WO1999029864A1 - Recombinant vaccinia constructs expressing feline il-2, compositions and methods of use thereof - Google Patents

Abstract

Recombinant poxvirus, specifically an avipox virus, preferably a canarypox virus, having therein a nucleic acid molecule encoding a biological response modifier, specifically a feline cytokine, or immunomodulating fragment thereof, in a non-essential region of the recombinant virus genome infect cells to express the biological immune response modifier. The recombinant poxvirus encoding a feline cytokine can be used to enhance or augment the immune response of said feline to antigenic components of infectious agents or tumors. The infected cells can be used to treat cancer in animals by intratumor administration or by in vivo treatment of tumor cells and reintroduction of the treated tumor cells. The cells also are used, in a non-replicating form, to protect felines against tumor formation.

Description

RECOMBINANT VACCINIA CONSTRUCTS EXPRESSING FELINE IL-2, COMPOSITIONS AND METHODS OF USE THEREOF

BACKGROUND OF THE INVENTION A number of strategies are being pursued in the search for effective immunotherapy for cancer and infectious diseases in companion animals as well as in humans. Many of these approaches are based on stimulating the host immune response against the tumor or infectious agent. The administration of cytokines to promote this type of antitumor or protective immune response is one such method of achieving this effect. Interleukin-2 (IL-2) or T Cell Growth Factor (TCGF) stimulates T and B cell growth

(Morgan et al, 1976), augments interferon-γ production and immunoglobulin production by B cells (Ruscetti et al, 1977; Grimm et al, 1983), induces IL-6 production by monocytes (Ceuppens et al, 1985) and modulates expression of the IL-2 receptor (Robb et al, 1984). lnterferon-γ upregulates MHC expression to enhance antigen presentation, while IL-6 enhances CTL differentiation and IgG secretion by B cells. IL-2 is necessary for complete activation of the immune response to a foreign antigen. The inclusion of exogenous IL-2 at the time of immunization ensures adequate amounts of IL-2 are present locally for a complete and efficient activation of the immune system.

IL-2 has demonstrated varying levels of modulation of antitumor activity. However, further improvements are necessary if this strategy is to be applied in the veterinary clinic. In order to circumvent toxicity issues associated with the systemic administration of cytokines, a method of localized delivery of cytokines to the tumor microenvironment at appropriate concentrations is required. Therefore, the delivery system employed to achieve this goal is a key to success.

SUMMARY OF THE INVENTION In accordance with one aspect of the present invention, there is provided a recombinant poxvirus containing therein a nucleic acid sequence encoding a biological response modifier or immunomodulating fragment thereof in a non-essential region of the recombinant virus genome. The poxvirus may be an avipox virus, particularly a canarypox virus, for example, an

ALVAC virus expressing the biological response modifier. Alternatively, the poxvirus may be a vaccinia virus, for example, a NYVAC virus expressing the biological response modifier. The recombinant poxvirus used herein generally has non-essential virus-encoded genetic function inactivated therein. The biological response modifier preferably is a cytokine or a chemokine, such as a feline cytokine. Examples of cytokines useful in the present invention, include interleukins, such as IL-2 or IL-12, or granulocyte macrophage colony stimulating factor (GM-CSF).

The present invention includes use of the recombinant poxvirus described herein to augment or enhance the immune response of an animal, preferably a feline, to an antigen. The antigen may be co-administered with the recombinant poxvirus. In a preferred embodiment, the recombinant poxvirus may be engineered to contain, in addition to the biological response modifier of the present invention, a foreign gene sequence encoding an antigen of interest, such that the biological response modifier and the antigen are co-expressed. Alternatively, the antigen may be administered, by conventional means known to those having skill in the art, to an animal that has been previously or contemporaneously infected with the recombinant poxvirus of the present invention. By administering to an animal in this way the biological response modifier together with the antigen of interest, an enhanced immune response to the antigen of interest may be obtained relative to that observed were the antigen of interest to be administered without the biological response modifier.

The present invention further includes, in another aspect thereof, cells infected by a recombinant poxvirus as provided herein and wherein the biological response modifier is expressed in the cells. The cells usually are tumor cells including feline tumor cells. As discussed above, the biological response modifier that is expressed in the cells preferably is a cytokine or chemokine.

The recombinant poxvirus provided herein as well as the cells infected thereby may be used in the treatment of a biological response modifier-susceptible condition in an animal. Accordingly, in a further aspect of the invention, there is provided a method of treatment of a biological response modifier-susceptible condition in an animal, preferably a feline, which comprises administering to the animal an effective amount of a recombinant poxvirus provided herein, which may be in the form of the infected cells provided herein.

