyr Val Ser Pro Thr Glu Met Val Asp Val Ser Met Met Ser Met Tyr Gly Lys Ala Phe Asn His Ala Ser Val Lys Glu Ser Phe Gly Asn Phe Ser Ile Ile Glu Leu Pro Tyr Val Gly Asp Thr Ser Met Met Val Ile Leu Pro Asp Lys Ile Asp ' 555 560 565 20 Gly Leu Glu Ser Ile Glu Gln Asn Leu Thr Asp Thr Asn Phe Lys Lys Trp Cys Asn Ser Leu Glu Ala Thr Phe Ile Asp Val His Ile Pro Lys Phe Lys Val Thr Gly Ser Tyr Asn Leu Val Asp Thr Leu Val Lys Ser Gly Leu Thr Glu Val Phe Gly Ser Thr Gly ASp Tyr Ser Asn Met Cys Asn Leu Asp Val Ser Val Asp Ala Met Ile His Lys Thr Tyr Ile Asp 35 Val Asn Glu Glu Tyr Pro Glu Ala Ala Ala Ala Thr Ser val Leu Val GCA GAC TGT GCA TCA ACA GTT ACA AAT GAG TTC TGT GCA GAT CAT CCG g60 Ala Asp Cys Ala Ser Thr Val Thr Asn Glu Phe Cys Ala Asp His Pro Phe Ile Tyr Val Ile Arg His Val Asp Gly Lys Ile Leu Phe Val Gly Arg Tyr Cys Ser Pro Thr Thr Asn Cys ~ 45 695 700 ~2) INFORMATION FOR SEQ ID NO:4:
(i) ~yU~:N~ CHARACTERISTICS:
(A) LENGTH: 346 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) M~T~crTTlT~ TYPE: protein (xi) S~QUENCE D~SCRIPTION: SEQ ID NO:4:
Met ASp Ile Phe Arg Glu Ile Ala Ser Ser Met Lys Gly Glu Asn Val l 5 10 15 Phe Ile Ser Pro Ala Ser Ile Ser Se~ Val Leu Thr Ile Leu Tyr Tyr Gly Ala Asn Gly Ser Thr Ala Glu Gln Leu Ser Lys Tyr Val Glu Lys Glu Glu Asn Met Asp Lys val Ser Ala Gln Asn Ile Ser Phe Lys Ser 10 Ile Asn Lys Val Tyr Gly Arg Tyr Ser Ala Val Phe Lys Asp Ser Phe Leu Arg Lys Ile Gly Asp Lys Phe Gln Thr Val Asp Phe Thr Asp Cys Arg Thr Ile Asp Ala Ile Asn Lys Cys Val Asp Ile Phe Thr Glu Gly loo 105 110 Lys Ile Asn Pro Leu Leu Asp Glu Gln Leu Ser Pro Asp Thr Cys Leu Leu Ala Ile Ser Ala Val Tyr Phe Lys Ala Lys Trp Leu Thr Pro Phe Glu Ly3 Glu Phe Thr Ser ASp Tyr Pro Phe Tyr Val Ser Pro Thr Glu Met Val ASp Val Ser Met Met Ser Met Tyr Gly Lys Ala Phe Asn His Ala Ser Val Lys Glu Ser Phe Gly Asn Phe Ser Ile Ile Glu Leu Pro Tyr Val Gly Asp Thr Ser Met Met Val Ile Leu Pro Asp Lys Ile Asp Gly Leu Glu Ser Ile Glu Gln Asn Leu Thr Asp Thr Asn Phe Lys Lys Trp Cys Asn Ser Leu Glu Ala Thr Phe Ile Asp Val His Ile Pro Lys Phe Lys Val Thr Gly Ser Tyr Asn Leu Val Asp Thr Leu Val Lys Ser Gly Leu Thr Glu Val Phe Gly Ser Thr Gly Asp Tyr Ser Asn Met Cys Asn Leu Asp Val Ser Val Asp Ala Met Ile His Lys Thr Tyr Ile Asp Val Asn Glu Glu Tyr Pro Glu Ala Ala Ala Ala Thr Ser Val Leu Val Ala Asp Cys Ala Ser Thr Val Thr Asn Glu Phe Cys Ala Asp His Pro Phe Ile Tyr Val Ile Arg His Val Asp Gly Lys Ile Leu Phe Val Gly Arg Tyr Cys Ser Pro Thr Thr Asn Cys

Claims (35)

What is claimed is:

1. A method for inhibiting cell-mediated killing of a mammalian target cell, themethod comprising introducing into the target cell a nucleic acid consisting essentially of a nucleic acid expressing a poxvirus serpin proteinase inhibitor (SPI)-1 polypeptide, thereby inhibiting cell-mediated death of the target cell.

