Title of Invention

POXVIRUS VECTOR ENCODING PROSTATE SPECIFIC ANTIGENS FOR TREATMENT OF PROSTATE CANCER

Abstract A genetic vaccine construct comprising a poxvirus vector which incorporates and, on administration to a subject, expresses in a cell of said subject a sequence of nucleotides encoding a prostate-specific polypeptide, wherein said poxvirus vector does not productively infect said subject. The genetic vaccine construct is for treatment of prostate cancer. The prostate specific polypeptide is preferably rat prostatic acid phosphatase, and is preferably xenogenic to the subject. The proxvirus vector is an avipox virus vector, preferably a fowl poxvirus vector. The genetic vaccine construct may also encode for one or more cytokines.
Full Text Poxvirus vector encoding prostate specific antigens for treatmmt of prostate cancer
FIELD OF THE INVENTION
The present invention relates generally to the field of genetic vaccinations and
particularly to genetic immunotherapy and/or immunoprophylaxis of prostate cancer.
More particularly, the present invention provides a genetic construct capable of
stimulating a selective immune response to prostate cells including prostate cancer cells.
The present invention also provides, inter alia, compositions for the immunotherapy
and/or immunoprophylaxis of prostate cancer, antibodies thereto and diagnostic
reagents therefor and methods for the treatment and/or prophylaxis of prostate cancer.
BACKGROUND OF THE INVENTION
Bibliographic details of the publications referred to by author in this specification are
collected at the end of the description.
Reference to any prior art in the specification is not, and should not be taken as, an
acknowledgment or any form of suggestion that this prior art forms part of the common
general knowledge in any country.
Advances in molecular biology and informatics in the last decade have greatly enhanced
our broad understanding, and potential to gain a greater understanding, of biological
events and have created enormous potential for pharmaceutical and related industries to
develop strategies for the prevention and treatment of diseases and other disorders. A
particularly important problem relates to the prevention and treatment of prostate cancer
and other prostate related diseases or conditions.
Prostate cancer is the second most common cause of cancer death in males. Prostate
cancer is only potentially curable when it is confined to the prostate gland using one of
two local modalities of treatment: surgery (radical prostatectomy) or radical

radiotherapy (external beam or brachytherapy). However, approximately 40% of men
who have had apparently curative treatment for localised disease will subsequently
develop metastatic disease. About 70% of men have metastases at some time during the
course of their disease.
For men with metastatic disease, medical or surgical castration often produces a
remission but inevitably lethal androgen-resistant disease supervenes that is relatively
resistant to chemotherapy, as reviewed in Logothetis, C.J. et al, Semin. Oncol. 21:620,
1994. Metastatic prostate cancer is incurable and the terminal hormone-refractory phase
of the disease is least responsive to any kind of treatment.
Surgical castration is achieved through the operation of bilateral orchidectomy and its
therapeutic effects are approximately equivalent to complete androgen blockade using a
combination of a LHRH agonist/antagonist and an anti-androgen drug (Santen R.J., J
Clin. Endocrinal Metab. 75:685-689, 1992; Thenot, S. et al, Mol. Cell Endocrinol.
156:85-93, 1999). Depending upon the operator, either procedure may be associated
with considerable morbidity with incontinence and impotence rates in some series that
approach 50%. About 70-80% of men with metastatic disease respond to either kind of
hormonal treatment and are palliated effectively for a median duration of approximately
two and a half years. Hormonal treatments have side effects of their own, which
include lethargy, weakness and cognitive impairment. In time, "androgen-independent"
growth of the cancer supervenes, which is usually fatal {Thenot S. et al, supra). This
hormone-resistant phase of the disease has a median survival of 40-50 weeks.
Combination chemotherapy may produce clinical benefit in approximately 25% of cases
but without prolongation of survival.
There is some evidence that cancer patients make spontaneous albeit ineffective
immune responses to their own cancers {Lee, P.P. et a!., Nature Medicine 5(6):677-685,
1999; Albert, M. L, et al, Nature Medicine 4:1321-1324, 1998). Most of these
immune responses are made against normal components of the tissue from which the
cancer originates, and are known as differentiation antigens. This has been well

demonstrated for melanocyte differentiation antigens, which comprise the major class of
defined melanoma tumor antigens (Rosenberg, S.A. et al, Immunity 10:281-287, 1999),
Moreover, melanocyte differentiation antigens have been defined as tumor rejection
antigens by the adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes
(Rosenberg, S.A. et al, J Am MedAssoc 271:903. 1994).
In prostate cancer, on the other hand, the evidence that prostate differentiation antigens
are recognized by t! e immune system of cancer patients is limited. In particular, none
of these antigens has been defined as a tumor rejection antigen. However, T cell
proliferative responses to human prostate specific antigen (hPSA) and human prostatic
acid phosphatase (hPAP) were detected in 6% and 11% of prostate cancer patients,
respectively, together with human PAP-specific production of the T helper cytokine,
interferon-y. These findings suggest that an immune environment, which can support
PAP-specific cytotoxic T lymphocytes, may exist in prostate cancer patients (McNeel
D.G. et al, Cancer Research 61:5161-5167, 2001). Further evidence in support of pre-
existing immunity to human PAP, which is T helper cell-dependent, is the discovery of
human PAP-specific antibodies in approximately 5% of prostate cancer patients and
male controls (McNeel D.G. et al, J. Urinol. 164(5):1825-1839, 2000). Further
investigations identified a number of T helper epitopes, which may represent naturally
processed human PAP-specific MHC class II epitopes (McNeel et al, 2001, supra).
Moreover, antitumor responses were observed in prostate cancer patients who were
immunized with dendritic cells loaded either with human prostatic acid phosphatase
(hPAP) (Peshwa, M.V. et al, Prostate 36:129-138, 1998) or a peptide derived from
human prostate specific membrane antigen (hPSMA) (Lodge, P.A. et al, Cancer
Research 60:829-833, 2000; Murphy et al, Prostate 38:73-78, 1999(a); Murphy G.P. et
al, Prostate 39:54-59, 1999(b)).
Considerable effort has consequently been expended in developing therapeutic
strategies targeting prostate specific antigens, the most well characterised of these
antigens being PSA (Prostate Specific Antigen), PSMA (Prostate Specific Membrane
Antigen) and PAP (Prostatic Acid Phosphatase). Prostate cancer is an attractive

candidate for immunotherapy because tumors grow slowly and patients have generally
been spared immunosuppressive doses of chemoradiotherapy. Fong el al, in particular,
have shown anti-PAP T cell proliferative responses in human subjects administered
with antigen loaded dendritic cells. Dendritic cells were enriched from peripheral blood
mononuclear cells and loaded with mouse PAP to provide xencgeneic stimulation of the
immune response {Fong, L. et al, J Immunol 167:7150-7156, 2001).
There is a need, however, for an efficacious, specific and safe immunotherapeutic
and/or immunoprophyiactic strategy for the treatment or prevention of prostate cancer.
In accordance with the present invention, the inventor has developed such a strategy
based on genetic vaccination with a recombinant poxvirus construct expressing a
prostate specific polypepu'de such as prostatic acid phosphatase preferably together with
a immunostimulatory molecule, for example, an immunosttmulatory cytokine such as,
in particular, IL-2.
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word
"comprise", and variations such as "comprises" and "comprising" will be understood to
imply the inclusion of a stated integer or step or group of integers or steps but not the
exclusion of any integer or step or group of integers or steps.
Nucleotide and amino acid sequences are referred to by sequence identifier numbers
(SEQ ID NO:). The SEQ DD NOs: correspond numerically to the sequence identifiers
1, 2, etc. A summary of SEQ ED NOS: is provided in Table 1. A sequence
listing is provided after the claims.
The present invention provides inter alia a genetic vaccine construct comprising a
poxvirus vector which incorporates and, on administration to a subject, expresses in a
cell of said subject, a sequence of nucleotides encoding a prostate-specific polypeptide,
or a homologue or derivative or analogue thereof, wherein said poxvirus vector does not

productively infect said subject.
In some embodiments, the expression products of said genetic vaccine construct
stimulate a PAP- specific immune response. In other embodiments, the expression
products of said genetic vaccine construct stimulate a prostate cell specific immune
response. In other embodiments, expression products of said genetic vaccine construct
stimulate autoimmune prostatitis.
Other embodiments of the present invention provide a genetic vaccine construct
comprising a poxvirus vector which incorporates and, on administration to a subject,
expresses in a cell of said subject, a sequence of nucleotides encoding a prostate-
specific polypeptide or a homologue, derivative or analogue thereof and a sequence of
nucleotides encoding an immunostimulatory polypeptide, wherein said poxvirus vector
does not productively infect said subject.
In some embodiments, the expression products of said genetic vaccine construct
stimulate a prostate cell specific immune response. In other embodiments, the
expression products of said genetic vaccine construct stimulate autoimmune prostatitis.
Preferred poxvirus vectors are avipox or orthopox vectors. A particularly preferred
poxvirus vector is a fowlpox virus vector.
In a related aspect, antibodies, nucleic acid probes and/or other reagents which
specifically bind to or are otherwise capable of distinguishing the present genetic
vaccine construct or one or more of its expression products are contemplated within the
scope of the present invention.
Preferably, the prostate specific polypeptide is a prostatic acid phosphatase, or a
homologue, derivative or analogue thereof.
In a further preferred embodiment, the prostatic acid phosphatase is a xenogeneic

homologue thereof. In some embodiments xenogeneic homologues tor use in human
subjects are rodent and more particularly a rat homologue. In particular, rat prostatic
acid phosphatase is preferred.
Accordingly, other embodiments of the present invention contemplate a genetic vaccine
construct comprising a poxvirus vector which incorporates and, on administration to a
subject, expresses in a cell of said subject, a sequence of nucleotides encoding a
xenogeneic prostatic acid phosphatase and a sequence of nucleotides encoding an
immunostimulatory polypeptide, wherein said poxvirus vector does not productively
infect said subject and wherein an expression product of said genetic vaccine construct
stimulates a prostate cell specific immune response.
In still other embodiments of the present invention, the immunostimulatory polypeptide
is an immunostimulatory cytokine. For example, said cytokine is preferably a Th-1 or
Th-2 type cytokine.
Suitable cytokines are one or more of IFNv, IL-12, IL-2, TNFa, IL-4, IL-7, GM-CSF,
IL-6, IL-15, IL-18 or flt-3 ligand.
In preferred embodiments the cytokines are one or more of IL-2, IFNy or IL-12.
A particularly preferred cytokine is IL-2.
Accordingly, yet other embodiments of the present invention contemplate a genetic
vaccine construct comprising a fowlpox virus vector which incorporates and, on
administration to a subject, expresses in a cell of said subject, a sequence of nucleotides
encoding a xenogeneic prostatic acid phosphatase and a sequence of nucleotides
encoding an IL-2 polypeptide, wherein said fowlpox virus vector does not productively
infect said subject and wherein an expression product of said genetic vaccine construct
stimulates a prostate cell specific immune response.