The susceptible condition generally is presence of, or susceptibility to, infectious agents or the presence of tumor cells in the animal. In cases where the susceptible condition involves an infectious disease, the biological response modifier may be delivered in conjunction with a conventional vaccine. In cases where the susceptible condition involves the presence of tumor cells, the recombinant poxvirus or infected cells may be administered intratumorally in vivo. Alternatively, the recombinant virus may be administered by removing tumor cells from the animal, infecting the tumor cells with the recombinant virus and administering the infected tumor cells to the animal. The biological response modifier may be a cytokine or a chemokine, preferably an interleukin, and more preferably IL-2.

Tumor cells which have been infected with the recombinant virus of the present invention may be used, in a non-replicating form, to protect an animal against tumor formation. In an additional aspect of the present invention, therefore, there is provided a method of protecting an animal against tumor formation which comprises administering to the animal an effective amount of non-replicating tumor cells which have been infected by a recombinant poxvirus as provided herein.

Non-replicating tumor cells may be prepared by irradiating cells as provided herein. The invention extends to the recombinant poxvirus provided herein when used as a medicament, as well as to the use of the recombinant poxvirus provided herein in the manufacture of a medicament for the treatment of a biological response modifier-susceptible condition in an animal.

Further, the invention extends to the cells provided herein when used as a medicament, as well as to the use of the cells in the manufacture of a medicament for the treatment of a biological response modifier-susceptible condition in an animal.

Recombinant poxvirus expressing a biological response modifier, such as cytokine and chemokines, augment or enhance an immune response to an antigen or antigens. The antigens can be provided independently of the recombinant poxvirus or, preferably, they are encoded by the recombinant poxvirus to ensure optimal spatial and temporal delivery of the antigen together with the biological response modifier.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 demonstrates expression of functional feline IL-2 from the recombinant virus vCP1338.

GENERAL DESCRIPTION OF THE INVENTION The construction of the recombinant poxviruses is described in a number of granted United States patents. U.S. Patents Nos. 4,603,112, 4,769,330, 4,722,848, 5,110,587 and 5,364,773 relate to various recombinant virus constructs, including ALVAC and NYVAC. U.S.

Patents Nos. 5,453,364, 5,225,336 and 5,155,020 relate to attenuated recombinant vaccinia virus constructs. U.S. Patents Nos. 5,174,993 and 5,505,941 relate to recombinant avipox vims constructs and methods of use thereof. (Throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Despite having promising attributes as a "universal" immunization vehicle, safety issues have provided a concern for the re- introduction of vaccinia virus as an immunizing agent. These concerns stem from complications observed during the Smallpox Eradication Program (Fenner et al, 1988). From one perspective, the safety issues surrounding the use of vaccinia-based vaccine candidates have been addressed with the development of the NYVAC and ALVAC vectors.

The NYVAC strain was derived from the vaccinia virus Copenhagen strain by the precise deletion of 18 ORFs encoding functions implicated in the pathogenicity of orthopoxviruses, as well as host-range regulatory functions governing the replication competency of these viruses on cells from certain species (Tartaglia et al, 1992). General biological properties of NYVAC include: 1) a highly debilitated replicative capacity on cells derived from mice, swine, equids, and humans; 2) the ability to replicate with wildtype efficiency on primary chick embryo fibroblasts; and 3) a highly attenuated phenotype in immunocompetent and immunocompromised animal systems used historically to assess the virulence of vaccinia virus strains (Tartaglia et al, 1992). These attributes are described in detail in U.S. Patent No. 5,494,807. Despite these highly attenuated properties, NYVAC has been shown to function effectively as an immunization vehicle (Tartaglia et al, 1992; Konishi et al, 1992). These properties are consistent with NYVAC providing a safer alternative to existing vaccinia virus vaccine strains for developing vector-based vaccine candidates. Due to the attenuation profile of NYVAC, the Recombinant DNA Advisory Committee of the National Institutes of Health has reduced the biological containment level of this virus from BSL-2 to

BSL-1. It is the only member of the Orthopoxvirus genus accorded a BSL-1 biocontainment level.

