Note: Descriptions are shown in the official language in which they were submitted.
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
-- 1 --
"CELLULAR IMMUNOGENS USEFUL AS CANCER VACCINES"
Cross-Reference to Related Application
Priority from U.S. provisional patent application No. 60/010,262,
filed January 19, ~996 is claimed.
Field of the Invention
The invention relates to the field of cancer vaccination and
immunotherapy.
Back~round of the Invention
A current goal of cancer research is the identification of host
factors that either predispose to tumor formation or serve to enhance tumor
growth.
Genes that confer the ability to convert cells to a tumorigenic
state are known as oncogenes. The transforming ability of a number of
retroviruses has been localized in individual viral oncogenes (generally v-onc).Cellular oncogenes (generally c-onc) present in many species are related to viral
oncogenes. It is generally believed that retroviral oncogenes may represent
escaped and/or partially metamorphosed cellular genes that are incorporated intothe genomes of tr~n~mi.~ible, infectious agents, the retroviruses.
Some c-onc genes intrinsically lack oncogenic properties, but may
be converted by mutation into oncogenes whose transforming activity reflects
the acquisition of new properties, or loss of old properties. Amino acid
CA 022419~2 1998-07-14
W O 97/25860 PCTnUS97/00582
-- 2 --
substitution can convert a cellular proto-oncogene into an oncogene. For
example, each of the members of the c-ras proto-oncogene family (H-ras, N-ras
and K-ras) can give rise to a transforming oncogene by a single base mutation.
Other c-onc genes may be functionally indistinguishable from the
5 corresponding v-onc, but are oncogenic because they are expressed in much
greater amounts or in ina~prOpLiate cell types. These oncogenes are activated
by events that change their expression, but which leave their coding sequence
unaltered. The best characterized example of this type of proto-oncogene is c-
myc. Changes in MYC protein sequence do not appear to be essential for
10 oncogenicity. Overexpression or altered regulation is responsible for the
oncogenic phenotype. Activation of c-myc appears to stem from insertion of a
retroviral genome within or near the c-myc gene, or translocation to a new
environment. A common feature in the translocated loci is an increase in the
level of c-myc expression.
Gene amplification provides another m~ch~ni~m by which
oncogene expression may be increased. Many tumor cell lines have visible
regions of chromosomal amplification. For example, a 20-fold c-myc
amplification has been observed in certain human leukemia and lung carcinoma
lines. The related oncogene N-myc is five to one thousand fold amplified in
20 human neuroblastoma and retinoblastoma. In human acute myeloid Icukemia
and colon carcinoma lines, the proto-oncogene c-myb is amplified five to ten
fold. While established cell lines are prone to amplify genes, the presence of
known oncogenes in the amplified regions, and the consistent amplification of
particular oncogenes in many independent tumors of the same type, strengthens
25 the correlation between increased expression and tumor growth.
~ mmunity has been successfully in~ ce~ against tumor formation
by inoculation with DNA constructs cont~ining v-onc genes, or by inoculation
with v-onc proteins or peptides. A series of reports describe a form of
"homologous" challenge in which an animal test subject is inoculated with either30 v-src oncoprotein or DNA constructs cont~inin~ the v-src gene. Protective
immnnity was induced against tumor formation by subsequent challenge with v-
CA 022419~2 1998-07-14
i
W O 97/25860 PCT~US97/0058
- 3 -
src DNA or v-src-in-lnced tumor ce}ls. See, Kl-7llm~ki et al., JNCI (1988),
80:959-g62; Wisner et al., J. Virol. (1991), 65:7û20-7024; Halpern et al.,
Virology (1993), 197:480-484: Taylor et al., Virology (1994), 205:569-573;
Plachy et al., Immunogenetics (1994), 40:257-265. A challenge is said to be
5 "homologous" where reactivity to the product of a targeted gene is in~ ced by
immllni7~3tion with the same gene, the corresponding gene product thereof, or
fragment of the gene product. A challenge is "heterologous" where reactivity
to the product of a targeted gene is indllce(l by immlmi7~tion with a different
gene, gene product or fragment thereof.
WO 92/14756 (1992) describes synthetic peptides and oncoprotein
fragments which are capable of eliciting T cellular immllnity, for use in cancervaccines. The peptides and fragments have a point mutation or translocation as
compared to the corresponding fragment of the proto-oncogene. The aim is to
induce immunoreactivity against the mllt~tPd proto-oncogene, not the wild-type
lS proto-oncogene. WO 92/14756 thus relates to a forrn of homologous challenge.
EP 119,7()2 (1984) describes synthetic peptides having an amino
acid sequence corresponding to a deterrninant of an oncoprotein encoded by an
-- oncogenic virus, which determinant is vicinal to an active site of the
oncoprotein. The active site is a region of the oncoprotein required for
20 oncoprotein function, e.g., catalysis of phosphorylation. The peptides may beused to i..,l--ll"i~ hosts to elicit antibodies to the oncoprotein active site. EP
119,702 is thus directed to a form of homologous challenge.
The protein product encoded by a proto-oncogene constitutes a
self antigen and, depending on the pattern of its endogenous expression, would
25 be tolerogenic at the level of T cell recognition of the self peptides of this
product. Thus, vaccination against cancers which derive from proto-oncogene
overexpression is problematic.
Recent attempts have been made to induce im ml-nity in vitro or
in vivo to the product of the HER-2/neu proto-oncogene. The proto-oncogene
30 encodes a 185-kDa transmembrane protein. The HER-2/neu proto-oncogene is
overexpressed in certain cancers, most notably breast cancer. In each report
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582
-- 4 -
discussed below, the immllnogen selected to induce immllnity comprised a
purified peptide of the pl85HER-2~neU protein, and not a cellular immunogen.
Disis et al., Cancer Res. (1994) 54:16-20 identified several
breast cancer patients with antibody immllnity and CID4+ helper/inducer T-cell
S immllnity responses to pl85HER-2~ne" protein. Antibodies to pl85HER-2~neU were
identified in eleven of twenty premenopausal breast cancer patients. It was
assumed prior to this work that patients would be immlln~logically tolerant to
HER-2/neu as a self-protein and that i~ y would be difficult to generate.
Disis et al, Cancer Res. (1994~ 54:1071-1076 constructed
synthetic peptides identical to pl85HER-~neU protein segments with amino acid
motifs similar to the published motif for HLA-A2. l-binding peptides. Out of
four peptides synthesized, two were shown to elicit peptide-specific cytotoxic
T-lymphocytes by primary in vitro i~ i7~tion in a culture system using
peripheral blood Iymphocytes from a normal individual homozygous for HLA-
A2. Thus, it was concluded that the pl85HE~-2~n~U proto-oncogene protein
contains immunogenic epitopes capable of generating human CD8~ cytotoxic T-
lymphocytes.
~ The cytotoxic T cells elicited in the latter report were not,
however, shown to recognize tumor cells, but only targets that bound the
synthesized peptides. Other work (Dahl et al., J. Immunol. (1996), 157:239-
246) has demonstrated that cytotoxic cells may recognize targets that bind
peptide but fail to recognize targets that endogenously synth~si7~ peptide. It is
thus unclear whether the cytotoxic cells elicited by Disis et al. would be capable
of recognizing tumor cells. In any event, no protection against tumor growth
was demonstrated by Disis et al.
Peoples et al., Proc. Natl. Acad. Sci. USA (1995), 92:432-436,
report the identification of antigenic peptides presented on the surface of ovarian
and breast cancer cells by HLA class I molecules and rccognized by tumor-
specific cytotoxic T Iymphocytes. Both HLA-A2-restricted breast and ovarian
tumor-specific cytotoxic T Iymphocytes recognized shared antigenic peptides.
CA 022419~2 1998-07-14
,
W O 97/25860 PCTAUS97/00582
-- 5 --
T cells senciti7e~1 against a nine-amino acid sequence of one of the peptides
demonstrated significant recognition of HL~-A2 HER~/neu tumors.
It remains unclear whether Peoples et al. have successfully
attacked proto-oncogene-encoded self, as the imml-ni7.ing peptide which is
5 expressed in the tumor cells contained an isoleucine at position 2, whereas the
peptide expressed in normal tissue contains valine residue at this position.
Moreover, although stim~ tion of T cells occurred in vitro, this stimulation
does not le~ sell~ a true primary immlm~ response insofar as the starting T cellpopulation represented tumor infiltrating Iymphocytes.