Still other embodiment of the present invention contemplate a genetic vaccine construct
comprising a fowlpox virus vector which incorporates and, on administration to a
subject, expresses in a cell of said subject, a sequence of nucleotides encoding a rat
prostatic acid phosphatase and a sequence of nucleotides encoding an IL-2 polypeptide.
wherein said fowlpox virus vector does not productively infect said subject and wherein
an expression product of said genetic vaccine construct stimulates a prostate cell
specific immune response.
Suitably, the prostate cell specific immune response comprises proliferation of T cells
which enhance inhibition, lysis, or other forms of downregulation of the number or
proliferation of prostate derived cells in a subject.
Other embodiments of the present invention provide a composition comprising a genetic
vaccine construct comprising a poxvirus vector which incorporates and, on
administration to a subject, expresses in a cell of said subject, a sequence of nucleotides
encoding a prostate-specific polypeptide, or a homologue or derivative or analogue
thereof, together with one or more pharmaceutically acceptable carriers, wherein said
poxvirus vector does not productively infect said subject and wherein expression
products of said genetic vaccine construct stimulate a prostate cell specific immune
response.
Still other embodiments of the present Invention provide a composition comprising a
genetic vaccine construct comprising a poxvirus vector which incorporates and, on
administration to a subject, expresses in a cell of said subject, a sequence of nucleotides
encoding a prostate-specific polypeptide, or a homologue or derivative or analogue
thereof, and a sequence of nucleotides encoding an immunostimulatory polypeptide, and
one or more pharmaceutically acceptable carriers, wherein said poxvirus vector does not
productively infect said subject and wherein expression products of said genetic vaccine
construct stimulate a prostate cell specific immune response.
Yet other embodiments of the instant invention provide a method for stimulating or

otherwise enhancing a prostate cell specific immune response in a subject comprising
administration to said subject of an effective amount of a composition comprising a
genetic vaccine construct comprising a poxvirus vector which incorporates and, on
administration to a subject, expresses in a cell of said subject, a sequence of nucleotides
encoding a prostate-specific polypeptide, or a homologue, derivative or analogue
thereof, for a time and under conditions sufficient for expression products of said
genetic vaccine construct to stimulate or otherwise enhance a prostate cell specific
immune response, and wherein said poxvirus vector does not productively infect said
subject.
Still another related aspect of the present invention provides a method for
irnmunotherapy and/or immunoprophylaxis of prostate cancer comprising
administration of an effective amount of a composition comprising a genetic vaccine
construct comprising a poxvirus vector which incorporates and, on administration to a
subject, expresses in a cell of said subject, a sequence of nucleotides encoding a prostate
specific polypeptide, or homologue, derivative or analogue thereof, wherein said
poxvirus vector does not productively infect said subject, and wherein expression
products of said poxvirus vector stimulate a prostate cell specific immune response
effective in the treatment and/or prophylaxis of prostate cancer.
A further related aspect of the present invention contemplates the use of a genetic
vaccine construct in the manufacture of a medicament for the immunotherapy and/or
immunoprophylaxis of prostate cancer, wherein said construct comprises a poxvirus
vector which incorporates and, on administration to a subject, expresses in a cell of said
subject, a sequence of nucleotides encoding a prostate-specific polypeptide, or a
homologue, derivative or analogue thereof wherein said poxvirus vector does not
productively infect said subject, and wherein said expression products of said poxvirus
vector stimulate a prostate cell specific immune response effective in the treatment or
prophylaxis of prostate cancer.
Still further embodiments of the present invention contemplate the use of a genetic

vaccine construct in the manufacture of a medicament for the immunotherapy an&or
immunoprophylaxis of prostate cancer, wherein said construct comprises a pox virus
vector which incorporates and, on administration to a subject, expresses in a cell of said
subject, a sequence of nucleotides encoding a prostate-specific polypeptide, or a
homologue, derivative or analogue thereof, and a sequence of nucleotides encoding an
immunostimulatory polypeptide, wherein said poxvirus vector does not productively
infect said subject, and wherein said expression products of said poxvirus vector
stimulate a prostate cell specific immune response effective in the treatment or
prophylaxis of prostate cancer.
In some embodiments, the prostate-specific polypeptide is a prostatic acid phosphatase
or a homologue or derivative or analogue thereof.
A particularly preferred immunostimulatory polypeptide in this embodiment of the
invention is an immunostimulatory cytokine. For example, said cytokine is preferably a
Th-1 or Th-2 type cytokine.
Preferred examples of cytokine is one or more of IFNy, IL-12, IL-2, TNFa, IL-4, IL-7,
GM-CSF, IL-6, IL-15, IL-18 or flt-3 ligand.
More preferably, the cytokines are one or more of IL-2, IFNy or IL-12.
A particularly preferred cytokine is IL-2.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a photographic representation of elecirophoreticaily resolved PCR-fragments.
VIR501 and VIR502 were subjected to PCR to test for correct gene insertion. PCR
amplifications using human PAP, rat PAP and human IL-2 specific primers for the
detection of human IL-2 and rat PAP insertion into YIR501 and human IL-2 and human
PAP insertion in VIR502. Negative control experiments where carried out using water
instead of DNA template.
Figure 2 is a photographic representation of ELISA wells showing the results of tests for
secretion of human IL-2 by VIR501 and VIR502. After three rounds of plaque purification
a number of plaque clones were amplified by infecting CEF cells. After infection, a small
sample of culture medium was removed and tested for the presence of human IL-2 using a
human IL-2-ELISA kit. A visible colour change indicates the presence of IL-2 in the test
sample.
Figure 3 is a photographic representation of a Western Blot showing FPV-mediated
expression of human PAP. TK'143B cells were infected with FPV vectors at MOI of 10
and the cells harvested for immunoblot analysis 48h and 72h post-infection (p.i.). FPV-HA
was used as a negative vector control. The lack of cross-reactivity of the anti-human PAP
polyclonal antibody was demonstrated by the absence of a band for recombinant rat PAP.
The negative control for the method of detection was probing of the immunoblot with the
secondary antibody alone. 1, FPV-HA; 2, VIR501 (48h p.i.); 3, VIR502 (48h p.i.); 4,
VIR501 (72h p.i.); 5, V1R502 (72h p.i.); 6, bacterial recombinant rat PAP (pQE system); 7,
molecular weight marker; 8, FPV-HA; 9, VIR501 (48h p.i.); 10, VIR502 (48h p.i.). The
49.1kDa marker together with the location of the specific human PAP bands is indicated.
Figure 4 is a graphical representation of results of ELISA showing immunogenicity of
FPV expressing human PAP. Three NZ White rabbits were immunised by intramuscular
injection with 2xlO8 plaque forming units (PFU) each of empty vector control, FPV-M3, or
VIR501 (FPV encoding rat PAP and human IL-2) or VIR502 (FPV encoding human PAP

and human IL-2). The animals were boosted according to the same schedule three weeks
after the priming immunisation. Two weeks after the boost immunisation the animals were
bled again. Reactivities of the immune sera were tested against bacterial recombinant
proteins that were made in the pQE system: A. human PAP, or B. rat PAP. Shown are
dilution curves for pre-immune sera (light-grey dashed line); po?t-immune sera, three
weeks after priming immunisation (medium grey line with filled triangles); post-immune
sera, two weeks after boost immunisation (thick black line with filled diamonds). Negative
controls included plates coated with irrelevant pQE-derived proteins, human La/SS-B
autoantigen or staphylococcal exotoxin B (SEB) for which OD (at 405nm) values were obtained (data not shown).
Figure 5 is a representation of the nucleotide sequence of the insertion site of VIR501
containing human IL-2 and rat PAP sequences.
Figure 6 is a representation of the nucleotide sequence of the insertion site of VIR502
containing human IL-2 and human PAP sequences.
Figure 7 is a representation of the aligned amino acid sequences of rat PAP from VIR501
with human PAP from VIR502 as indicated.
Figure 8 is a graphical representation of ELISA showing immunogenicity of FPV
expressing rat PAP. Two wethers (castrated male sheep) were immunised by intramuscular
injection with either 2xlO8 plaque forming units (PFU) of VIR501 (FPV encoding rat PAP
and human IL-2) [upper panels] or 3x10* PFU of VIR502 (FPV encoding human PAP and
human IL-2) flower panels]. Four weeks after immunisation, sera were collected for
ELISA. The reactivity of the immune sera was tested against recombinant proteins that
were made either in the pQE bacterial expression system: A. human PAP, B. rat PAP, or
. the InsectSelect expression system: C. rat PAP (upper panel) or human PAP (lower panel).
Shown are dilution curves for pre-immune sera (light-grey dashed line) and post-immune
sera, four weeks after the priming immunisation (medium grey line with filled triangles).