The basic vaccinia virus vector technology has been extended to other members of the poxvirus family. Extension to the Avipoxvirus genus, in particular fowlpoxvirus (FPV), was targeted for species-specific applications in the poultry industry (Taylor et al, 1988a). Studies with a FPV recombinant expressing an immunogen from a mammalian pathogen (the rabies virus glycoprotein G), however, demonstrated the ability of this recombinant to elicit immune responses in a number of non-avian species (Taylor et al, 1988b), thus establishing these viruses as viable candidates for developing non-replicating vector-based vaccine candidates for veterinary and human application. The inability of the Avipoxviruses to productively replicate in non-avian species provides an exquisite safety barrier against the occurrence of vaccine- associated and vaccine-induced complications. Subsequent studies with canarypoxvirus (CPV)-based recombinants in non-avian species also demonstrated their utility as immunization vehicles (Taylor et al, 1991 ; Taylor et al, 1992). In this regard the canarypoxvirus-based recombinants were found superior to similar FPV recombinants and equivalent to thymidine-kinase mutants of replication-competent vaccinia virus recombinants (Tartaglia et al, 1992, Taylor et al, 1992). A plaque-cloned isolate of CPV was derived from the vaccine strain Kanapox and designated ALVAC (Tartaglia et al. , 1992).

ALVAC, like NYVAC, has demonstrated a highly attenuated phenotype in a number of animal systems comparing existing vaccinia virus vaccine strains (Tartaglia et al, 1992). The Recombinant DNA Advisory Committee has reduced the biological containment for ALVAC to BSL-1. The concept of using a non-replicating vector in humans was supported by the results of phase I clinical trials using an ALVAC-based rabies G (Cadoz et al, 1992) and an ALVAC- HIV-1_MN env (Pialoux et al, 1995) recombinant.

In this invention, the poxvirus vectors, particularly the ALVAC and NYVAC vectors, are used in a novel manner to modulate an immune response. Several biological response modifiers have been cloned and assessed in the treatment of various conditions including cancer, autoimmunity and transplantation. The effectiveness of many of these treatments has been limited by the large doses required and the associated toxicity. To avoid these issues, the recombinant poxvirus vector, particularly the ALVAC vector, is used to deliver immunomodulatory molecules, such as cytokines or chemokines, to the appropriate environment in an animal, particularly a feline, at appropriate concentrations. Thus, the use of ALVAC or

NYVAC expressing immunomodulatory molecules is a new and useful approach for the treatment of certain conditions, particularly cancer, where the use of biological response modifiers has shown promise.

The present invention employs recombinant poxvirus as the delivery system for cytokines in tumor therapy. In this regard, we have evaluated a canarypox (ALVAC) virus encoding Interleukin-2 (IL-2) as a candidate cancer immunotherapeutic.

These results indicate that ALVAC-feline IL-2 expresses functional feline IL-2 which can be used to treat biological response-modifier susceptible conditions such as infection or tumor formation in animals. In particular, ALVAC-feline IL-2 may be used as an anticancer immuno therapeutic in cats.

BIOLOGICAL DEPOSITS

The recombinant pox virus that is described and referred to herein has been deposited with the American Type Culture Collection (ATCC) located at 12301 Parklawn Drive, Rockville, Maryland 20852, USA pursuant to the Budapest Treaty and prior to the filing of this application. Samples of the deposited plasmid will become available to the public and all restrictions imposed on access to the deposit will be removed upon grant of a patent on this application. The invention described and claimed herein is not to be limited in scope by recombinant virus deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar recombinant virus is within the scope of the invention.

Deposit Summary

EXAMPLES

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations. EXAMPLE 1 :

This Example illustrates construction of the pNVQH6C5LSP insertion plasmid (C5 locus). A genomic library of canarypox DNA was constructed in the cosmid vector pVK102 (Knauf and Nester, 1982)probed with pRW764.5 (a pUC9 based plasmid containing an 880 bp canarypox PvuII fragment which includes the C5 ORF) and a cosmid clone containing a 29 kb insert was identified (pHCOSl). A 3.3 kb Clal fragment from pHCOSl containing the C5 region was identified (SEQ ID NO: 1). The C5 ORF is initiated at position 1537 and terminated at position 1857.

The C5 insertion vector was constructed in two steps. The 1535 bp upstream sequence was generated by PCR amplification from purified genomic canarypox DNA using oligonucleotide primers C5A (SEQ ID NO: 2)(5'- ATCATCGAATTCTGAATGTTAAATGTTATACTTTG-3') and C5B (SEQ ID NO: 3)(5'- GGGGGTACCTTTGAGAGTACCACTTCAG-3'). This fragment was digested with EcoRI and ligated into pUC8 digested with EcoRI/Smal to yield pC5LAB. The 404 bp arm was generated by PCR amplification using oligonucleotides C5C (SEQ ID NO: 4)(5'- GGGTCTAGAGCGGCCGCTTATAAAGATCTAAAATGCATAATTTC-3') and C5DA (SEQ ID NO: 5)(5'-ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG-3'). This fragment was digested with PstI and cloned into Smal/PstI digested pC5LAB to yield pC5L. pC5L was digested within the MCS with Asp718/NotI and ligated to kinased and annealed oligonucleotides CP26 (SEQ ID NO: 6)(5'- GTACGTGACTAATTAGCTATAAAAAGGATCCGGTACCCTCGAGTCTAGAATCGAT CCCGGGTTTTTATGACTAGTTAATCAC-3') and CP27 (SEQ ID NO: 7)(5'-