The research accounts of Disis et al. and Peoples et al. re~uired
a forrn of in vitro stimulation, either priming as described by Disis et al., orrestim~ tion as described by Peoples et al. The in vitro protocols of Disis et
al. and Peoples et al. require a mutant cell line to aid in selection of the peptide
which will serve to induce reactivity. Non-mllt~nt peptide antigen-presenting
15 cells have their HLA class I molecules already loaded with endogenous
péptides, a phenomenon which precludes exogenous loading from without. The
value of the mutant lines is that they lack the TAP genes (encoding the
transporters associated with antigen presentation). Class I binding of internally-
derived peptides is significantly lowered, and "empty" class I molecules are
20 present on the cell surface and available for binding of exogenously added
peptides. This availability of peptide binding sites on membrane-bound class
I allows e~c~min~tion of whether a given peptide will (i) even bind to class I,
and (ii) function as a target in cytotoxic T cell assays. However, the need for
a mutant cell line for deduction of c~ntlici~te immllni7.ing peptide sequences
25 limits the usefulness of peptide-based i~ ion schemes.
Fendly et al., J. Biol. Response Modifiers (1990), 9:449-455
present an account of a polypeptide-based imml~notherapy. Purified polypeptide
corresponding to the extracellular domain of the pl85HER-~'ne" protein was
obtained from a transfected cell line. The purified peptide was employed in the
30 immlmi7~tion of guinea pigs. The immnni7ecl ~nim~l.c developed a cellular
imm~mP response, as monitored by delayed-type hypersensitivity. Antisera
CA 022419~2 1998-07-14
W O 97/2~860 PCT~US97/00582 -- 6 --
derived from immnni7ed ~nim~l~ specifically inhibited the in vitro growth of
human breast tumor cells overexpressing p185HER-2'ne". There is no indication
by Fendly et al. of induction of self versus non-self reactivity. It is likely that
the guinea pigs were chiefly responding to non-self determin~ntc (as defined in
5 terrns of the guinea pig host) on the human polypeptide immunogen.
The use of peptides for immunization is of necessity limited to
immnni7~tion with a single haplotype. There are approximately thirty HLA
types in man. In each case of peptide immllni7~tion, one must be careful to
select peptides which match the host HLA type. The selected peptide must be
10 immunogenic in the host and be capable of presentation to host immnn-o system cells.
What is needed is an immllni7~tion method for immllni7.ing
hllm~n~ and ~nim~ against self-encoded proto-oncogenes which are associated
with the development of cancer, which dispenses with the need for isolating
15 irnmunogenic, HLA host-matched peptides for immunization.
Summarv of the Invention
It is an object of the invention to induce reactivity to self-
determin~nl.~ of the product of an overexpressed proto-oncogene.
It is an object of the invention to provide for a forrn of therapy
2Q or prophylaxis based upon the capacity to induce immlm~ reactivity to proto-- oncogene-encoded self as overexpressed in tumor cells.
It is an object of the invention to provide a cellular immunogen
for use in immnni7.~tion against self proto-oncogene determin~ntc.
It is an object of the invention to provide for a method for
2~ vaccinating a host against disease associated with the overexpression of a proto-
oncogene.
These and other objects will be apparent from the following
disclosure.
A method of vaccinating a host against disease associated with the
30 overexpression of a target proto-oncogene is provided. The method comprises:
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
-- 7 --
(a) excising cells from the host;
(b) transfecting the excised cells with at
least one transgene construct comprising at least
one transgene cognate to the target proto-oncogene
and a strong promoter to drive the expression of
the transgene in the transfected cells, the
transgene encoding a gene product which induces
host immllnoreactivity to host self-determin~nt~ of
the product of the target proto-oncogene gene;
(c) returning the excised cells transfected
with the transgene construct to the body of the
host to obtain expression of the transgene in the
host.
According to one principal embodiment of the invention, the
15 transgene comprises wild-type or mutant retroviral oncogene DNA. According
to another principal embodiment of the invention, the transgene comprises wild-
type or mutant proto-oncogene DNA of a species different from the host
species. Where the transgene comprises mutant retroviral oncogene DNA or
mutant proto-oncogene DNA, the mutant DNA is preferably nontransforming.
20 The mutant DNA preferably comprises a deletion mutation in a region of the
DNA which is essential for transformation. Preferably, the host cells are
transfected with a plurality, most preferably at least five, different transgeneconstructs, each construct encoding a different deletion mutation.
In one preferred embodiment of the invention, the mutant DNA
25 has at least about 75 % homology, more preferably at least about 80 %
homology, most preferably at least about 90% homology, with the
corresponding wild-type oncogene or proto-oncogene DNA.
The invention is further directed to a cellular immunogen for
immllni~.ing a host against the effects of the product of a target proto-oncogene,
30 the overexpression of which is associated with a cancer. The cellular
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/0058 -- 8 --
immllnogen comprises the host cells which have been transfected with at least
one transgene construct, as described above.
The invention is also directed to a method of preparing the
cellular imml~nogen, by (a) excising cells from the host, and (b) transfecting the
S excised cells with at least one transgene construct, as described above.
The cells transfected with the transgene are preferably rendered
non-dividing prior to return to the body of the host.
The term "corresponds to" is used herein to mean that a
polynucleotide sequence is homologous (i. e., is identical, not strictly
evolutionarily related) to all or a portion of a reference polynucleotide sequence,
or that a polypeptide sequence is identical to a reference polypeptide sequence.The term "cognate" as used herein refers to a gene sequence that
is evolutionarily and functionally related between species. For example but not
limitation, in the human genome, the human c-myc gene is the cognate gene to
the mouse c-myc gene, since the sequences and structures of these two genes
indicate that they are highly homologous and both genes encode proteins which
are functionally equivalent.
By "homology" is meant the degree of sequence similarity
between two different amino acid sequences, as that degree of sequence
similarity is derived by the FASTA program of Pearson and Lipman, Proc.
Natl. Acad. Sci. USA (1988), 85:2444-2448~ the entire disclosure of which is
incorporated herein by reference.
As used herein, the term "operably linked" refers to a linkage of
polynucleotide elements in a functional relationship. A nucleic acid is "operably
linked" when it is placed into a functional relationship with another nucleic acid
sequence. For instance, a promoter or enhancer is operably linl~ed to a coding
sequence if it affects the transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and, where
n~cess~ry to join two protein coding regions, contiguous and in reading frame.
The word "transfection" is meant to have its ordinary meaning,
that is, the introduction of foreign DNA into eukaryotic cells.
CA 02241952 1998-07-14
W O 97/Z5860 PCTrUS97/00582
- _ g
By "transgene" is meant a foreign gene that is introduced into one
or more host cells.
By "transgene construct" is meant DNA cont~ining a transgene
and additional regulatory DNA, such as promoter elements, necessary for the
e~,ession of the transgene in the host cells.
Description of the Fi3~ures
Figs. lA and lB are plots of the mean tumor diameter over time
following subcutaneous wing web inoculation of 1-day-old line TK (Fig. lA)
and line SC (Fig. lB) chickens with 100 ,ug of tumorigenic plasmids pcsrc527
(--~--), pVSRC-C1 ~--~--) or pMvsrc (~ ). The mean tumor diameter
(mm) at a particular time point and for any one group of TK or SC line
chickens inoculated was computed as the sum of the diameters of the primary
tumors divided by the number of chickens surviving to that point. The ratios
at each time point show, for a particular group, the number of chickens bearing
palpable tumors to the total number of survivors to that point (standard typeface
for pcsrc527, italics for pVSRC-C1, bold typeface for pMVsrc). Error bars
(unless obscured by the symbol) inrli~te standard error.
Figs. 2A and 2B are plots of the growth of challenge (wing web)
tumors in test and control line TK chickens under conditions of (i) priming and
homologous challenge with plasmid pcsrc527 (Fig.2A~ --, test; -----,
control), or (ii) priming and homologous challenge with plasmid pVSRC-C1
(Fig. 2B: --O--, test; ------, control). Test chiçk~n~ were primed at 1 day
posthatch with 100 ,ug of construct; test and control chickens were challenged
at five weeks posthatch with 200 ,ug of construct. The mean challenge diameter
was computed as in Figs. lA and lB. At each time point the ratio of chickens
~ bearing palpable challenge tumors to total number of survivors to that point is
jn(1ic~t~.~1 (standard typeface for control group, bold typeface for test group).
The statistical comparison between the mean challenge tumor diameters of the
test versus the control group at a particular time point was made using a two-
tailed student's t test, *(p<0.05), **(p<0.01), ***(p<0.001). The statistical
S~S 111 IJTE SHEET (RULE 26)
CA 022419~2 1998-07-14
WO 97125860 PCT/US97/00582
- 10 -
comparison between the ratios of chickens bearing palpable challenge tumors to
total number of survivors of the test versus the control group at a particular
time point was made using a chi-s~uared test; the paired ratios are underlined
for only those time points where p C 0.05. Error bars in-lic~te standard error.