Figure 9 is a schematic represcntion showing, for VIR501 an VIR502, where the single
insertion of human IL-2 and PAP sequences is located in the towlpox. virus genome. The
fowlpox virus genome used to construct VIR50] and VIR502 has not yet been sequenced
so the fowlpox virus Genbank sequence (AF198100) has been used as a reference for
location of insertion site relative to the thvmidine kinase (FPV086R) ORF.
Figure 10 is a schematic representation showing a map of the plasmid integration vector
pVHL04, used to construct VIR50I.
Figure 11 is a schematic representation showing a map of the plasmid integration vector
pVHL05, used to construct VIR502.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present, invention is predicated, in part, on the determination that a genetic vaccine
construct based on a live poxviral vector which incorporates and expresses in a cell of a
subject a sequence of nuclcotides encoding a polypeptide which is normally only
expressed on or near the surface of prostate cells, preferably together with an
immunostimulntory polypeptide, is capable of selectively inducing immune prostatitis
in a subject,
Accordingly, the present invention provides inter alia a genetic vaccine and methods for
treating or preventing prostate related diseases or conditions such as prostate cancer.
Without being limited by any particular theory or mode of operation, by using a
poxviral vector which does not productively infect the subject, the risk of an on-going
viral infection and/or expression of prostate specific polypeptide in a wide range of
possibly inappropriate cells is minimised. Furthermore, by using a prostate specific
polypeptide which exhibits a low level of similarity to other polypeptides in the subject,
the risk of generating an inappropriate immune response is also reduced.
Accordingly one aspect of the present invention contemplates a genetic vaccine
construct comprising a poxvirus vector which incorporates and, on administration to a
subject, expresses in a cell of said subject, a sequence of nucleotides encoding a
prostate-specific polypeptide, or a homologue or derivative or analogue thereof, wherein
said poxvirus vector does not productively infect said subject and wherein expression
products of said genetic vaccine construct stimulate a prostate cell specific immune
response.
By "genetic vaccine construct" is meant a composition comprising a recombinant
nucleic acid molecule which, for the purpose of immunisation is administered to a
subject in whom one or more antigenic polypeptides, encoded by at least a part of said
nucleic acid molecule, are expressed.

In accordance with the present invem;on, the poxvirus vector does noi "productively
infect" the subject. The phrase "does not productively infect" or no "productive
infection" means that the vector may infect cells of the subject, for instance near the
zone of administration, however, the virus does not propagate and the risk of an on-
going viral infection and/or prostate specific polypeptide expression in a wide range of
possibly inappropriate cells is minimised. In particular this may occur because viral
replication is inefficient, incomplete, or limited.
For example, it would be inappropriate or undesirable for the poxviral vector to
propagate and spread in cells of important non-prostate ceil organs thus making them
targets of immune destruction. Of course, initial infection and expression of proteins by
the vector is required and engenders an immune response.
Those skilled in the art will know that poxviruses comprise a diverse group of viruses
classified traditionally according to their host range. For example, wild type avipox
viruses do not replicate in the cells of non-avian species. The limiting step in
replication is inefficient late gene expression or inefficient maturation of viral particles
(Somogyi P. et al, Virology 197:439-444, 1993). However, genes under the control of
early poxviral promoters are expressed in the cells of non-avian species such as man and
heterologous genes are routinely expressed in this way {Taylor, J. et al, Vaccine 6:497-
503, 1988; Cox, W. et al, Virology 195:845-850, 1993). In immunocompetent hosts,
some poxviral infections such as, for example, infection with certain strains of vaccinia
in man are generally limited, nevertheless, man is a host species for vaccinia virus and
at least initially, substantial viral replication would be expected.
The measure for absence of productive infection in accordance with the present
invention is the inability of the poxvirus vector to propagate in or spread from the cells
initially infected. In a preferred embodiment, the absence of productive infection in a
subject is less than approximately 10% of total viral replication observed in a
permissive host, preferably less than 5%, more preferably less than 1%, even more
preferably less than 0.1% and still more preferably less than 0.01%. The choice of

poxvirus vector will therefore depend, inter alia, on the subject species.
For the avoidance of any doubt, in accordance with the present invention, ihc
recombinant avipcx including fowlpox vectors of the present invention do not
productively infect non-avian hosts.
Alternatively, a conditionally replication defective poxvirus vector may be engineered
by methods known in the art not to productively infect a host. For example, some
aspects of the genetic basis for host specificity in vaccinia poxvirus strains are
understood and replication defective vaccinia viruses have been generated by deletion
of "host range" genes {Perkus, M.E. et a!., Virology 179:276-28, 1990). Also
replication deficient or attenuated viruses such as modified vaccine virus (MVA) are
examples of poxviruses which do not productively infect a human subject. Such
modified or attenuated poxvirus vectors may be obtained by repeated passage of viruses
in cells in vitro, for example in chicken embryo fibroblasts.
Reference to "poxvirus" includes viruses selected from, for example, avipox (eg,
fowlpox, canarypox, penguinpox, pigeonpox) orthopox (eg, vaccinia) capripox (eg,
sheep, goats) and suipox (eg, swinepox). Avipox vectors are preferred vectors. A
particularly preferred vector is fowlpox.
Although human subjects are primarily contemplated, reference to a "subject" should be
understood to include mammals including primates (eg, humans, monkeys), livestock
animals (eg, sheep, cows, horses, donkeys, goats, pigs), laboratory test animals (eg,
mice, rats, ducks, dogs, guinea pigs, rabbits, hampsters), companion animals (eg, dogs,
cats, birds), and captive wild animals (eg, kangaroos, deer, foxes). Preferably said
subject is a primate and even more preferably a human subject.
Reference to a "cell" in "expresses in a cell" includes expression in antigen presenting
cells such as dendritic cells.

The general principles and procedures for generating and using recombinant poxvirus
vectors are well known in the art. Briefly, homologous recombination between a donor
recombination vector and a poxvirus within a host cell permits correct introduction of
the desired sequences. The donor vector comprises a sequence of nucleotides
permitting site specific homologous recombination with a poxvirus vector, a sequence
of nuclcotides encoding a prostate specific polypeptide together with one or more of any
other elements required for amplification in a prokaryotic host, selection of transfected
cells, and transcription of nucleic acid sequences. Double and further recombinants
such as a vector further comprising a sequence of nucleotides encoding an
immunostimulatory polypeptide or peptide are generated in essentially the same
manner, however different promoters and selection markers may be employed.
In a preferred aspect, the present invention contemplates a genetic vaccine construct
comprising an avipox vector which incorporates and, on administration to a subject
expresses in a cell of said subject, a sequence of nucleotides encoding a prostate-
specific polypeptide, or a homologue or derivative or analogue thereof, wherein said
avipox vector does not productively infect said subject and wherein expression products
of said genetic vaccine construct stimulate a prostate cell specific immune response.
A fowlpox vector is a preferred avipox vector. Fowlpox viruses are preferred inter alia
because, they express appropriate levels of heterologous protein. The use of fowlpox
virus in man may also be preferred because immunity to fowlpox would not generally
be present. In contrast, a large proportion of the human population has been exposed to
vaccinia virus as a result of prior vaccination regimes. As a result, the introduction of
vaccinia virus into a human patient can provoke an immune response to the vaccinia
viral vector. In this situtation, the vector may be neutralised before any antigenic
proteins are expressed.
The genetic vaccine construct of the present invention may also comprise a sequence of
nucleotides which is a useful marker for detection by nucleic acid based assays, or
expressed and useful for detection such as by protein assays including enzyme or

antibody based assay?.
The present vectors may be identified using any suitable protocol such as heteroduplex
analysis, polymerase chain reaction (PCR), ligase chain reaction (LCR), sequence specific
hybridization probes (SSO), single-stranded conformational polymorphism (SSCP),
sequencing, mass spectrometry, enzyme cleavage, protein probes including antibody,
enzyme or immunoreactive based assays and combinations of these.
Another aspect of the present invention contemplates an isolated antibody which is
determined by epitopes which are uniquely formed in expression products of die subject
genetic vaccine construct.
Isolated antibodies may be monoclonal or polyclonal. Alternatively, fragments of
antibodies may be used such as Fab fragments. Furthermore, the present invention extends
to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is
considered herein to include fragments and hybrids of antibodies.
In one embodiment, specific antibodies can be used to screen a sample from a subject for
the presence of expression products of the vaccine construct,
Alternatively, the ability of a subject to mount a specific antibody response to a
proteinaceous form of the vaccine construct may be used to determine whether a subject
has previously been vaccinated with the subject vaccine construct. Techniques for the
assays contemplated herein are known in the art and include, for example, sandwich assays
and ELISA.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the
enzyme or protein and either type is useful for immunoassays. The methods of obtaining
both types of sera are well known in the art. Polyclonal sera are less preferred but are
relatively easily prepared by injection of a suitable laboratory animal with an effective
amount of a proteinaceous form of a molecular marker, or antigenic parts thereof,

collecting serum from the animal, and isolating specific sera by any of the known
immunoadsorbcnt techniques. Although antibodies produced by this method an- utilizable
in virtually any type of immunoassay, they are generally less favoured because of the
potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of
the ability to produce them in large quantities and the homogeneity of the product. The
preparation of hybridoma cell lines for monoclonal antibody production derived by fusing
an immortal cell line and lymphocytes sensitized against the immunogenic preparation can
be done by techniques which are well known to those who are skilled in the art.
Another aspect of the present invention contemplates a method for detecting a
proteinaceous form of the instant genetic poxviral vaccine construct in a subject said
method comprising contacting a biological sample from said subject with an antibody
specific for a proteinaceous form of the genetic poxviral vaccine construct for a time and
under conditions sufficient for an antibody-antigen complex to form, and then detecting
said complex.
The presence of a complex may be detected in a number of ways such as by Western
blotting and ELISA procedures. A wide range of immunoassay techniques are available as
can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These
includes both single-site and two-site or "sandwich" assays of the non-competitive types,
as well as in the traditional competitive binding assays. These assays also include direct
binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are favoured
for use in the present invention. A number of variations of the sandwich assay technique
exist and all are intended to be encompassed by the present invention. Briefly, in a typical
forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample
to be tested brought into contact with the bound molecule. After a suitable period of
incubation, for a period of time sufficient to allow formation of an antibody-antigen

complex, a second antibody specific to the antiren, labelled with a reporter molecuic
capable of producing a detectable signal is then added and incubated, allowing time
sufficient for the formation of another complex of antibody-antigen-labclled antibody, Any
unreacted material is washed away, and the presence of the antigen is determined by
observation of a signal produced by the reporter molecule. The results may either be
qualitative, by simple observation of the visible signal, or may be quantitated by
comparing with a control sample containing known amounts of hapten. Variations on the
forward assay include a simultaneous assay, in which both sample and labelled antibody
are added simultaneously to the bound antibody. These techniques are well known to those
skilled in the art, including any minor variations as will be readily apparent.
The sample is generally a biological sample comprising biological fluid but also includes
to supernatant fluid such as from a cell culture. Methods of sample preparation arc well
known to those skilled in the art.
"Reporter molecule" as used in the present specification, means a molecule which, by its
chemical nature, provides an analytically identifiable signal which allows the detection of
antigen-bound antibody. Detection may be either qualitative or quantitative. The most
commonly used reporter molecules in this type of assay are either enzymes, fluorophores
or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody,
generally by means of glutaraldehyde or periodate. As will be readily recognized, however,
a wide variety of different conjugation techniques exist, which are readily available to the
skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase,
p-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with
the specific enzymes are generally chosen for the production, upon hydrolysis by the
corresponding enzyme, of a detectable colour change. Examples of suitable enzymes
include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic
substrates, which yield a fluorescent product rather than the chromogenic substrates noted
above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten
complex, allowed to bind, and then the excess reagent is washed away. A solution