GGCCGTGATTAACTAGTCATAAAAACCCGGGATCGATTCTAGACTCGAGGGTACC GGATCCTTTTTATAGCTAATTAGTCAC-3') to yield pC5LSP. The vaccinia H6 promoter (Guo et al., 1989; Perkus et al., 1989) was derived by PCR using pRW823 as template (a plasmid containing the H6 promoter linked to an irrelevant gene) and oligonucleotides CP30 (SEQ ID NO: 8) (5'-TCGGGATCCGGG-TTAATTAATTAGTCATCAGGCAGGGCG-3') and CP31 (SEQ ID NO: 9) (5'-

TAGCTCGAGGGTACCTACGATACAAACTTAACGGATATCG-3'). The PCR product was digested with BamHI and Xhol (sites present at the 5'end of CP30 and CO31, respectively) and ligated to BamHI/XhoI digested pC5LSP generating plasmid pVQH6C5LSP. This plasmid was digested with EcoRI, ligated with kinased and self- annealed oligonucleotide CP29 (SEQ ID NO: 10)(5'-AATTGCGGCCGC-3') and digested with Notl. The linearized plasmid was purified and self-ligated to generate pNVQH6C5LSP- 5. This C5 insertion plasmid contains 1535 bp of canarypox DNA upstream of the C5 ORF, translation stop codons in six reading frames, vaccinia early transcription termination signal, an MCS with BamHI, vaccinia H6 promoter, Kpnl, Xhol, Clal and Smal restriction sites, vaccinia early termination signal, translation stop codons in six reading frames and 404 bp of downstream canarypox sequence (31 bp of C5 coding sequence and 373 bp of downstream canarypox sequence).

Example 2: This Example illustrates the preparation of ALVAC-feline IL-2 recombinant canarypox vims.

The feline IL-2 gene was cloned as follows. Total RNA was isolated from phytohemagglutinin (PHA) stimulated feline peripheral blood mononuclear cells (PBMCs)(Berger, 1979) using TRI reagent (Molecular Research Center, Inc. Cincinnati, OH). First strand cDNA was synthesized from this RNA using AMV reverse transcriptase (Life Sciences, St. Petersburg, FL) and random hexamer primers (Watson and Jackson, 1985). Using the published feline IL-2 sequence (Cozzi et al 1993) PCR primers were designed to amplify the IL-2 coding region. Primers JP311 (SEQ ID NO. 11)(5'- TATGCGGATATCCGTTAAGTTTGTATCGTAATGTACAAAATT-CAACTCTTG-3'), which contains the 3' end of the vaccinia H6 promoter (Guo et al., 1989; Perkus et al., 1989) coupled to the 5' end of the feline IL-2 coding region, and JP312 (SEQ ID NO. 12) (5'- TACTACCTCGAGTACAGTCAGCGTTGAGAAG-3'), which contains the 3' end of the feline IL-2 coding region, were used to amplify a 500 bp fragment from the first strand cDNA template. This fragment was gel purified and reamplified with the above primers. The fragment was digested with EcoRV/XhoI and ligated into pNVQH6C5LSP (see Example 1 ) also digested with EcoRV/XhoI. pNVQH6C5LSP is an ALVAC insertion plasmid which contains the H6 promoter. The resulting plasmid, pC5FIL2H6, was confirmed by sequence analysis (SEQ ID NO. 13). It contains the vaccinia H6 promoter coupled to the feline IL-2 coding region flanked by the left and right arms of the C5 insertion locus. The sequence of the IL-2 gene (SEQ ID NO. 14) was identical to that published by Cozzi et al, 1993.

This donor plasmid, pC5FIL2H6 , was used in in vivo recombination (Piccini et al, 1987) with the ALVAC vims vector to generate the recombinant virus vCP1338.

EXAMPLE 3: This Example illustrates expression of functional feline IL-2 from vCP1338.