2. The method of claim 1, further comprising introducing into the target cell a nucleic acid consisting essentially of a nucleic acid expressing a poxvirus SPI-2 polypeptide.

3. The method of claim 1, further comprising introducing into the target cell a nucleic acid consisting essentially of a nucleic acid expressing a therapeutic polypeptide.

4. The method of claim 1, wherein the poxvirus is an orthopoxvirus.

5. The method of claim 4, wherein the orthopoxvirus is cowpox virus.

6. The method of claim 4, wherein the orthopoxvirus is rabbitpox virus.

7. The method of claim 4, wherein the orthopoxvirus is a smallpox virus.

8. The method of claim 4, wherein the orthopoxvirus is a vaccinia virus.

9. The method of claim 1, wherein SPI-1 has the amino acid sequence of SEQ
ID NO: 2 or conservative variants thereof.

10. The method of claim 2, wherein SPI-2 has the amino acid sequence of SEQ
ID NO: 3 or conservative variants thereof.

11. The method of claim 1, further comprising introducing into the cell a nucleic acid consisting essentially of a nucleic acid expressing SPI-3 or conservative variants thereof.

12. The method of claim 1, wherein introducing occurs in vitro.

13. The method of claim 1, wherein introducing occurs in vivo.

14. The method of claim 1, further comprising administering the target cell to apatient.

15. The method of claim 14, wherein the target cell is heterologous to the patient.

16. The method of claim 14, wherein the target cell is autologous to the patient.

17. The method of claim 1, wherein the introduced nucleic acid is contained within a liposome.

18. The method of claim 1, wherein the introduced nucleic acid comprises a viralvector.

19. The method of claim 18, wherein the viral vector is derived from a mammalian virus.

20. The method of claim 19, wherein the mammalian virus is selected from the group consisting of retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses.

21. The method of claim 1, wherein the target cell is a human cell.

22. The method of claim 1, wherein the target cell is a pancreatic islet cell.

23. The method of claim 1, wherein the target cell is a bone marrow cell.

24. The method of claim 1, wherein the target cell is a CD4+ lymphocyte.

25. The method of claim 24, wherein the CD4+ lymphocyte is obtained from a patient infected with a human immunodeficiency virus.

26. The method of claim 24, wherein introducing comprises perfusing the a tissue with a liquid comprising a nucleic acid encoding the SPI-1.

27. An isolated target cell that is resistant to cell-mediated death, the cell comprising a nucleic acid consisting essentially of a nucleic acid expressing a poxvirus serpin proteinase inhibitor (SPI)-1 polypeptide.

28. The cell of claim 27, further comprising a nucleic acid consisting essentially of a nucleic acid expressing an SPI-2 polypeptide.

29. A method for determining whether a test nucleic acid encodes a polypeptide that inhibits cell-mediated killing of a target cell, the method comprising introducing into a target cell a poxvirus that is defective in the expression of at least one serpin proteinase inhibitor;
expressing in the target cell the test nucleic acid; and detecting inhibition of killing of the target cell as an indication that the test nucleic acid encodes a polypeptide that inhibits cell-mediated killing.

30. The method of claim 29, wherein the poxvirus is an orthopox virus.

31. The method of claim 29, wherein the proteinase inhibitor is selected from the group consisting of SPI-1 and SPI-2.

32. The method of claim 29, wherein detecting is in vitro.

33. The method of claim 29, wherein detecting is in vivo.

34. The cell of claim 27 further comprising a therapeutic gene.

35. The cell of claim 28 further comprising a therapeutic gene.