containing the appropriate substrate is thu, added to the complex of antibody-antigen-
antibody. The substrate will react with the enzyme linked to the second antibody, giving a
qualitative visual signal, which may be further quantitated, usually spectrophotometrically,
to give an indication of the amount of hapten which was present in the sample. "Reporter
molecule" also extends to use of cell agglutination or inhibition of agglutination such as
red blood cells on latex beads, and the like.
Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be
chemically coupled to antibodies without altering their binding capacity. When activated
by illumination with light of a particular wavelength, the fluorochrome-labelled antibody
adsorbs the light energy, inducing a state of excitability in the molecule, followed by
emission of the light at a characteristic colour visually detectable with a light microscope.
As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-
hapten complex, After washing off the unbound reagent, the remaining tertiary complex is
then exposed to the light of the appropriate wavelength. The fluorescence observed
indicates the presence of the molecule of interest. Immunofluorescence and EIA
techniques are both very well established in the art and are particularly preferred for the
present method. However, other reporter molecules, such as radioisotope,
chemiluminescent or bioluminescent molecules, may also be employed.
The phrase "expression products" includes the products of transcription and/or translation.
Accordingly proteins are preferred products but the activity of transcripts in RNA form is
not excluded from the scope of the present invention.
The phrase "prostate specific polypeptide" is used in a broad sense and includes a
polypeptide which is expressed on or near the surface of prostate cells, Including
prostate cancer cells, and is not substantially expressed on the surface of non-prostate
cells. In this way, an immune response is directed specifically to prostate cells and not
other self cells of the subject,
A preferred prostate specific polypeptide is a polypeptide which has a low level of

similarity to other polypcptides in a subject. This aspect provides that an immune
response is directed preferentially to prostate cells expressing a prostate specific
polypeptide and rather than cells expressing a cross reactive cpitope not determined by a
prostate specific polypeptide.
By way of further clarification a prostate specific polypeptide is not substantially
expressed on or near the surface of non-prostate cells if it is expressed at a level of less
than about 10% of that determined on or near the surface of prostate cells and more
preferably less than 5%, more preferably less than 1%, even more preferably less than
0.1%, even still more preferably less than 0.01% or even yet still more preferably less
than 0.001%.
In a particularly preferred embodiment, the subject prostate specific polypeptide is a
prostatic acid phosphatase. Advantageously, prostatic acid phosphatase (PAP) is
expressed specifically in the prostate cells including prostate cancer cells and it has been
used widely as a marker for prostate cancer. Additionally PAP exhibits a low level of
amino acid and nucleotide sequence similarity to known proteins and their encoding
nucleic acids, PAP also has a range of homotogues which exhibit a high level of amino
acid and nucleotide sequence similarity.
Homologues, derivatives or analogues of prostate specific polypeptides and their
encoding nucleotide sequences are clearly contemplated. Generally, such forms exhibit
comparable or enhanced function in the present invention, relative to sequences from
which they are derived or based.
For the purpose of the present invention a derivative of the subject nucleic acid
sequences may be a functional part or fragment which achieves the advantage of the
present invention or it may comprise one or more mutations or modifications.
Mutations include one or more nucleotide deletions, insertions or substitutions.
Mutations may be silent, conservative, missense or frameshift mutants provided that the
antigenic function of the polypeptide expressed therefrom is retained or enhanced.

Preferably, derivatives have at least 50% similarity to the prc-derivatised or parent
molecule, preferably at least, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%
similarity to the pre-derivatised or parent molecule. Sequence comparisons are
preferably the whole molecule but may also be part thereof, preferably the comparisons
are made in a continuous series of at least about 21 nuclcotides. The nucleotide
sequences of prostate specific polypeptides such as PAP, PSMA and PAP and are
published in Genbank. Homologues from other species are readily obtained by well
known screening and cloning methods.
Functional derivatives may be obtained by any route and may be synthetic or
recombinant. A straightforward but random route is to use mutagenesis followed by
testing or expression and testing of the expression products such as by testing its ability
to induce anti-polypeptide immune response. Additionally, derivatives may be
modified to have other useful properties such as to enhance processing and presentation
of the expressed peptide in order to enhance the immune response thereto.
Alternatively, or in addition, derivatives may maintain function whilst having additional
features such as modifications which permit the polypeptide or peptide to be
distinguished over the wild type polypeptide.
Analogues are not a part or mutant form of the parent molecule but they have an
analogous function. Analogues may be recombinant or synthetic and preferably have
enhanced function over the parent molecule for instance by excluding
immunosuppressive epitopes. Analogues may be designed so that their expressed
proteins mimic certain immunological or physiochemical property of the prostate
specific polypeptide.
Homologues of prostate specific polypeptides include isoenzymes, splice variants,
tissue specific forms and species specific forms of the polypeptide. Species
homologues are also referred to as xenogeneic forms of prostatic acid phosphatase and
include, of course, primate, mammalian and rodent homologues. By ^xenogeneic" is
meant the use of forms derived from a different species compared to the species' origin

of the subject. Thus, for human subjects, a xenogeneic prostate specific polypeptide is
any form wherein it is not derived from humans. Preferably, homologues exhibit a high
level of sequence or immunological similarity. Derivatives and analogues of the instant
homologues are also contemplated herein.
Usefully, various algorithms are available in the art that permit analysis of peptide
sequences and homologues thereof to determine the likelihood that they will exhibit
enhanced function. For example, the Parker algorithm {Parker, K.C. et al, Journal of
Immunology 152:163-175, 1994) estimates half-times of dissociation for MHC class-I
peptide binding motifs.
Similarity at the nucleic acid level may be assessed in assays exploiting different
hybridisation conditions as is well known in the art and is, for example, described in
Ausubel et al, 2002. Preferably, a derivative nucleic acid molecule of the invention is
capable of hybridizing to a reverse complement of a nucleotide sequence encoding a
prostate specific polypeptide under low stringency conditions at 42°C.more preferably
under medium stringency and most preferably under high stringency conditions.
Low stringency hybridisation conditions includes and encompasses from at least about 0 to
at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for
hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
Generally, low stringency is at from about 25-30°C to about 42CC. The temperature may
be altered and higher temperatures used to replace formamide and/or to give alternative
stringency conditions.
Medium stringency includes and encompasses from at least about 16% v/v to at least about
30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for
hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions.
High stringency includes and encompasses from at least about 31% v/v to at least about
50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for
hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing

condiiions. In general, washing is carried out Tm = 6'-).3 + 0.41 (G+C)% (\farmv ei a!.. ./.
Mol Bio!. ;'. 109, 1962). However, the T,,. of a duplex DNA decreases by 1CC with every
increase of 1% in the number of mismatch base pairs (Bonner et ai, Eur. J. Biochem.
46(1): 83-88, 1974). Formamide is optional in these hybridization conditions. Accordingly,
particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC
buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1 % w/v SDS
at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v
SDS at a temperature of at least 65°C.
The term "similarity" as used herein includes exact identity between compared sequences
at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level,
"similarity" includes differences between sequences which result in different amino acids
that are nevertheless related to each other at the structural, functional, biochemical and/or
conformational levels.
Where there is non-identity at the amino acid level, "similarity" includes amino acids that
are nevertheless related to each other at the structural, functional, biochemical and/or
conformational levels. In a particularly preferred embodiment, nucleotide and sequence
comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or
polypeptides include "reference sequence", "comparison window", "sequence similarity",
"sequence identity", "percentage of sequence similarity", "percentage of sequence
identity", "substantially similar" and "substantial identity". A "reference sequence" is at
least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units,
inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides
may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide
sequence) that is similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of the two
polynucleotides over a "comparison window" to identify and compare local regions of

sequence similarity. A "comparison window" refers to a conceptual segment of typically
12 contiguous residues that is compared to a reference sequence. The comparison window
may comprise additions or deletions (i.e. gaps) of about 20% cr less as compared 10 the
reference sequence (which does not comprise additions or deletions) for optimal alignment
of the two sequences. Optimal alignment of sequences for aligning a comparison window
may be conducted by computerised Implementations of algorithms (GAP, BESTFJT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics
Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best
alignment (i.e. resulting in the highest percentage homology over the comparison window)
generated by any of the various methods selected. Reference also may be made to the
BLAST family of programs as, for example, disclosed by Altschul et al. A detailed
discussion of sequence analysis can be found in Unit 19,3 of Ausubel et al.
The terms "sequence similarity" and "sequence identity" as used herein refers to the extent
that sequences are identical or functionally or structurally similar on a nucleotide-by-
nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
Thus, a "percentage of sequence identity", for example, is calculated by comparing two
optimally aligned sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino
acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp,
Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total number of positions in the
window of comparison (i.e., the window size), and multiplying the result by 100 to yield
the percentage of sequence identity. For the purposes of the present invention, "sequence
identity" will be understood to mean the "match percentage" calculated by the DNASIS
computer program (Version 2.5 for windows; available from Hitachi Software engineering
Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the
reference manual accompanying the software. Similar comments apply in relation to
sequence similarity.