~igs. 3A and 3B are plots of the growth of challenge (wing web)
tumors in TK chickens under conditions of (i) priming with plasmid pVSRC-C1
and heterologous challenge with plasmid pcsrc527 (Fig. 3A~ , test;
-, control) or (ii) priming with pcsrc527 and heterologous challenge with
pVSRC-~1 (Fig. 3B: --O--, test; ------, control). Test chickens were
primed at 1 day posthatch with 100 ,ug of construct; test and control chickens
were challenged at five weeks posthatch with 20~ ,~bg of construct. The mean
ch~llenge tllmor diameter was computed as in Figs. lA and lB. At each time
point the ratio of chickens bearing palpable challenge tumors to total number ofsurvivors to that point is inflic~t~d (standard typeface for control group, boldtypeface for test group). St~ti~tic~l comparisons were made between test and
control groups at a particular time point as described for Figs. 2A and 2B.
[*(p < 0.05), **(p < 0.01), ***(p C 0.001), for the student's t test], and the
paired ratios are underlined for only those time points where, in the chi-squared
test, p<0.05. Error bars in~ te standard error.
Detailed Description of the Invention
A vaccination strategy is provided to prevent development of
cancers. The vaccination method may be carried out on a subject at risk for a
particular cancer, but before the development of the cancer. The practice of theinvention may serve for the immunoprevention of prevalent human cancers,
such as colon carcinoma, breast carcinoma, and various lymphomas whose
progress is accomp~n~ by the overexpression of a cellular proto-oncogene.
The vaccination strategy of the present invention relies on the
induction of an immllnP. response that targets tumor cells by virtue of the
recognition of the proto-oncogene-specific antigenicity. The aim of the vaccine
protocol is to induce reactivity to self-determin~ntc of an ovele,~ ssed proto-
SUBSTITUTE SHEET (RULE 26)
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
oncogene product. The strategy exploits the structural relatedness between the
product of the cellular proto-oncogene and that of the product of genes cognate
to the target proto-oncogene. The cognate gene may comprise a wild-type or
mutant cognate retroviral oncogene or a wild-type or mutant proto-oncogene
5 of a species different from the host species. The starting point of the vaccine
strategy is the high degree of primary sequence homology that exists between
the protein product of a targeted proto-oncogene and that of its cognate
retroviral oncogene, or between the proto-oncogene product and the product of
a cognate proto-oncogene from a different species. However, in contrast to
10 other proposed vaccine strategies, the present invention is not based on the
immune recognition of a determinant defined by a cancer specific mutation.
For those tumors showing proto-oncogene overexpression, this
sequence homology permits application of the following strategy, which can be
employed either prophylactically or therapeutically under conditions of cell-
15 surface expression, or other forms of adjuvanicity, as chosen to enhanceimmunogenicity: (a) immunization of host biopsied cells with a DN~ construct
comprising a transgene cognate to the target proto-oncogene, which transgene
encodes a gene product which induces host immunoreactivity to host self-
determin~nt~ of the product of the target proto-oncogene; (b) return of the
20 transfected cells to the body of the host to obtain expression of the transgene in
the host, and thus immllnity against the proto-oncogene product. The invention
relies on the targeting of a self-determinant found on an overexpressed or
overabundant proto-oncogene-encoded product. The foreign peptide elements
of the immunizing oncogene product will trigger peripheral Iymphocytes
25 exhibiting a weak cross reactivity for the self peptides of the targeted proto-
oncogene product. Although such self peptides would be present in normal cells
expressing the proto-oncogene, targeting of the tumor cells is favored in view
of their overexpression of the proto-oncogene.
The immune strategy exploits the antigenicity of two alternative
30 types of determin~3nt~: (13 tumor-associated antigenic deterrninant(s) induced as
a consequence of the activity of the oncogene product, e.g., an enzymatic
CA 0224l9~2 l998-07-l4
W O 97/25860 PCTrUS97/00~82
- 12 -
modification of a cellular protein effected by the oncogene product, or (2) tumor
associated antigenic determinant(s) intrinsic to the oncogene-encoded product
itself. The difficulty in exploiting the first alternative by traditional means, i.e.,
antigen purification, is that at present little or no systematic information exists
5 bearing on the properties of an antigen that, though oncogene-induced, is not
oncogene-encoded. This situation makes purification of any such antigen
problematic. However, this problem is obviated from the outset by the present
invention which utilizes biopsied cells which, as transfected in culture by the
cognate retroviral oncogene, would express the relevant antigenicity.
In terms of exploiting the second alternative, that of an
antigenicity intrinsic to the proto-oncogene product, a relevant consideration is
that the protocol of immunization according to the present invention primes the
host to determin~nt~ of the oncogene product itself. A consequence of this
immunization is induction of T-cell reactivity to the divergent, i.e foreign,
15 peptide determin~nts of the retroviral oncogene product, i.e., those peptide
determin:lnt~ that show sequence ~lirl~lcnces with the positionally homologous
determin~nt~ of the cellular proto-oncogene product. The induction of this
reactivity does not in itself have vaccine potential, since the foreign
determin~nt~ specific to the retroviral oncogene product are normally absent
20 from the cellular proto-oncogene product. Nevertheless, the foreign peptide
elements, notably those that differ by only a single amino acid from the
positionally homologous self peptides, trigger peripheral T-lymphocytes
exhibiting a weak cross-reactivity for the self peptides. Although such self
peptides are present in normal cells expressing the proto-oncogene, targeting of25 the tumor cells is favored in view of their overexpression of the proto-oncogene.
It is possible that many tumor-associated and overexpressed
proto-oncogenes might possess mutations. In some cases, overexpression may
very well arise as a direct conse~uence of one or more of the mutations.
However, the present vaccination method does not have as its object the
30 deliberate targeting of non-self determin:~nf~ generated by proto-oncogene
mutations. Unlike prior vaccination methods designed to target such mutation-
CA 022419=,2 1998-07-14
.
WO 97/25860 PCT/US97/0058
- 13 -
driven non-self determin~nt~, it is the aim of the present invention to induce
reactivity for self-determin~nt.s in the overexpressed product of tumor associated
and overexpressed proto-oncogenes.
Prior efforts attempting to elicit reactivity to proto-oncogene self
5 determin~nt~ have relied on in vitro protocols lltili7ing mutant cell lines toidentify individual self peptide immllnogens (Disis et al., Cancer Res. (1994)
54: 1071-1076; Peoples et al., Proc. Natl. Acad. ~ci USA (1995), 92:432-436).
According to the present invention, the host immlln~ system is presented with
the full array of naturally-derived class I binding peptides. The vaccine strategy
10 of the present invention obviates the need for any a prior~ assessment of the immunogenicity of individual peptides.
While the cellular immlmogens of the invention display self
peptides, non-self peptides would also be presented which may serve as more
effective tolerance breakers. The value of a non-self, but closely related to self,
15 peptide is that it may more readily activate those T cells that have both a weak
cross reactivity for the cognate self peptide and an activation threshold
(determined by the tightness of binding to the T cell receptor) too high to be
~ triggered by the self peptide. Moreover, cognate non-self is inductive of a good
immune response, simply because it does in fact constitute nonself. The non-
20 self immune response is expected to predispose the induction of the inevitably
weaker response to the self determin~nt~ on the same protein product, since the
resultant cytokine release provides local help to initiate the weaker anti-self
response.
As hereinafter exemplified in a model of src-oncogene-based
25 tumor formation, immllni7~ion with cells transfected with a transgene construct
expressing the v-src oncogene product induces reactivity to the product of the
c-src proto-oncogene, thereby conferring protection against the growth of
tumors displaying overexpression of the c-src proto-oncogene.
CA 0224l9~2 l998-07-l4
W 097/25860 PCT~US97/00582
- 14 -
Tar~et Proto-Onco~enes
According to the present invention, patients with a family history
of a cancer characterized by the overexpression of a particular proto-oncogene
are selected for immuni7~tion. Alternatively, patients whose tumors can be
5 shown to overexpress the proto-oncogene are selected. Overexpression of a
proto-oncogene may derive from an increase over a basal level of transcription.
Overexpression may also derive from gene amplification, that is, an increase in
gene copy number, coupled with a basal or elevated level of t~ cli~3tion.
Proto-oncogene overexpression may be assayed by conventional probing tech-
10 niques, such as described in Molecular Cloning: A Laboratory Manual J.Sambrook et al., eds., Cold Spring Harbor Laboratory Press, 2nd ed. 1989.
The level of target proto-oncogene expression may be determined by probing
total cellular RNA from patient cells with a complementary probe for the rele-
vant mRNA. Total l~NA from the patient cells is fractionated in a glyox-
15 al/agarose gel, transferred to nylon and hybridized to an apl)lopliately labellcdnucleic acid probe for the target mRNA. The number of relevant mRNA tran-
~ scripts found in the patient cells is compared to that found in cells taken from
the same tissue of a normal control subject.