To determine if IL-2 was being expressed by vCP1338, a functional IL-2 assay (Gillis et al 1978) was conducted using the supematent from vCP1338 infected CEF cells. IL-2 activity was determined by the maintenance of proliferation of the IL-2 dependent cell line CTLL-2 of serial dilutions of each supematent fluid. Proliferation of the CTLL2 cell line was assessed by measuring [3H]-thymidine incorporation. One unit of IL-2 activity is defined as being capable of sustaining 50% of maximum proliferation of the cell line. NIH IL-2 standards (human and murine) were included in each assay. Since CTLL2 cells are also capable of proliferating in response to murine IL-4, the presence of IL-4 was ruled out by assessing IL-4 activity against the IL-4 dependent cell line CT.4S. The results show that a significant level of functional IL-2 is present in the vCP1338 supematent, while no activity was detected in uninfected or ALVAC infected cell supernatents (Figure 1).

The present invention provides a novel manner of augmenting or enhancing the immune response of a feline to antigens as components of vaccines. The present invention also provides a novel therapy for tumors or infectious agents in felines as well as a novel delivery system employing cytokines and other biological response modifiers, specifically feline IL-2. Modifications are possible within the scope of the invention. REFERENCES

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Guo, P., S. Goebel, S. Davis, M.E. Perkus, B. Languet, Ph. Desmettre, G. Allen and E. Paoletti. 1989. Expression in recombinant vaccinia vims of the equine herpesvirus 1 gene encoding glycoprotein gpl3 and protection of immunized animals. J. Virol. 63:4189-4198.

Hsieh, C.-S., S.E. Macatonia, C.S. Tripp, S.F. Wolf, A. O'Garra and K.M. Murphy. 1993. Development of Thl CD4+ cells through IL-12 produced by Listeria-infected macrophages. Science 260:547.

Knauf, V.C. and E.W. Nester. 1982. Wide Host Range Cloning Vectors: a cosmic clone bank of an agrobacterium Ti plasmid. Plasmid 8:45-54. Konishi, E., S. Pincus., E. Paoletti., W.W. Laegried., R.E. Shope and P.W. Mason. 1992. A highly attenuated host-range restricted vaccinia vims strain, NYVAC, encoding the prM, E, and NS1 genes of Japanese encephalitis vims prevents JEV viremia in swine. Virology 190,

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Claims

CLAIMS We claim:

1. A recombinant poxvims comprising a nucleic acid sequence encoding a feline cytokine, or immunomodulating fragment thereof, in a non-essential region of the recombinant vims genome.

2. The recombinant poxvims of claim 1 wherein said poxvims is an avipox vims.

3. The recombinant poxvims of claim 2 wherein said recombinant vims has non-essential vims-encoded genetic functions inactivated therein.

4. The recombinant vims of claim 3 wherein said avipoxvims is a canarypox virus.

5. The recombinant vims of claim 4 wherein said nucleic acid sequence encodes a feline cytokine.

6. The recombinant poxvims of claim 5 wherein said nucleic acid sequence encodes feline interleukin-2 (feIL-2).

7. Cells infected by a recombinant poxvims of claim 1 and wherein said feline cytokine is expressed in the cells.

8. The cells of claim 7 wherein said cells are feline tumor cells.

9. The cells of claim 7 wherein said feline cytokine is feline IL-2 (feIL-2).

10. A method of augmenting or enhancing an immune response in a feline, comprising administering to said feline, either alone or in combination with an antigen of interest, the recombinant poxvims of claim 1.

11. The method of claim 10, wherein said antigen is coexpressed with the feline cytokine.

12. A method of treatment of a cytokine-susceptible condition in an animal, which comprises administering to said animal a recombinant poxvims of claim 1.

13. The method of claim 12 wherein said susceptible condition is the presence of tumor cells in the animal.

14. The method of claim 13 wherein said recombinant poxvims is administered intratumorally in vivo.

15. The method of claim 13 wherein said recombinant vims is administered by removing tumor cells from the animal, infecting said tumor cells with said recombinant vims, and administering the infected tumor cells to the animal.

16. A method of treatment of a cytokine-susceptible condition in an animal, which comprises administering to said animal an effective amount the cells of claim 7.

17. The method of claim 16 wherein said susceptible condition is the presence of tumor cells in the animal.

18. The method of claim 17 wherein said cytokine is feline IL-2 (feIL-2).

19. The method of claim 16 wherein said susceptible condition is susceptibility to infectious disease.

20. A method of protecting an animal against tumor formation which comprises administering to said animal non-replicating tumor cells which have been infected by a recombinant poxvims of claim 1.