il'J
In accordance with one aspect of the present invention, the prostate specific polypernde
has no more than about 70% amino acid similarity to other antigenic proteins in the
subject. More preferably, the prostate specific polypepiide has no more than 60%, even
more preferably no more than about 50% amino acid similarity.
Another aspect of the present invention consequently provides a genetic vaccine
construct comprising an avipox vector which incorporates and, on administration to a
subject expresses in a cell of said subject, a sequence of nucleotides encoding a prostatic
acid phosphatase, and/or a homologue or derivative or analogue thereof, wherein said
avipox vector does not productively infect said subject and wherein expression products
of said genetic vaccine construct stimulate a prostate cell specific immune response.
In a particular embodiment, a xenogeneic homologue of the prostatic acid phosphatase
is a preferred homologues which exhibits potentially higher binding affinity for more
HLA molecules than an indigenous prostatic acid phosphatase homologue.
In accordance with one aspect of the present Invention the inventor has determined that
rat PAP-derived motifs exhibit higher binding affinities for more HLA molecules than
human PAP-derived motifs. Accordingly xenogeneic administration is proposed for
some applications. A preferred xenogeneic form of prostatic acid phosphatase for
particular use in human subjects is rat PAP. Without intending to be limited by any one
particular mechanism or mode of action, the use of a xenogenic homologue is provided
to assist in overcoming self tolerance and to illicit effective effector cells such as, for
example, CD4+ and CD8+ T cells and Natural Killer (NK). Effective T cells are
generally high affinity and/or high avidity immune effector cells. Combinations of
xenogeneic and indigenous prostate specific polypeptides are also contemplated.
Another aspect of the present invention provides a genetic vaccine construct comprising
a poxvirus vector which incorporates and, on administration to a subject expresses in a
cell of said subject, a sequence of nucleotides encoding a xenogeneic homologue of
prostatic acid phosphatase, or a further derivative or an analogue thereof, wherein said

poxvirus vector docs not productively infect said subject and wherein expression
products of said genetic vaccine construct stimulate a p. state cell specific immune
response.
The phrase "stimulates a prostate cell specific immune response" includes reference to
inducing or enhancing or otherwise stimulating a cellular and/or humoral immune
response in a subject to one or more antigenic components of a prostate specific
polypeptide which is expressed on or near the surface of prostate cells including
prostate cancer cells. In a preferred embodiment, the immune response comprises
cellular and humoral responses sufficient to generate immune prostatitis including
antigen specific cylotoxic cells which inhibit, lyse or otherwise down regulate the
number or proliferation of prostate cells including prostate cancer cells, if present, in a
subject. Even more preferably, the immune response is directed selectively towards
prostate cells including prostate cancer cells, if present, and is not directed to other cells
in the subject. As previously canvassed, in exploiting self antigens in vaccines, the
present inventor has selected polypeptides which are essentially specific to the prostate
and which furthermore exhibit a low level of amino acid or nucleotide sequence
similarity to other proteins in the subject.
Various algorithms and assays including in vitro and in vivo assays are available to test
or predict the effectiveness and/or suitability of particular genetic vaccine constructs
within the scope of the present invention. In particular, various cellular and animal
models of prostate cancer in humans are available including primate, dog and rodent
models.
In a further related embodiment the prostate cell specific immune response is enhanced
by co-expressing the prostate specific polypeptide with an immunostimulatory
molecule.
By "enhanced" is meant that administration of the present composition results in a
prostate cell specific immune response which is more effective in treating or preventing

prostate-related diseases or conditions in a subject than the immune response, if anv. in
that subject prior to administration of the present composition.
Accordingly, another aspect of the present invention provides a genetic vaccine
construct comprising a poxvirus vector which incorporates and, on administration to a
subject expresses in a cell of said subject, a sequence of nucleotides encoding a
prostate-specific polypeplide or a homologue, derivative or analogue thereof and a
sequence of nucleotides encoding an immunostimulatory molecule, wherein said
poxvirus vector does not productively infect said subject and wherein expression
products of said genetic vaccine construct stimulate a prostate eel] specific immune
response.
The term "immunostimulatory' molecule" is used in its broadest sense and includes
polypeptides or functional parts thereof which stimulate or enhance a prostate cell
specific immune response generated by the immune system in response to the herein
described genetic vaccine construct. The immunostimulatory molecule may, in the case
of particular prostate specific polypeptides or particular poxviral vectors described
herein, be required in order to generate immune prostatitis. In other embodiments, the
immunostimulatory molecule modulates and/or enhances the immune response.
Preferred immunostimulatory polypeptides include all or a functional part of
polypeptides including cytokines, chaperokines, chemokines, accessory or adhesion
molecules such as B7 and 1CAM. Polypeptides which down regulate immunoinhibitory
molecules are also encompassed by the present invention.
In a preferred embodiment the immunostimulatory molecule is a cytokine. In
accordance with the present invention it is contemplated that the cytokine is co-
expressed with one or more prostate specific polypeptides. During antigen processing,
the cytokine modulates the immune response to enhance its effectiveness. Preferred
cytokines are one or more of IFNy, IL-12, IL-2, TNFcc, IL-4, IL-7, GM-CSF or IL-6.
Even more preferred cytokines are one or more of IL-2, IFNy or IL-12. A particularly

prefeircd cytokine is IL-2.
IL-2 :$ a preferred cytokine, inter alia, because of its ability to enhance the immune
response to the instant vector and because of its documented safety in humans ■. cier
controlled conditions, In treating human subjects, human-derived cytokincs are
preferred.
Accordingly, yet another aspect of the present invention contemplates a genetic vaccine
construct comprising a fowlpox virus vector which incorporates and, on administration
to a subject, expresses in a cell of said subject, a sequence of nucleotides encoding a
xenogeneic prostatic acid phosphatase and a sequence of nucleotides encoding an IL-2
polypeptide, wherein said fowlpox virus vector does not productively infect said subject
and wherein expression products of said genetic vaccine construct stimulates a prostate
cell specific immune response.
Still another aspect of the present invention contemplates a genetic vaccine construct
comprising a fowlpox virus vector which incorporates and, on administration to a
subject, expresses in a cell of said subject, a sequence of nucleotides encoding a rat
prostatic acid phosphatase and a sequence of nucleotides encoding an IL-2 polypeptide,
wherein said fowlpox virus vector does not productively infect said subject and wherein
expression products of said genetic vaccine construct stimulates a prostate cell specific
immune response.
Yet another related aspect of the instant invention provides a method of stimulating or
otherwise enhancing a prostate cell specific immune response in a subject comprising
administration to said subject of an effective amount of a composition comprising a
genetic vaccine construct comprising a poxvirus vector which incorporates and, on
administration to a subject expresses in a cell of said subject, a sequence of nucleotides
encoding a prostate-specific polypeptide, or a homologue, derivative or analogue
thereof, for a time and under conditions sufficient to stimulate or otherwise enhance a
prostate cell specific immune response, and wherein said poxvirus vector does not

productively infect said subject.
Administration of the genetic vaccine construct composition may be optimised using
protocols which are well known in the art. In particular the dose and frequency will
vary with the mode of administration as well as various parameters relating to the
subject including size, previous exposure to the vaccine, stage of prostate cancer
development, The composition may be administered by any convenient route such as
by oral, intravenous, intranasal, intramuscular, intraperitoneal, subcutaneous,
intradermal, mucosal or suppository routes. Preferred modes of administration are
intravenous or intramuscular, however, the chosen route will be influenced by factors
such as cost and the stability of the dosage form.
An "effective amount" includes reference to a virus titre necessary to at least partly
obtain the desired immune response overall. This will of course vary with the status of
the subject and accordingly is optimised during pre-clinical and clinical investigations.
Various adjuvants may be used to enhance the efficacy of the subject vaccine.
Examples include alum, lecithins, BCG and saponins, or cellular adjuvants such as
dendritic cells.
The vaccine composition may be co-administered or administered as part of an overall
vaccination regime, with other molecules. For example, the subject vaccine constructs
and its expression products may be administered as part of a prime or boost vaccination
component in a "prime-boost" strategy wherein the immune response is enhanced by
presenting antigens to the immune system via various formats.
Still another related aspect of the present invention provides a method of
immunotherapy and/or immunoprophylaxis of prostate cancer comprising
administration of an effective amount of a composition comprising poxvirus vector
which incorporates and, on administration to a subject, expresses in a cell of said
subject, a sequence of nucleotides encoding a prostate specific polypeptide or

homologue, derivative or analogue thereof, wherein s?.:d pexvirus vector does not
productively infect said subject, and wherein expression products of said po.xvirus
vector stimulate a prostate cell specific immune response effective in the treatment
and/or prophylaxis of prostate cancer.
Reference to "immunotherapy" includes amelioration of the symptoms of prostate
cancer or a reduction in the number or proliferation of prostate cancer cells as well as
treatment to total recovery. Reference to "immunoprophylaxis" includes prevention of
developing prostate cance: or the symptoms of prostate cancer as well as a reduction in
the likelihood of developing the symptoms or more severe symptoms of prostate cancer.
If a subject were diagnosed as exhibiting a marker for progression or a marker for
susceptibility to prostate cancer, the poxvirus vector is administered before diagnosis of
prostate cancer.
Pharmaceutical forms of the composition may be suitable for injectable use such as sterile
aqueous solutions and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions.
The composition must be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a medium solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene
glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can
be maintained, for example, by the use of a coating such as lecithin. The prevention of the
action of microorganisms can be brought about by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
In many cases, it will be preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the viral particles ir. the required
Pinount in the appropriate medium with optionally various of the other ingredients
enumerated above, as required. Batches are tested for sterility contamination with protein,
virus concentrate prWml) virus stability, pH and fill volume.
A broad range of doses may be applicable depending on the subject, severity of condition
and proposed route and medium for administration.
ft is especiaDy advantageous to formulate parenteral compositions in dosage unit form
(pfu/ml) for ease of administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary dosages for subjects to be treated;
each unit containing a predetermined quantity of active material calculated to produce the
desired therapeutic or prophylactic effect in association with a pharmaceutical carrier. The
specification for the novel dosage unit forms of the invention are dictated by and directly
dependent on (a) the unique characteristics of the active material and the particular
therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding
such an active material for the treatment of disease in living subjects having a diseased
condition in which bodily health is impaired as herein disclosed in detail. Techniques foi
enterically locating live vaccine formulations are known in the art.
A further related aspect of the present invention contemplates the use of a genetic
vaccine construct in the manufacture of a medicament for the immunotherapy and/or
immunoprophylaxis of prostate cancer, wherein said construct comprises a poxvirus
vector which incorporates and, on administration to a subject expresses in a cell of said
subject, a sequence of nucleotides encoding a prostate-specific polypeptide, or a
homologue, derivative or analogue thereof wherein said poxvirus vector does not
productively infect said subject and wherein expression products of said poxvirus
vectors stimulate a prostate cell specific immune response effective in the treatment or
prophylaxis of prostate cancer.
A still further related aspect of the present invention contemplates the use of a genetic

vaccine construct in the manufacture of a medicament for the immunotherapv and or
immunoprophylaxis of prostate cancer, wherein said construct comprise? a poxvirus
vector which incorporates and, on administration to a subject expresses in a cell of said
subject, a sequence of nucleotides encoding a prostate-specific polypeptide, o; a
homologue, derivative or analogue thereof and a sequence of nucleotides encoding an
immunostirnulatory polypeptide, wherein said poxvirus vector does not productively
infect said subject and wherein expression products of said poxvirus vectors stimulate a
prostate cell specific immune response effective in the treatment or prophylaxis of
prostate cancer.
In a related aspect of this embodiment, the prostate-specific polypeptide is prostatic acid
phosphatase and/or a homologue or derivative or analogue thereof.
A particularly preferred immunostirnulatory molecule for this embodiment of the
invention is an immunostimulatory cytokine such as, for example, a cytokine selected
from one or more of IFNy, IL-12, IL-2, TNFa, IL-4, IL-7, GM-CSF or IL-6. Even more
preferred cytokines are one or more of IL-2, IFNy or IL-12. A particularly preferred
cytokine is IL-2.
The present invention further provides a genetic vaccine construct as described herein
for use in therapy. The present invention additionally provides use of a genetic vaccine
construct as described herein in the manufacture of a medicament for treatment or
prophylaxis of prostate cancer.
The present invention is now further described with reference to the following non-
limiting Examples.