As an alternative to measuring mRNA transcripts, the expression
20 level of a target proto-oncogene may be assessed by assaying the amount of
encoded protein which is formed. Western blotting is a standard protocol in
routine use for the determination of protein levels. See Molecular Cloning,
supra, Chapter 18, incorporated herein by reference. Accordingly, a cell Iysate
or other cell fraction cont~inin~ protein is electrophoresed on a polyacrylamide25 gel, followed by protein transfer to nitrocellulose, and probing of the gel with
an antibody specific for the protein in question. The probe step permits
resolution of the desired protein from all other proteins in the starting mixture.
The bound antibody may be prelabeled, e.g., by a radioisotope such as l25I, so
as to permit its detection on the gel. Alternatively, a secondary reagent (usually
30 an anti-immunoglobin or protein A) may be radiolabeled or covalently coupled
CA 022419~2 1998-07-14
,
W O 97/25860 PCT~US97/OOS8~
- 15 -
to an enzyme such as horseradish peroxidase or ~lk~linlo phosphatase. The
strength of the signal is proportional to the amount of the target protein. The
strength of the signal is compared with the signal from a sample analyzed in thesame manner, but tal~en from nor~nal as opposed to tumor tissue.
A description of the methodology and use of Western blotting to
determine the levels of the c-src-encoded protein pp60C-SrC in adenomatous polyps
(colonic epithelia) is provided by Cartwright et al., Proc. Natl. Acad. Sci. USA(1990), 87:558-562, the entire disclosure of which is incorporated herein by
reference.
An at least about eight-fold increase in that gene's expression in
the patient cells compared to expression in normal control cells from the same
tissue would indicate candidacy for vaccination.
Table 1 includes a partial list of representative proto-oncogenes,
the overexpression of which has been associated with one or more m~ n~nries.
15 Each listed proto-oncogene is a target proto-oncogene according to the present
invention. The corresponding oncogene, of which the target proto-oncogene is
the normal cellular homolog, is also identified. This list of target proto-
oncogenes is intended to be representative, and not a complete list.
Table 1
Represe-~l~live ~ist of Target Proto-Oncogenes
Proto-
Onco~eene Tumor Comments/References
AKT-2 ovarian v-Akt is the oncogene of the AKT8 virus, which
- induces Iymphomas in mice.
1. Bellacosa et al., (1995) Int. ~. Cancer
64(4):280-5: Southern-blot analysis has shown
AKT-2 amplification in 1 2 .1% of ovarian
carcinomas, while Northern bot analysis has
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
- 16-
revealed overexpression of AKT-2 in 3 of 25
fresh ovarian carcinomas which were negative for
AKT-2 amplification.
2. Cheng et al., (1996~ Proc. Natl. Acad. Sci.
USA 89(19): 9267-71): Amplification of AKT-2
has been ~l~tecte~l in 10% of pancreatic
carcinomas.
AKT-2 pancreatic Cheng et al., (1996) Proc. Natl. Acad. Sci. USA
93(8):3636-41: Amplification of AKT-2 has been
detected in 10% of pancreatic carcinomas.
c-erbB-2 bladder c-ErbB-2 is also known as HER2/neu. V-erbB is
the oncogene of the avian erythroblastosis virus.
1. Underwood et al., (1995) Cancer Res.
55(11):2422-30: Protein overexpressio-n was
observed in 45% of patients with non-recurrent
disease and 50 % of patients with recurrent
disease; 9% of bladder tumors analyzed shoed
gene amplification.
2. Coombs et al., (1993) Pa~hology 169(1):35-
42: c-ErbB-2 gene amplification was observed in
14% of bladder tumors analyzed.
3. Gardinerelal., (1992) Urolog. Res. 20(2):17-
20: Nineteen percent of primary transitional cell
bladder carcinomas showed c-erbB-2 gene
amplification.
c-erbB-2 breast 1. Molina et al., (1966) Anticancer Research
16(4B):2295-300: Abnormal c-erbB-2 levels were
found in 9.2% of patients with locoregional breast
CA 02241952 1998-07-14
W O 97/25860 PCTrUS97/00582
carcinoma, and in 45.4% of patients with
advanced disease. 2. DePotter et al., ~1995)
VirchowsArch. 426(2): 107-15: Overexpression of
the oncoprotein is observed in about 20 % of
invasive duct cell carcinomas of the breast. 3.
Bandyopadhyay et al., (1994) Acta Oncol
33(5):493-8: 35.4% of breast tumors showed c-
erbB-2 overexpression; 17.4% showed gene
amplification. 4. Fontana et al., (1994)
0 Anticancer Res. 14(5B):2099-104: 26% of
samples showed c-erbB-2 amplification. 5. Press
et al., (1993) Cancer ~esearch 53(20) :4960-70:
Amplified overexpression was identified in 38%
of primary breast cancers. 6. Berns et al.,
(1992) Cancer Res. 52~5):1107-13: 23% of
primary breast cancer tissues exhibited
amplification. 7. Delvenne etal., (1992) Eur. J.
of Cancer 28(2-3):700-5: c-erbB-2 mRNA was
overexpressed in 34% of breast tumor samples.
8. Inglehart, (1990) CancerRes. 50(20):6701-7:
Two to thirty-two-fold gene amplification was
found in multiple stages of tumor progression. 9.
Slamon et al., (1989) Science 244:707-12: A
28% incidence of amplification of c-erbB-2 was
found in 189 primary breast cancers. 10. Kraus
et al., (1987) EMBO J. 6(3):605-10: Eight cell
lines demonstrated c-erbB-2 mRNA levels ranging
from 4 to 128-fold overe~cpression. 60% of all
tumors analyzed showed elevated levels of c-erbB-
2 mRNA.
CA 0224l9~2 l998-07-l4
W O 97125860 PCTrUS97/00~82.
- 18 -
c-erbB-2 lung 1. Osaki et al., (1995) Chest 108(1):157-62:
Lung tissue overexpression of c-erbB-2 was
discovered in 42.5% of samples. 2. Lorenz et
al., (1994) Clin. Invest. 72(2):156-63: A 64-fold
increase in the amount of c-erbB-2 mRNA was
observed; 33 % of lung tumors showed
overexpression of c-erbB-2.
c-erbB-2 ovarian 1. Katsaros et al., (1995) Anticancer Res.
15(4): 1501-10: Abnormally high expression of c-
0 erbB-2 was found in 31% of tumor samples. 2.
Felip et al., (1995) Cancer 75(8):2147-52: 21.7 %
of ovarian tumors showed overexpression of c-
erbB-2. 3. Fan et al., (1994) Chin. Med. J.
107(8):589-93: c-erbB-2 amplification was found
in 30.8% (8 of 26) of human ovarian cancers. 4.
vanDam et al ., ( 1994) J. of Clin. Path .
~ 47(10):914-9: 24% of ovarian tumors showed c-
erbB-2 overexpression. 5. Csokay et al., (1993)
Eur. ~. of Surg. Oncology 19(6):593-9: c-erbB-2
amplification was found in 34% of fresh ovarian
tumor samples. 6. McKenzie et al., (1993)
Cancer 71(12):3942-5: 30% o~ ovarian tumor
samples in-lir~ted c-erbB-2 overexpression. 7.
Hung et al., (1992) Cancer Letters 61(2):95-103:
A 100-fold c-erbB-2 overexpression was
discovered in one human cell line. Two to four-
fold amplification was also discovered.
MDM-2 lellk~mi~ M~M-2 is the murine double minute-2 oncogene.
1. Bueso-Ramos et al., (1993) Blood 82(9):2617-
CA 02241952 1998-07-14
W O 97/25860 PCTrUS97/00582
- 19 -
23: 53 ~ of cases showed overexpression of
MDM-2 rr~RNA. The level of MDM-2 mRNA
overexpression in some cases of leukemias was
comparable to that observed in some sarcomas,
S which demonstrate more than 50-fold MDM-2
gene amplification. No evidence of gene
amplification was observed. 2. Watanabe et al.,
(1994) Blood 84(9):3158-65: 28% of patients
with B-cell chronic Iymphocytic leukemia or non-
Hodgkin's Iymphoma had 10-fold higher levels of
MDM-2 gene expression. MDM-2 overexpression
was found more frequently in patients at advanced
clinical stages.
c-myb colon V-myb is the oncogene of the avian
myeloblastoma virus. 1. Ramsay et al., (1992)
Cell Growth and Diff. 3(10):723-30: c-myb levels
were always higher in colon cancer samples than
normal tissue. 2. Alitalo et al., (1984) Proc.
Natl. Acad. Sci. 81(14):4534-8: c-myb levels
were always higher in colon cancer samples than
normal tissue.
c-myc breast V-myc is the oncogene of the avian myelocytoma
virus. 1. Lonn et al., (l99S) Cancer
75(11):2681-7: Amplification of c-myb occurs in
16% of patients with breast cancer. 2. Hehir et
al., (1993) J. of Surg. Oncolog~ 54(4):207-9: c-
myc overexpression was found in 60% of breast
carcinoma samples. 3. Kreipe et al., (1993)
CancerResearch 53(8): 1956-61: Amplification of
CA 022419~2 1998-07-14
W O 97/25860 PCTnUS97/00582
- 20 -
c-myc was found in 52.6% of samples that
displayed a Ki-S1 labelling index excee~ling 30%.
4. Watson et al., (1993) J. Nat. Cancer Inst.
85(11):902-7: Amplification of c-myc occurs in
S up to 20 - 30% of breast cancers. 5. Berns et
al., (1992) Cancer Research 52(5):1107-13:
Amplification was found in 20 % of primary breast
cancer patients; the range was 3-14 gene copies.
6. Watanabe et al., (1992) Cancer Research
52(19):5178-82: Expression of c-myc was
increased by 10-fold.
c-myc gastric/ 1. Rigas, (1990) Clin. Gastroent. 12(5):494-9:
colorectal Overexpression of c-myc is found in 80 of colon
cancers. 2. Erisman et al., (1988) Oncogene
2(4):367-78: Adenocarcinoma cell lines express
5-10-fold elevated levels of c-myc mRNA. Eight
to thirty-seven-fold higher levels of c-myc protein
was found in tumor cell lines compared to normal
cells. 3. Sikora et al., (1987) Cancer
59(7):1289-95: Up to 32-fold overexpression of
c-myc mRNA was observed in 12 to 15 tumors.
4. Tsuboi et al., (1987) Biochem. and Biophys.
Res. Comn~. 146(2):705-10: Gastric Cancer: A
2-3-fold overexpression was observed in gastric
cancer. A 2-10-fold overexpression was observed
in colorectal cancer.
c-myc lung 1. Lorenz et al., (1994) Clin. Invest. 72(2):156-
63: A 57-fold increase in c-mYc mRNA levels
was observed. 23% of samples indicated strong
CA 0224l952 l998-07-l4
.
W O 97/25860 PCTrUS97/00582.
- 21 -
expression of c-mvc. 2. Kato et al., (1993) Jap.
J. of Cancer Res. 84(4):355-9: Liver tissue
metastases from human small cell lung carcinoma
revealed 30-fold amplification of c-myc.
5 c-myc naso- Porter et al., (1994) Acta Oto-Laryng. 114(1):
pharn- 1105-9: 22% of samples showed intense staining
geal for c-myc.
c-myc ovarian 1. Bian et al., (1995) Chin. J. of Ob. Gyn.
30(7):406-9: 50% of samples showed
amplification of c-myc. 2. Katsaros et al.,
(1995) Anticancer Res. 15(4):1501-10: 26% of
samples exhibited c-myc amplification. 3. van
Dam et al., (1994) J. Clin. Path. 47(10):914-9:
Overexpression of c-myc was found in 35% of
ovarian carcinomas. 4. Xin et al., (1993) Chin.
J. of Ob. Gyn. 28(7):405-7: 54.5% of samples
showed amplification of c-myc. 5. Tashiro et al.,
(1992) Int. J. of Cancer 50(5):828-33:
Overexpression was found in 63.5% of all serous
adenocarcinoma tissues and 37.3% of all ovarian
carcinoma tissues. Signific~nt overexpression of
~ c-myc was observed at Stage III compared with
other stages.
C-7~ C prostate Nag et al., (1989) Prostate 15(2):115-22: A 10-
fold amplification of c-myc was observed. Fifty-
fold higher levels of mRNA transcripts of c-myc
were found.
CA 022419~2 1998-07-14
W O 97/2~860 PCTrUS97/00582
- 22 -
c-ras lung Ras oncogenes were first recognized as the
transforming genes of Harvey and Kirsten murine
sarcoma viruses . Lorenz et al., ( 1994) Clin.
Invest. 72(2): 156-63: a 13-fold increase in
overexpression of c-Ki-ras was observed. 18% of
tumors displayed strong overexpression of c-Ki-
ras.
c-ras ovarian 1. Katsaros et al., (1995) Anticancer Res.
15(4): 1501-10: Higher levels of ras protein than
in normal or benign ovarian tumors were found in
45 % of tumor samples. 2. vanDam et al.,
(1994) J. of Clin. Path. 47(10):914-9: 20% of
ovarian tumors exhibited c-ras overexpression.
The levels of expression of c-ras were much
higher in tumors of patients with recurrent or
persistent disease after chemotherapy, than in the
tumors of patients at initial presentation.
c-src breast V-src is the oncogene of the Rous sarcoma virus,
which induces sarcomas in chickens.
Muthuswamy et al., (1994) Mol. and Cell. Biol
14(1):735-43: c-erbB-2-inA~lcedm~mm;lrytumors
possessed 6-8-fold higher c-src kinase activity than
adjacent epithelium.
c-src colon/ 1. Cartwright et al., (1994) J. of Clin. Invest.
colorectal 93(2):509-15: c-srcactivity is 6-10-fold higher in
mildly dysplastic ulcerative colitis (a chromic
infl~mm~tory disease of the colon with a high on
incidence of colon cancer) than in non-dysplastic
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
- 23 -
epithelia. This data suggests that activation of c-
src is an early event in the genesis of UC colon
cancer. 2. Talamonti et al., (1993) J. of Clin.
In~est. 91(1):53-60: High level of c-src activity
from colorectal cancer is found in liver
metastases. 3. Termuhlen et al., (1993) J. of
Surg. Res. 54(4): 293-8: Colon carcinoma
metastases to the liver had significantly increased
activity of c-src with an average 2.2-fold increase.
Extrahepatic colorectal metastases demonstrated an
average 12.7-fold increase in c-src activity over
normal mucosa.
c-yes colon V-yes is the oncogene of two avian sarcoma
viruses, Esh sarcoma virus and Y73. 1. Pena et
al., (1995) Gastroent. 108(1):117-24: Twelve to
fourteen-fold higher expression of c-yes was found
in colonic transforming oncogene adenomas
compared to normal mucosa. Activity of c-yes
was elevated in adenomas that are at greatest risk
for developing cancer. 2. Park et al., (1993)
Oncogene 8(10):2627-35: A ten to 20-fold higher
than normal activity of c-yes was observed in 3
out of S colon carcinoma cell lines. A 5-fold
higher than normal activity was found in 10 out of
21 primary colon cancers, compared to normal
colonic cells.
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
- 24 -
Selection of Co,enate Trans~ene for Preparation of Cellular Immuno~en
According to the present invention, a transgene construct is
engineered comprising a transgene which is cognate to the target proto-oncogene
(hereinafter "cognate transgene" or "CTG"). The transgene is selected such that
5 it encodes a gene product which induces host immunoreactivity to host self-
determin~nt~ of the product of the target proto-oncogene. The transgene should
be expressed to very high levels in the transfectants. Thus, the construct should
contain a strong promoter.
The product encoded by the cognate gene must have a high
10 degree of sequence homology with the product of the target proto-oncogene, but
also must display some amino acid differences with the target proto-oncogene
product. Thus, there must be a subset of one or more amino acid differences
between the target proto-oncogene and its cognate in order to provide
immunogenic stimulus. Two classes of genes that satisfy these criteria are
15 retroviral oncogenes and xenogenic proto-oncogenes. The word "xenogenic"
is intended to have its normal biological me~ning, that is, a property or
characteristic referring or relating to a different species. Thus, a xenogenic
proto-oncogene is meant to include the a homologous proto-oncogene of a
species other than the host organism species. It may be appreciated that in the
20 case of a target proto-oncogene, e.g. MDM2, for which no retroviral homolog
is yet known, a xenogenic homologue is advantageously utilized as the source
of the DNA for the cognate transgene.
In principle, a more effective immllnC~genic stimulus would
depend on the particular sequence, and not on the distinction between a
25 retroviral oncogene and a xenogenic proto-oncogene in terms of their relativetransforming capacity. Thus, in certain cases, a retroviral oncogene may be
better at providing a tolerance-breaking immunogenic stimulus, and in other
cases, a xenogenic proto-oncogene may be more effective.