EXAMPLE 1
Construction of a genetic vector comprising a prostate specific polvpepride
Human and rat PAP nucleic acid sequences are publicly available, and their cDNAs may
be cloned amd sequcnced using routine methods well lenown to those skilled in the art.
Bacteria] recombinant rat PAP and human PAP plasmid vectors were obtained from Dr
Doug McNeel (Department of Medicine, division of Medical Oncology, University of
Washington, Seattle, Washington 98195, USA) and their products are used for coating
ELISA plates. Recombinant rat PAP and human PAP proteins have been made in the
InsectSelect system and scaled-up production of purified proteins is done so that they may
be used in both rat and human cellular immunological assays .
The recombinant fowlpox viruses expressing human PAP (FPV.hPAP) and rat PAP
(FPV.rPAP) can be generated using molecular biology techniques for shuttle vector
construction using procedures described by Sambrook et al. "Molecular Cloning: A
Laboratory Manual" Cold Spring Harbor Laboratory, 3rd Edition, 2001 and using
molecular virology technique to generate recombinant poxviruses using procedures
described by Boyle, D.B., et al, Gene 65(1): 123-8, 1988; Coupar, B.E. eta!., Gene 68(1):
1-10, 1988 and Smith G.L., Chapter 9, Expression of genes by vaccinia virus. In Molecular
Virology, A Practical Approach, Ed. AJ Davison and RM Elliott. Practical Approach
Series, 1RL Press at Oxford University Press., 257-283, 1993. Construction of FPV.hPAP
and FPV.rPAP is briefly outlined as follows.
i._____PAP expression cassette
The PAP protein coding sequence, being either human or rat origin, was operatively linked
to a fowlpox virus specific promoter sequence. The promoter sequence in this case does
not have to be fowlpox virus specific or vaccinia specific and other Avipox derived
promoters can be used and can be of any of the following classes: early, late or early/late
(constitutive) promoters. A preferred element for efficient early phase expression during
an infection is the presence of a poxvirus early transcriptional stop sequence of the motif

"TTTTTNT", where N can be any nucleotide sequence, e.g., A or T or G or C, which must
be located 3' downstream of the PAP translational stop ;odon. Addit: n of this earl',
transcriptional stop motif will not be necessary if such a motif occurs by chance some
distance down stream of the PAP translational stop codon. This motif can be conveniently
added to the PAP sequence by R7-PCR amplification (using RNA as template) or PCR
amplification (using cDNA as template) with a primer combination that includes ihis motif
in the PCR primer that targets the 3' end of the PAP nucleotide sequence.
ii. Homologous recombination vector (so called shuttle vector) for aiding insertion of
the PAP expression cassette into the fowlpox genome
The expression cassette described above in step (i) was cloned into a plasmid vector
termed "shuttle vector" or "homologous recombination vector" resulting in a configuration
described as follows.
The PAP expression cassette was cloned in between two short fowlpox nucleotide
sequences of determined length that are homologouc to nucleotide sequences present in
fowlpox genomic DNA that has been previously cloned into a standard commercial
bacterial plasmid vector used for cloning purposes. These short fowlpox nucleotide
sequences are often referred to as homologous recombination arms (left and right) of
flanking arms (flank 1 and flank 2). The key feature here is that the expression cassette is
located within (interior of) the two flanking arms and not exterior to these arms. The result
of homologous recombination between these arms and their homologous sequence within
the fowlpox genome will aid insertion of the expression cassette into the fowlpox genome.
Examples of suitable insertion sites include the TK coding region, 3' of the TK coding
region and the ORF7 to ORF9 region (US 5,180,675).
The shuttle vector also contained a "reporter" expression cassette (beta-galactosidase
protein coding sequence operatively link to a poxvirus specific promoter) and a "positive
selection" expression cassette (E coll xanthine-guanine phosphoribosyl transferase
(Ecogpt) operative linked to a poxvirus specific promoter) located exterior to the two

homologous recombination arms. This configuration enables "transdominant selection" oi"
recombinant viruses.
iii. Homologous recombination
The insertion of the PAP expression cassette into the fowlpox virus genome was carried by
homologous recombination between the fowlpox virus genomic DNA, present during an
infection of tissue culture cells, and the shuttle vector described above in (ii). Chicken
embryo derived cells were infected with fowlpox virus at low multiplicity of infection, for
example, 0.01 infectious units per cell. An hour or two after infection the shuttle vector
containing the PAP expression cassette was transfected into these infected cells using
commercially available transfection kits following the suppliers instruction. After
transfection, the cells and medium were harvested once the infection had reached
confluency. A viral extract was prepared by releasing the virus from the infected cells
cither by mechanical means or repeated cycles of freezing and thawing or by sonicatior:
Two homologous recombination setups were prepared, one for making a recombinant
fowlpox virus expressing the rat PAP and another to make a recombinant fowlpox virus
expressing human PAP.
iv. Clone purification of recombinant fowlpox viruses expressing PAP
The viral extracts from the homologous recombination step were subjected to multiple
rounds of plaque purification in chicken embryo derived cells until no "white" plaques
could be observed when Xgal was present in the tissue culture medium. Mycophenolic
acid, Xanthine and hypoxanthine (as described in Smith G.L 1993, supra) was also present
in the culture medium during the infection for the purpose of positively selecting
recombinant viruses that had a functional Ecogpt inserted into their genomes. The
mycophenolic acid will inhibit the replication of non-recombinant virus. This selection
procedure will select for viruses where a single recombination between one of the
homologous arms and the viral genome inserts the whole shuttle vector into the viral
genome as viruses without the Ecogpt will not replicate under this selection environment.

Viral clones that produce blue plaques in the presence of Xga! were then amplified with
out mycophenolic selection and tested for presence or absence of non-recombinant virus
(empty vector) by PCR analysis using PCR primers that target the flanking region of the
site of insertion.
Recombinant fowlpox virus clones that were tested negative for empty vector
contamination was then subjected to further rounds of plaque purification in the absence of
mycophenolic acid, Xanthine and hypoxanthine to encourage the second recombination
event that will result in the deletion of the reporter and positive selection cassettes from the
recombinant virus. Clones that resulted in white plaques after the addition of Xgal to the
culture medium were amplified and tested for empty vector contamination, removal of
reporter and positive selection cassettes and for functionality of PAP expression.
A recombinant fowlpox vector (M3) encoding human PAP (FPV.hPAP) is plaque purified
and amplified to a titre of 109 pru/mL. The presence of the human PAP insert is confirmed
by PCR. Absence of contaminating wild type fowlpox virus is also confirmed by PCR.
Western blot analysis demonstrates the presence of secreted PAP in the supernatant of
chicken embryo skin (CES) cells, which were infected with FPV.hPAP.
The FPV.rPAP preparation is plaque-purified twice and the presence of the rat PAP insert
confirmed by PCR.
The FPV.rPAP vector is subjected to a third and final round of plaque purification. Then
the plaque-purified vector is amplified to high titre. Expression of secreted recombinant rat
PAP is assayed by Western blot of FPV.rPAP-infected CES cells. The absence of
contaminating wild type FPV is confirmed by PCR. Western blot analysis of human
monocyte-derived dendritic cells (moDC), which have been infected in vitro with
FPV.rPAP or FPV.hPAP, is done to demonstrate that the fowlpox-vectored transgenes are
expressed by the cell type that is most likely to be the target for expression in vivo.