The retroviral oncogene or xenogenic proto-oncogene DNA
30 forming the CTG may comprise the wild type oncogene or proto-oncogene
DNA. More preferably, a mutant DNA is utilized, which is engineered so as
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582
-25 -
to be non-transforming in the host. The DN~ is mllt~t~-l to include one or
more nucleotide insertions, deletions or substitutions which will encode an
oncogene product which is nontransforming in the host, but retains the requisitedegree of sequence homology with respect to the target proto-oncogene. A
5 cognate transgene deletion mutant (hereinafter "dCTG") is preferred.
A protein sequence is generally considered "cognate" with respect
to the target proto-oncogene-encoded protein if it is evolutionarily and
functionally related between species. A more precise view of cognation is based
upon the following sequence comparison carried out utilizing the FASTA
program of Pearson and Lipman, Proc. Natl. Acad. Sci. U~A (1988), 85:2444-
2448~ the entire disclosure of which is incorporated herein by reference.
Cognation is ~tt~ined upon satisfying two criteria imposed by FASTA; (i)
~lignment of segments corresponding to at least 75% of the target proto-
oncogene's encoded amino acid sequence; ~ii) at least 80% amino acid identity
15 within the aligned sequences. The segments of the target proto-oncogene
protein sequence and protein test sequence satisfying the two criteria are
referred to as "homology regions". ~ccordingly, at least 75% of the target
proto-oncogene protein sequence is alignable with the test sequence. The
alignable segments or homology regions may, however, represent less than 75 %
20 of the total test polypeptide chain for the case of test sequences that may
significantly exceed the target proto-oncogene protein in length.
One skilled in the art, armed with the FASTA program, may
survey existing sequence data bases (either protein sequences or DNA
sequences, insofar as the amino acid sequence is determined by FASTA for all
25 reading frames) for test sequences which are cognate with respect to the target
proto-oncogene. At the same time, one can isolate and then sequence what are
very likely to be cognate test sequences (e.g. feline MDM-2~ as likely to be
cognate to human MDM-2) and use FASTA to verify the presumed cognation,
according to the criteria set above. One may obtain the sequences of
30 presumptive cognate proto-oncogenes from a large number of m~mm~ n
CA 022419~2 1998-07-14
W O g7/25860 PCT~US97/00582.
- 26 -
sequences and screen these sequences with FASTA according to the aforesaid
formulation of cognation.
Because the product encoded by a CTG differs at a small number
of amino acid positions from the product encoded by the target proto-oncogene,
S an immllnogenic stimulus is provided that (i) is directed against the foreign
protein and (ii) with a lower probability, induce an anti-self response. The
CTG is selected such that the gene product will yield the greatest immunogenic
stimulus to induce anti-self reactivity. Provided that overall sequence homology(preferably greater than about 75%) is m~int~inP~l, the presence of scattered
amino acid differences is desired, since any one residue would likely have a
relatively low probability of inducing self-reactivity. Moreover, the greatest
number of residue differences would be advantageous, consistent with
m~int~ining the requisite degree of general sequence homology.
The selection of amino acid modifications for the CTG may be
facilitated by resort to available computer-based models used to identify
immunogenic peptide fragments of polypeptides. These models could be
employed to select CTGs which would possess the maximum number of
immunogenic peptides for a given HLA haplotype.
Screenin~ Procedure for CTG Selection
Notwithstanding the availability of computer-based algorithms
which have some predictive value, it is desirable to design CTGs with resort to
a screening procedure based on an actual experimental assay that can be Hl_A-
haplotype specific. Accordingly, cells are biopsied from a nor~nal volunteer of
particular haplotype. The cells are transfected with a CTG construct, preferablya dCTG construct, satisfyin~ ~he criteria set for cognition. More preferably, the
cells are transfected with multiple dCTGs, preferably at least five dCTGs,
satisfying the criteria for cognition. The at least five dCTGs are selected to
display amino acid differences that essentially extend throughout the polypeptide
chains of the encoded se~uences. The transfected cells are then used to
immunize the volunteer in accordance with the immunization method of the
CA 022419~2 1998-07-14
.
W O 97/25860 PCTrUS97/OOS82
- 27 -
present invention. After immllni7~rion, the human subject is tested in a standard
delayed hypersensitivity (DH) reaction with 104-106 irradiated, autologous
fibroblasts, as transfected with the same dCTG (or series of dCTGs) as used for
the immllni7in~ preparation. A positive DH reaction (induration) would verify
S the induction of reactivity. The induction of reactivity in this assay is readily
demonstrable because of the priming to the non-self determin~nts on the dCTG-
encoded protein and the readout in the DH reaction of the same nonself
determinzln~. Once DH reactivity is demonstrated in a DH reaction that
directly tests the antigenicity of the non-self determin~n~.~ encoded by the dCTG
(i.e., priming with a non-self construct, DH testing with the same non-self
construct), the subject can be then tested in a DH reaction based on testing with
the autologous cells transfected with a dCTG derived from the human proto-
oncogene itself (i.e., priming with a non-self construct, testing with the humanself construct). Testing of a battery of human volunteers will lead to a
15 catalogue of HLA-matched dCTGs, such that, for individuals of the same HLA
haplotype, the use of the particular dCTG would be inductive of reactivity to
proto-oncogene-encoded self. Different CTGs may thus be tested so as to
- correlate maximal secondary stim~ ion with a particular HLA haplotype.
At the same time, this procedure may be used with patients
20 undergoing tumor resection (if post-operative immuno-suppressive protocols are
not m~nflzltory), such that prior to resection, a course of immllni7~tion would
have been initi~t~d, the endpoint of which would represent the development of
a DH reaction.
Any given amino acid difference between the CT~-encoded
25 product and the proto-oncogene-encoded product has a low probability of beinga "tolerance-breaker". Thus, it is preferable to transfect the host cells with amixture of multiple different CTGs, preferably dCTGs. The number of
different dCTGs is preferably five or more. Moreover, it is preferred that,
among themselves, the multiple dCTGs show amino acid differences that
30 essentially extend throughout the polypeptide chains of the encoded sequences.
The dCTGs would be selected to maximize amino acid differences and, at the
CA 022419~2 1998-07-14
WO 97/25860 PCT/US97/00582
- 28 -
same time, make sure that differences are found all along the polypeptide chain.It would thus not be preferable to select a battery of deletions all from withinthe same domain of the polypeptide chain.
According to a protocol which utilizes 107 irradiated cells for
5 immlmi7:~tion Cont~inin~ five separate dCTGs, five groups of 2 X 106 cells areincluded in one inoculate, each group of 2 X 106 having been transfected with
a separate dCTG from the total set of five CTGs that are cognate to a particularproto-oncogene.
Selection of Non-Transformin~ Co~nate Trans~enes
10Non-transforming cognate transgene variants are most
advantageously derived via deletion of a sequence essential for transformation.
Unlike point mutations which are potentially reversible due to back mutations,
deletion mutations are irreversible. Furthermore, deletion mutations do not
possess the inherent disadvantage attaching to point mutations, namely, even
15 though the requirement for generation of an acceptable cognate transgene is for
a qualitative difference with the wild type, i . c ., non-transforming versus
~ transforming, any given point mutation may be neutral or else quantitative in
its effect, that is, the mutation may reduce but not totally elimin~te
transformability. Thus, according to a preferred embodiment of the invention,
20 a deletion is created in a region of the cognate transgene which encodes an
amino acid sequence required for transformation. Consonant with non-
transformability, the smallest deletion possible so as to leave intact the bullc of
the antigenicity of the transgene product is selected.
The engineering of a cognate transgene deletion mutant that
25 satisfies these criteria is facilitated by reports of structure-function relationship
in oncogene-encoded proteins. Such reports serve to identify regions of
oncoproteins that are essential for transformation, as opposed to regions ~vhichare either neutral or serve merely to modulate transformability. Although such
reports are usually based on in vitro transformation assays, and are therefore
30 independent of immun~ effects, these studies can be exploited to aid in the
CA 022419~2 1998-07-14
.
W 097/25860 rCT~US97/00582
- 29 -
.
construction of non-transforming dCTGs for use in the practice of the present
invention.
The deletion mutant is engineered to include at least a part of the
region identified as critical for transformation. In those cases where essentialS amino acids have been identified, the deletion will span these residues. The
engineering of any desired deletion can be readily accomplished by polymerase
chain reaction (PCR) according to conventional PCR techniques, based upon the
known nucleotide sequence of the unmutated cognate transgene.