EXAMPLl. 2
Construction of genetic vectors that co-express an immunostimulatur> molecule
The human IL-2 (hIL-2) cDNA has been cloned by RT-PCR from human peripheral blood
lymphocytes (PBL), which were activated for 24 h by PMA and ionomycin. The presence
of tlie correct DNA sequence was confirmed by DNA sequence analysis.
Insertion of a fowlpox virus specific hur.ian IL-2 expression cassette into FPV.hPAP and
FPV.rPAP
The human IL-2 cDNA was operatively linked to a fowlpox virus specific promoter.
Alternatives to fowlpox specific promoters can be vaccinia specific promoters of other
Avipox virus specific promoters. To this promoter plus hIL2, a poxvirus early
transcriptional stop sequence was added downstream of the IL-2 translationa] stop codon.
This expression cassette was cloned into a fowlpox shuttle vector with the same
configurations and features as described in step ii) of Example 1, except that the
homologous recombination arms were homologous to a different area of the fowlpox virus
genome than used for the PAP shuttle vectors.
Homologous recombination and viral selection were carried out as described in Example 1.
The end result was two recombinant fowlpox viruses both expressing human IL-2 but one
expressing human PAP (FPV.hPAP/hIL-2) and the other expressing rat PAP
(FPV.rPAP/hIL-2). ELISA was used to measure the in vitro production of human IL-2
upon infection of tissue culture cells by any of these two vectors.
EXAMPLE 3
In vivo immunogeuicity of xenogeneic genetic vaccine viral construct

The immunogcnicity of viral constructs is determined in appropriate animal models and in
illustrative embodiments, the immunogenicity of FPV.rPAP and FPV.rPAP/hlL-2 is
determined in mice and rabbits.
For detection of anti-rat PAP antibodies, rabbits axe immunized with 1 x 107 pfu
FPV.rPAP or FPV.rPAP/hIL-2 IMI then bled 28 d post-immuni7.ation for direct EL1SA of
serum for rat PAP-specific antibodies, Where rat PAP-specific antibodies are not detected
at 28 d post-immunization then animals are boosted with FPV.rPAP. As a positive control
for both antibody production and the ELISA, rabbits are immunized with recombinant rat
PAP in CFA and boosted with recombinant rat PAP in 1FA at day 21. Blood is drawn and
serum prepared for ELISA 14 days after boosting.
For detection of cellular responses to rat PAP, mice are immunized with 1 x 107 pfu
FPV.rPAP or FPV.rPAP/hlL-2 IMI. Cytolytic and proliferative cellular responses are
measured using spleens harvested from mice killed 6 days and 14 days post-immunization,
respectively. For detection of rat PAP-specific cytotoxic T lymphocytes (CTL), either
intracellular expression of IFNγ or cytolytic function by chromium release assay is
measured. Nylon-wool purified splenic T cells are incubated for 6 hours with either
irradiated syngeneic antigen presenting cells (APC): EL-4 cells that have been transfected
with rat PAP or EL-4 cells as a negative control. Surface staining for CDS and intracellular
staining for IFNy is assayed by flow cytometry. Alternatively, purified splenic T cells are
incubated for 4 hours with 51Cr-labelled EL-4 cell transfectants or EL-4 cells and antigen-
specific chromium release measured. For detection of rat PAP-specific proliferative
responses, splenic T cells are purified over a nylon wool column and incubated for 3 days
with irradiated syngeneic splenocytes, which have been loaded with recombinant rat PAP
or chicken ovalbumin as a negative control. In the final 18 hours of culture, tritiated
thymidine will be added and its incorporation measured as an index of antigen-specific
proliferation.

EXAMPLE 4
In vitro immunogcnicity of genetic vaccine viral construct
PAPS is a HLA-A2.1-binding peptide epitope of human PAP that is identical in rat PAP.
Peshwa et al., describe how PAP5-specific CTL can be derived in vitro and propagated as
ceil lines that lyse both PAP5-loaded T2 cells or the HLA-A2.1+ and PAP+ prostate cancer
cell line, LNCaP. Peripheral blood mononuclear cell (PBMC) cultures from HLA-A2+
donors are obtained and stimulated with PAP5 peptide.
PBMC cultures that continue to grow in response to PAPS peptide are cloned and
expanded. PAPS peptide-specificity is tested by IFNγ-ELISPOT assay. Where clones are
positive, they are propagated on PAPS-loaded T2 cells. The cytolytic activity of PAP5-
specific CTL is tested in a chromium release assay using LNCaP cells as targets.
Appropriate antigen processing and presentation of FPV-vectored rat PAP is assayed using
PAP5-specific CTL. MoDC from HLA-A2.1+ donors are infected with rFPV.rPAP and
antigen-specific reactivity determined by IFNγ-ELISPOT assay.
EXAMPLE 5
Xenoimmunization in a rat model
Each experimental group will comprise five eight week-old rat male Copenhagen rats. Rats
are immunized with 2 x 107 pfu of recombinant viral vectors intravenously (IV) or
intramuscularly (IM). Four weeks later, rats are killed and tissues harvested. Sera are
analysed by direct ELISA for the presence of anti-PAP antibodies. Prostate glands are
examined histologically for evidence of autoimmune prostatitis. Single cell suspensions are
prepared from spleens for in vitro recall proliferation and cytotoxicity assays as described
in the study by Fong et al. We have obtained from Dr Fong the AT-1 and AT-3 cells,
which are syngeneic to Copenhagen rats and are PAP-negative and PAP-positive,
respectively.

As a positive control for the induction of autoimmune prostatitis, rats are immunizes with
the recombinant vaccinia vectors (rVV) that express human PAP (rVV.hPAP). Wild type
virus and the recombinant virus that encodes rat PAP are used as negative controls. Virus
vectors are available from: (i) Dendreon Corp. (Seattle, WA, USA) and published by Fong
et al.; (ii) Dr Doug McNeel, which remain unpublished.
The recombinant fowlpox virus vector that encodes (rFPV.hPAP) is tested for its ability to
induce anti-PAP immune responses and autoimmune prostatitis.
The recombinant fowlpox virus vector that co-expresses human PAP and human IL-2
(rFPV.rPAP/hIL-2) is also tested.
Rats may also be primed with plasmid DNA that encodes human PAP (pcDNA3.1-hPAP)
100 µg IM three weeks before boosting with fowlpox virus vectors. Analysis is performed
approximately four weeks after boosting.
EXAMPLE 6
VIR501 comprising rat PAP and IL-2 and VIR502 comprising human PAP and IL-2
Further recombinant FPV vectors were constructed and tested as follows, Specifically,
VIR501 comprising rat PAP and human IL-2 and VIR502 comprising human PAP and
human IL-2 were generated in recombinant Fowlpox M3 essentially as described
hereinbefore except that the integration vector comprised both inserts under the control of
separate promoters. Figure 9 shows the insertion in FPV relative to the FPV thymidine
kinase gene. Integration vectors comprising cassettes for PAP and IL-2 were constructed
with expression under the control of vaccinia virus p7.5 (human IL-2) and fowlpox virus
early late promoter (rat PAP and human PAP). Plasmid maps of the integration vectors are
set out in Figures 10 and 11. The nucleotide sequences of the insertion sites for VER501
and VIR502 are set out in Figures 5 and 6 and in SEQ ID NO: 1, 2, 3 and 4. The amino
acid sequences of rat and human PAP encoded by the vectors are set out in SEQ ID NO; 5

and 6 and are aligned in Figure 7. The amino acid sequence of human IL-2 encoded by
both vectors is set out in Figures 5 and 6 and in SEQ ID NO: 7.
EXAMPLE 7
VIR501 and VIR502 express IL-2
The vectors were tested, by ELISA, for their ability to express human IL-2. After three
rounds of plaque purification, clones were amplified in CEF cells. After infection, culture
media were tested for the presence of IL-2 using a human IL-2 ELISA kit. The results are
shown in Figure 2 where a visible colour change in the well indicates the presence of IL-2
in test wells.
EXAMPLE 8
TK143B cells infected with VIR501 or VIR502 express PAP
One million cells of the thymidine kinase (TK)-deficient human osteosarcoma cell line,
143B, were plated in a 25 cm2 flask overnight in complete DMEM with 10% FCS. Cells
were infected with FPV vectors at a multiplicity of infection (MOI) of 10 for 48h in a
humidified 37°C incubator in 5% CO2 in air. The vectors used for infection were VIR501
(FPV encoding rat PAP and human IL-2), VIR502 (FPV encoding human PAP and human
IL-2), FPV encoding influenza hamagglutinin (FPV-HA) as a negative control. At 48h and
72h post infection, the infected cells were harvested and lysed with lysis buffer (0.15M
NaCl, 5mM EDTA, 1% Triton X100, 10mM Tris pH 8, 5mM DTT and 100µM PMSF). A
clear lysate was collected after centrifugation at 12000g for 15 min at 4°C. The samples
were boiled in sample buffer and separated using 12% SDS-PAGE. The blot was
transferred to transferred to a PVDF membrane (Amersham Pharmacia Biotech,
Buckinghamshire, England), which was blocked with 2% bovine serum albumin (BSA) in
PBS for 1h at room temperature. After discarding the blocking buffer, the membrane was
probed with polyclonal rabbit anti-human PAP (Signet Pathology System, MA, USA) at
1:500 dilution in blocking buffer overnight at 4°C. After washing with 0,05% Tween-20 in
PBS, a F(ab')2 fragment of goat anti-rabbit antibody conjugated to alkaline phosphatase

(Jackson Immunoresearch, PA, USA) was applied at a dilution of 1:2000 and inculeted for
1h at room temperature. The protein bands were detected with the ECL substrate
(Amersham Pharmacia Biotech, Buckinghamshire.England) and the fluorescent products
were scanned using a Molecular Dynamics Fluorlmager.
The Western blot (see Figure 3) shows expression of human PAP from a VIR502
(FPVhPAP)-infected human cell line. The polyclonal anti-human PAP antibody used for
its detection does not cross-react with rat PAP (lane 6). However, the specific reaction of
sheep immunised with VIR501 as described in Example 9 indicated that V1R501
successfully expresses rat PAP.
EXAMPLE 9
Immunogenicity of FPV vectors expressing human PAP
Bacterial recombinant proteins, pQE-hPAP or pQE-rPAP, or insect cell-derived human
and rat PAP recombinant proteins (produced by stable transfection of the Sf21 Drosophila
cell line using the InsectSelect™ expression system; Invitrogen, CA, USA) were diluted in
0.03M bicarbonate buffer (pH 9.6) at a concentration of 5µg/mL and used to coat
Maxisorp microtitre plates (Nunc, Roskilde, Denmark) overnight at 4°C. In each case,
recombinant proteins were hexa-his tagged and purified on a Nickel affinity column. The
plates were blocked with 2% BSA in PBS for 1h at 37°C (Figure 4) or with 5% normal
horse serum in PBS-azide for 1h at 37°C (Figure 8) and then incubated with serial dilutions
of rabbit sera in blocking buffer for 2h at 37°C (Figure 4) or with serial dilutions of sheep
sera in blocking buffer for 3h at 37°C (Figure 8). After washing with 0.05% tween-20 in
PBS, 1:2500 dilutions of alkaline phosphatase-conjugates of either goat anti-rabbit IgG
[F(ab')2] (Figure 4) or donkey anti-sheep IgG [F(ab')z] (Figure 8) were added and
incubated for a further 2h. Bound antibodies were detected via hydrolysis of p-nitrophenyl
phosphate substrate, and the developed colour was measured at OD 405nm.
ELISA data (see Figure 4) show a humoral immune response of male rabbits primarily to
human PAP only after immunisation with VIR502. Presumably, because they are rodents,

male rabbits are tolerant to the more closely related rat PAP encoded by VIR501. Figure 8
shows the breakdown of tolerance when castrated male sheep were immunised with
VER501 or VIR502. Sheep immunised with V1R501 (rat PAP, human IL-2) responded to
human PAP and rat PAP and the response was detectably enhanced when sera were tested
against recombinant protein generated in insect cells. Over the time course assessed, sheep
immunised with VIR502 (human PAP, human IL-2) did not generate as great a response to
human PAP or rat PAP as that shown by sheep immunised with VIR501 (rat PAP, human
IL-2). These data complement the data in rabbits, indicating that male sheep may be
tolerant to human PAP rather than rat PAP. As PAP is prostate specific, a prostate specific
immune response has been demonstrated after xenogeneic immunisation with VIR501 or
VIR502.
ELISpot data confirm a human cellular immune response in vitro to VIR501, Human
HLA-A2.1+ human monocyte-derived dendritic cells (MoDC) were generated by standard
means in GM-CSF and IL-4. MoDC were matured using lipopolysaccharide for 24 hours
and then infected for 5 days with VIR501 at a multiplicity of infection (MOI) of 10.
Peripheral blood mononuclear cells (PBMC) from the same donor were co-cultured for 7
days with the VIR501-infected MoDC and then Ficoll-purified before an 18-hour
interferon-γ QFNγ) ELISpot assay using as antigen presenting cells (APC) either peptide-
pulsed HLA-A2.1 -expressing murine T2 cells or vaccinia virus-infected HLA-A2.1+
PBMC. In comparison with control APC (autologous PBMC without APC; T2 cells
without peptide; T2 cells pulsed with an irrelevant HLA-A2 binding peptide from HTLV-
1), T2 cells pulsed with HLA-A2.1 -restricted PAPS peptide (Peshwa et al. Prostate 129-
138, 1998), which is identical between rat and human PAP, elicited a four-fold increase in
the number of lFNγ+ spot forming cells (SFC). Moreover, in comparison with control APC
(PBMC infected with wild type vaccinia), PBMC infected with vaccinia expressing rat or
human PAP elicited an approximate three-fold increase in the number of IFNγ+ SFC.
These data support the notion that after VIR501 infection, human APC correctly process
and present endogenously expressed PAP protein to autologous T cells.

BIBLIOGRAPHY
Albert, M. L. et al., Nature Medicine 4:1321-1324, 1998.
Altschul et al., Nucleic Acids Res. 25(17); 3389-3402, 1997.
Ausubei et al., "Current Protocols in Molecular Biology", 5th Edition, John Wiley &
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McNeel D.G. et al., J. Urinol. 154(5): 825-1839, 2000.
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Murphy et al., Prostate 35,73-78, 1999(a).
Murphy, G.P. et al., Prostate 39:54-59, 1999(b).
Parker, K.C. et al., Journal of Immunology 152:163-175, 1994.
Perkus, M.E. et al., Virology 179:276-28, 1990.
Peshwa, M.V. et al., Prostate 36:129-138, 1998.
Rosenberg, S.A. et al., J Am Med Assoc 271:903, 1994.
Rosenberg, S.A. et al., Immunity 70:281-287, 1999.
Sambrook et al. "Molecular Cloning: A Laboratory Manual" Cold Spring Harbor
Laboratory, 3rd Edition, 2001.
Santen R.J., J. Clin. Endocrinal. Metab. 7.5:685-689, 1992.
Smith G.L., Chapter 9, Expression of genes by vaccinia virus. In Molecular Virology, A
Practical Approach. Ed. AJ Davison and RM Elliott. Practical Approach Series, IRL
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Taylor, J. et al. Vaccine 6:497-503, 1988.
Thenot, S. et al. Mol. Cell Endocrinol. 156:85-93, 1999.
Those skilled in the art will appreciate that the invention disclosed herein is suceptible
to variations and modifications other than those specifically described. It is to be
understood that the invention includes all such variations and modifications. The
invention also includes all steps, features, compositions referred to or indicated in this
specification, individually or collectively, and any and al! combinations of any two or
more steps or features.
It would he also appreciated that in view of the sequence of the vaccine construct having
been clearly described hereinbefore, as also the method of producing the construct, the
applicant has not filed a biological deposit in any "depository", and persons skilled in the
art ought to be able to perform the invention, as claimed hereinafter, having regard to the
details provided in the preceding description.

WE CLAIM:
1. A genetic vaccine construct comprising an avipox virus vector which incorporates
and, on administration to a subject, expresses in a cell of said subject, a sequence
of nucleotides encoding a xenogeneic prostate specific polypeptide or a derivative
or analogue thereof, wherein said avipox virus vector does not productively infect
said subject.
2. A genetic vaccine construct comprising an avipox virus vector which incorporates
and, on administration to a subject, expresses in a cell of said subject, a sequence
of nucleotides encoding a xenogeneic prostate specific polypeptide or a derivative
or analogue thereof, and a sequence of nucleotides encoding an
immunostimulatory polypeptide, wherein said avipox virus vector does not
productively infect said subject.
3. The genetic vaccine construct as claimed in claim claim 1 or 2, wherein the
prostate specific polypeptide is prostatic acid phosphatase or a derivative or
analogue thereof.
4. The genetic vaccine construct as claimed in any one of claims 1 to 3, wherein the
subject is a human subject.
5. The genetic vaccine construct as claimed in claim 4, wherein the xenogeneic
prostate specific polypeptide is rodent prostatic acid phosphatase.
6. The genetic vaccine construct as claimed in claim 5, wherein the rodent prostatic
acid phosphatase is rat prostatic acid phosphatase.
7. The genetic vaccine construct as claimed in claim 2, wherein the
immunostimulatory polypeptide is a cytokine.
8. The genetic vaccine construct as claimed in claim 7, wherein the cytokine is one or
more of IL-2, IL-12, TNFα, IFNγ, IL-6, IL-4, IL-7 or GM-CSF.
9. The genetic vaccine construct as claimed in claim 8, wherein the cytokine is one or
more of IL-2, IFNγ or IL-12.
10. The genetic vaccine construct as claimed in claim 9, wherein the cytokine is IL-2.

11. The genetic vaccine construct as claimed in any one of claims 1 to 10, wherein the
avipox virus vector is a fowlpox virus vector.
12. A composition comprising the genetic vaccine construct as claimed in any one of
claims 1 to 11.
13. A composition consisting essentially of the genetic vaccine construct as claimed in
any one of claims 1 to 11.
14. The composition as claimed in claim 12 or 13, wherein expression products of said
genetic vaccine construct stimulate a prostate cell specific immune response.
15. The composition as claimed in claim 14, wherein prostate cell specific immune
response is a PAP specific immune response.
16. The composition as claimed in claim 14 or 15, wherein the expression products of
the genetic vaccine construct stimulate autoimmune prostatitis.
17. A recombinant vector for use in making the genetic vaccine construct as claimed in
any one of claims 1 to 11 comprising:
i) avipox virus vector nucleic acid sequences comprising sites for
homologous recombination with an avipox virus vector;
ii) one or more promoters; and
iii) a sequence of nucleotides encoding a xenogeneic prostate specific
polypeptide.
18. A recombinant vector for use in making the genetic vaccine construct as claimed in
any one of claims 2 to 11 comprising:
i) avipox virus vector nucleic acid sequences comprising sites for
homologous recombination with an avipox virus vector;
ii) one or more promoters;
iii) a sequence of nucleotides encoding a xenogeneic prostate specific
polypeptide; and
iv) a sequence of nucleotides encoding an immunostimulatory polypeptide.

19. An isolated antibody capable of acting as a marker for the genetic vaccine
construct which antibody recognises epitopes uniquely formed in expression
products of the genetic vaccine construct as claimed in any one of claims 1 to 11.
20. A nucleic acid probe which under appropriate hybridisation conditions, such as
herein described, specifically recognises the genetic vaccine construct as claimed
in any one of claims 1 to 11, said nucleic acid probe comprising a contiguous
sequence of nucleotides complementary to all or part of the nucleotide sequence of
said genetic vaccine construct.

A genetic vaccine construct comprising a poxvirus vector which incorporates and, on administration to a subject,
expresses in a cell of said subject a sequence of nucleotides encoding a prostate-specific polypeptide, wherein said poxvirus vector
does not productively infect said subject. The genetic vaccine construct is for treatment of prostate cancer. The prostate specific
polypeptide is preferably rat prostatic acid phosphatase, and is preferably xenogenic to the subject. The proxvirus vector is an avipox
virus vector, preferably a fowl poxvirus vector. The genetic vaccine construct may also encode for one or more cytokines.

Documents:

631-kolnp-2006-granted-abstract.pdf

631-kolnp-2006-granted-assignment.pdf

631-kolnp-2006-granted-claims.pdf

631-kolnp-2006-granted-correspondence.pdf

631-kolnp-2006-granted-description (complete).pdf

631-kolnp-2006-granted-drawings.pdf

631-kolnp-2006-granted-examination report.pdf

631-kolnp-2006-granted-form 1.pdf

631-kolnp-2006-granted-form 18.pdf

631-kolnp-2006-granted-form 3.pdf

631-kolnp-2006-granted-form 5.pdf

631-kolnp-2006-granted-gpa.pdf

631-kolnp-2006-granted-reply to examination report.pdf

631-kolnp-2006-granted-specification.pdf


Patent Number 230146
Indian Patent Application Number 631/KOLNP/2006
PG Journal Number 09/2009
Publication Date 27-Feb-2009
Grant Date 25-Feb-2009
Date of Filing 20-Mar-2006
Name of Patentee VIRAX DEVELOPMENT PTY LTD
Applicant Address SUITE 220, KEW JUNCTION TOWER, 89 HIGH STREET, KEW, VICTORIA, 3101
Inventors:
# Inventor's Name Inventor's Address
1 BROWN, MICHAEL, PAUL 83 ANGLESEY AVENUE, ST. GEORGES, SOUTH AUSTRALIA, 5064
2 HOWLEY, PAUL, MICHAEL 60 ROSE AVENUE GLEN WAVERLEY, VICTORIA 3150
PCT International Classification Number C12N 15/863
PCT International Application Number PCT/AU2004/001129
PCT International Filing date 2004-08-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2003904496 2003-08-21 Australia