The following describes a representative protocol for deriving a
10 non-transforming dCTG of the smallest possible deletion, for use in the practice
of the present invention. A test dCTG, engineered on the basis of known or
ascertained transformation-specific domains, and driven by the strongest possible
promoter, is used to transfect murine 3T3 cells. A sister culture of 3T3 cells
is also transfected, with non-deleted CTG. Each CTG or dCTG cell culture is
15 inoculated into nude mice, in the absence of any treatment to render the cells
non-dividing. Those dCTGs which do not yield tumors in the mice even after
prolonged observation are then utilized as transgenes for the biopsied human
~ cells which, upon transfection with the transgene, will serve as a cellular
vaccine according to the practice of the present invention. The dCTGs are
20 selected with the smallest deletion mutant consonant with non-transformability.
Some CTGs representing xenogenic proto-oncogenes may not be
tumorigenic in the 3T3/nude mouse assay. For any such non-transforming
CTG, it is not essential to generate a dCTC. However, even given non-
tumorigenicity in nude mice, it may be desirable to opt for generation of a
25 deletion mutant when the transgene is based upon a xenogenic proto-oncogene.
In such cases, the deletion would be engineered so as to remove the
homologous region to that deleted in the particular dCTG that corresponds to
the deletion in the corresponding retroviral oncogene dCTG.
Even though the transgene construct may comprise mutant
30 oncogene or proto-oncogene DNA which is nontransforming, it is nevertheless
preferable, as a safety measure, to treat the transfected cells to render them non-
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582
- 30 -
dividing before inoculation back into the host. The cells are irradiated with a
radiation dosage sufficient to render them non-dividing.
Onco~enicity Assay of Co~nate Trans~enes
As a further safety measure, the oncogenicity of a given dCTG
5 is preferably thoroughly tested prior to infection of the human host cells which
are used as cellular immunogens according to the practice of the present
invention. For example, an oncogenicity testing regimen may take the form of
three separate assays: (i) dCTC~ transfection of NIH 3T3 cells, followed by
inoculation into nude mice; (ii) dCTG transfection of human fibroblasts,
10 followed by inoculation into nude mice; and (iii) dCTG transfection of human
fibroblasts, followed by an in vitro test of anchorage-dependent growth. In
principle, all three should be negative to validate the use of any given dCTG inthe vaccination method of the present invention.
According to the oncogenicity assay (i), after stable transfection
15 of NIH 3T3 cells with the test dCTG, the transfectants are inoculated into nude
mice. Tumorigenicity of the transfectants in the mice is then evaluated
according to standard protocols.
According to oncogenicity assay (ii), human fibroblasts are
transfected with the test dCTG as proposed in the above human imml~ni7~tion
20 protocol. After stablc dCTG transfection of human fibroblasts, however, rather
than carrying out X-irradiation of the transfectants to render them non-dividing,
followed by inoculation of the irradiated transfectants back into the human host,
the transfectants are directly inoculated into nude mice as a direct test of
tumorigenicity. Given the greater susceptibility of murine 3T3 cells to
25 oncogenic transformation, vis a vis primary human or murine transfectants
fibroblasts, assay (ii) is probably much less sensitive than assay (i), but doeshave the advantage of offering a direct test of dCTG oncogenicity in human
cells.
According to oncogenicity assay (iii), non-irradiated dCTG-
30 transfected human fibroblasts are assayed for anchorage-dependent growth, i. e.
CA 0224l9~2 l998-07-l4
W O 97/25860 PCTrUS97/00582
- 31 -
colony formation in soft agar, as a test of dCTG transforming potentia~ in
human cells. Anchorage independence, as defined by the ability of cells to
grow when suspended in semisolid medium, is a common phenotype acquired
by human tumor cells, particularly those tumor cells of mesenchymal origin,
such as fibrosarcomas. While assay (iii) has no in vivo readout, it offers an
independent test of the critical issue of dCTG oncogenicity in human cells.
The oncogenicity assays are performed according to published
protocols. Assay (i), comprising dCTG transfection of NIH 3T3 cells followed
by inoculation into nude mice, may be performed according to the protocol of
Stevens et al., Proc. Natl. Acad. Sci. US~ (1988), 85:3875-3879, including
DNA transfection by the calcium phosphate coprecipitation method of
Manohaven et al., Carcinogenesis (1985), G: 1295-1301. Accordingly, NIH 3T3
cells (7.5 X 105 cells per 100-mm dish) are exposed to a calcium phosphate-
DNA coprecipitate (40 ~g of genomic DN~ plus 3 ,llg of pSV2neo per dish) for
4 hours. Two days later, each dish is trypsinized and reseeded into a 175-cm2
flask. For the next 10 days, cultures are selected in G418 (400 ,ug/ml), and theflasks are then trypsinized and cells are replated in the same flask to dispersethe G418-resistant colonies into a diffuse lawn of cells. Two days later, the
cells are harvested and washed with serum-free medium prior to injection. One
injection of 5 ~ 106 cells into the right flank and one injection of 1 X 107 cells
into the left flank, each in a volume of 200 ,~bl, are done on each nude mouse.
Injection sites are monitored at 3- or 4-day intervals for 100 days. The sites are
scored for the number of tumors induced per injection site.
Oncogenicity assay (ii), whereby dCTG transfection of human
fibroblasts followed by inoculation into nude mice, is carried out in the same
manner as assay (i) except that for assay (ii) the human fibroblast transfectants
are substituted for the murine 3T3 transfectants.
Assay (iii), involves a test of the in vitro anchorage-dependent
growth of dCTG-transfected human fibroblasts. The assay is carried out as
described in Stevens et al., J. Cancer Res. and Clin. Oncol. 1989, 115:118-
128. 1 x 105 cells are seeded per 60-mm dish into 0.33% Noble agar over a
CA 022419~2 1998-07-14
W O 9712S860 PCTrUS97/00582
- 32 -
6-ml 0.5% agar base layer in Hams ~10 supplemented with 6% fetal bovine
serum. A portion of the agar suspension is diluted with Hams F10 plus 6%
fetal calf serum to 200 cells/5 ml to deterrnine the cloning efficiency of thesecells when seeded into plastic 60-mm dishes. Agar dishes are fed with 1 ml
5 Hams F10 supplemented with 6% fetal bovine serum on the 1st and l5th day
after seeding. Four weeks after seeding, all agar colonies ~ 75 ,llm in diameterare counted and the colony counts are norm"li7f d to the plating efficiencies
which aliquots of the initially seeded cells showed on plastic. This comparison,or norrn~li7~ion, of the agar colony counts to the plastic dish co~ony counts is10 useful in identifying and correcting for any mechanical artifacts which mightresult from the seeding into agar of dead cells that had persisted from the initial
transfection treatment or from heat-in~ ce-1 cell death, which might have
occurred while suspending cells in molten agar during the process of seeding theagar dishes.
The following is a partial list of various deletions which, based
upon published accounts of experiments with human or animal cells, are
believed to render the identified CTG non-tumorigenic.
.
CA 022419=.2 1998-07-14
wo 97/25860 PcT/US97/00582
- 33 -
Table 2
Deletion Mutations Renderin~ In~ ted Gene Non-Transformin~
CTG G~nh~nk Number Amino acids References
accession of ~lelete~l,
number for amino rendering
se~uence acids in CTG non-
gene transforming
,4kt-2 (c-akt) M95936; 480 148-234 Bellacosa et
(mouse) SEQ ID al., Science
NO:3 (Mus (1991),
musculus 254:274-
serine/threon 278;
ine kinase) Bellacosa et
al.,
Oncogene
(1993),
8(3):745-54.
CA 02241952 1998-07-14
W O 97/25860 PCT~US97/00582.
- 34 -
CTG G~nh~nk Number Amino acids References
accession of ll~lete~l,
number for amino rendering
sequence acids in CTG non-
gene transforming
c-neu (c- M11730; 1255 1-731 Bargmann
erbB-2) (rat) SEQ ID et al.,
NO:4 EMBO
(human (1988),
tyrosine 7(7) :2043-
kinase-type 5~;
receptor Bernards et
(HER2) gene al., Proc.
Natl. Acad.
Sci. USA
(1987),
84(19):6854
-8.
CA 0224l952 l998-07-l4
.
W O 97/25860 PCT~US97/OOS82
- 35 -
CTG Genbank Number Amino acids References
~ccç~ n of ~1~1ete-1,
number for amino rendering
sequence acids in CTG non-
gene transforming
mdm-2 U33199; 489 9-155 Dubs-
(human) SEQ ID Poterszman,
NO:5 Oncogene
(human (1995),
mdm2-A 11 (11) :2445
mRNA); -50.
U33200;
SEQ ID
NO:6
(human
mdm2-B
mRNA);
U33201;
SEQ ID
NO:7
(human
mdm2-C
mRNA);
U33202;
SEQ ID
NO:8
(human
mdm2-D
mRNA);
U33203;
CA 02241952 1998-07-14
WO 97/2S860 PcT/Us97/00582
- 36 -
CTG GPnh~nk Number Amino acids References
~ccç~iQn of deleted,
number for amino rendering
sequence acids in CTG non-
gene transforming
c-myb J02012; SEQ 640 275-327 Kalkbrenner
(human) ID NO: 10 et al.,
(proviral Oncogene
oncogene v- (1990),
myb) 5(5):657-61.
c-myc X00364; 439 129-144 Sarid et al.,
(human) SEQ ID Proc. Natl.
NO: 11 Acad. Sci.
(human c- USA (1987),
myc 84(1): 170-3.
oncogene)
v-ras M77193; 189 32-44 Zhang et
(Harvey SEQ ID al., Science
Murine NO: 12 (Rat (1990),
Sarcoma sarcoma 249: 162-5
Virus) virus v-ras (1990)
oncogene)
CA 02241952 1998-07-14
W O 97/25860 PCTrUS97/00582
- 37 -
CTG G~nh: " Number Amino acids References
accession of (lP.I~te~l,
- number for amino rendering
sequence acids in CTG non-
gene transforming
v-src (Rous U41728; 526 430-433 Bryant et
Sarcoma SEQ ID al., Mol.
Virus) N0:13 (RSV Cell. Bio.
Schmidt- (1984),
Ruppin A 4(5):862-6.
clone SRA-
V; v-src
gene)
c-yes D00333; 541 438-441 Zheng et
~chicken) SEQ ID al.;
N0: 14 Oncogene
(human c- (1989),
yes-2 gene) 4(1): 99- 104.
En~ineerin~ of Vectors for Host Cell Transfection
The engineering of vectors for expression of a particular CTG,
preferably a dCTG, is based on standard methods of recombinant DNA
technology, i. e. insertion of the dCTG via the polylinker of standard or
5 commercially available expression vectors. The dCTG is operably linked to a
strong promoter. Generally speaking, a "strong" promoter is a promoter which
achieves constitutively high expression of the dCTG in the transfected cells.
- Each promoter should include all of the signals nf~cess:~ry for initi~ting
transcription of the relevant downstream sequence. These conditions are
10 fulfilled, for example, by the pBK-CMV expression vector available from
Stratagene Cloning Systems, La Jolla, CA (catalog no. 212209). The pBK~
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582.
- 38 -
CMV vector contains the cytomegalovirus (CMV) immediate early promoter.
dCTGs xenogenic with respect to a particular target proto-oncogene may be
isolated by conventional nucleic acid probing techniques, given the availabilityof a highly homologous probe represented by the cognate retroviral oncogene
5 and/or the human proto-oncogene itself.
Collection of Host Cells for Transfection
The host cells which may be transfected to derive the cellular
immnnogens of the present invention must express class I MHC and be
susceptible to isolation and culture. Fibroblasts express class I MHC and may
10 be cultured. Accordingly, punch biopsies of host human skin are performed to
harvest fibroblasts. Punch biopsies can be performed by a competent physician
as a standard clinical procedure. Each biopsy yields a starting population of 1-2
X 107 cells that would proliferate in culture. Methods for the preparation of
tissue cultures of human fibroblasts are well developed and widely used. See,
15 Cristofalo and Carpenter, J. Tissue Cultllre Methods (1980), 6:117-121, the
entire disclosure of which is incorporated herein by reference. Essentially, slcin
. obtained by punch biopsy is washed using an appropriate wash medium, finely
minced and cultured in a suitable culture medium, such as Dulbecco's Modified
Eagle Medium (DMEM), under CO2 at 37~C. The cells are trypsinized with
20 a trypsin solution and transferred to a larger vessel and incubated at 37~C in
culture fluid.
Host Cell Transfection
The expression vector carrying the dCTG is used to transfect
biopsied host cells according to conventional transfection methods. One method
25 of transfection involves the addition of DEAE-dextran to increase the uptake of
the naked DNA molecules b~ a recipient cell. See McCutchin and Pagano, J.
Natl. Cancer Inst. (1968) 41:3~1-7. Another method of transfection is the
calcium phosphate precipitation technique which depends upon the addition of
Ca~ ~ to a phosphate-cont~ining DNA solution. The resulting precipitate
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582
- 39 -
apparently includes DNA in association with calcium phosphate crystals. These
crystals settle onto a cell monolayer; the resulting apposition of crystals and cell
surface appears to lead to uptake of the DNA. A small proportion of the DNA
taken up becomes expressed in a transfectant, as well as in its clonal descen-
S dants. See Graham et al., Virolog;y (1973), 52:456-467 and Virology (1974), 54:536-539.
Preferably, transfection is carried out by cationic phospholipid-
me~ tt~d delivery. In particular, polycationic liposomes can be formed from
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)
or related liposome-forming materials. See Felgner et al., Proc. Natl. Acad.
Sci. USA (1987) 84:7413-7417 (DNA-transfection); Malone et a~., Proc. Natl.
Acad. Sci. USA (1989), 86:6077-6081) (RNA-transfection). One preferred
technique utilizes the LipofectAMINETM Reagent (Cat. No. 18324-012, Life
Technologies, Inc., Gaithersburg, MD) which is a 3: 1 (w/w) liposome
formulation of the polycationic lipid 2,3-dioleyloxy-N-
[2(spermint-c~rboxamido)ethyl-N,N-dimethyl-~-prop~n;lminiumtrifluoroacetate
(DOSPA) (Chemical Abstracts Registry name: N-[2-(~2,5-bis[(3-
aminopropyl)amino]-1-oxypentyl}amino)ethyl]-N,N-dimethyl-2,3-bis(9-
octadecenyloxy)-l-prop~ minillm trifluoroacetate), and the neutral lipid
dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water.
Transfectiorl lltili7ing the LipofectAMINETM Reagent is carried out according tothe manufacturer's published protocol. The protocol (for Cat. No. 18324-012)
provides for either transient or stable transfection, as desired.
The advantage of transient expression is its rapidity, i.e. there is
no requirement for cellular proliferation to select for stable integration events.
This rapidity could conceivably be of major clinical importance, in cases of an
already metastatic tumor burden, wherein the weeks required for selection of
stable transfectants may simply not be available to the clinician.
There are, nonetheless, two general disadvantages to the use of
transient transfection. The first is that expression usually peters out after a few
days, in contrast to the continual expression in the case of stable transfection.
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
- 40 -
This is not particularly crippling in terms of our immnni7:~tion protocol. The
inoc-ll"tf~-l, irradiated cells used for immnni7~tion would likely not survive in
vivo for more than 4 or S days, in any case. Thus the nominal advantage
accruing to stable transfection, that of a long-duration expression by the progeny
5 of the parental inoculated cell, is not of particular relevance in the case of the
immlmi7.ing regime described herein, which is based on the use of non-dividing,
probably short-lived cells.
A second disadvantage of transient transfection resides in the fact
that it yields a cell population, only a subset of which has actually been
10 transfected and thus expresses the protein encoded by the transgenc. This
problem is obviated in the case of stable transfection, wherein over time one can
develop a pure population of transfectants via selection for a resistance marker,
such as neo, under conditions of clonal proliferation of the initial stable
transfectants, i. e. ~ ghter cells of transiently transfected cells lack the
15 transgene, in contrast to the case with stable transfectants. In the situation
where there is sufficient time to effect immllni7~tion based on stably transfected
cells, the progeny of all transfected clones would be utilized, not just the
progeny of a single clone, as is sometimes done for detailed biochemical and
molecular analyses of gene expression. Clearly the more clones utilized, the
20 more quickly one can arrive at the requisite number of cells to be used for
immllni7~tion.
Percenta~e of Cells Exhibitin~ dCTG Expression
The percentage of cells exhibiting dCTG expression may be
determined by an immunohistology assay. In this procedure, a small number
25 of cells ( ~ 500) from the harvested pellet following centrifugation of transfected
cells are deposited on a cover slip and fixed with cold acetone. At this point,
a standard immunohistological assay is carried out with the cells on the cover
slip, i.e. addition of a primary monoclonal antibody reactive to the dCTG-
encoded protein, followed by the addition of a developing antibody, e.g. a
30 fluorescent tagged antibody reactive to the primary monoclonal antibody.
CA 0224l9~2 l998-07-l4
W O 97125860 PCTAUS97/00582
- 41 -
Measurement of the percentage of cells scoring as dCTG-positive in the
fluorescent assay allows a determination of the number of positive transfectantsin the starting culture, and thus the number of total cells to be used for
immllni7~tion to arrive at the desired number of dCTG-positive cells to be
inoculated in the patient.
If, as would be almost certain, the percentage of cells scoring as
dCTG-positive is less than one hundred percent, one can simply increase the
number of cells to be used for immllni7~tion, so as to include the desired
number of transfectants. The non-transfected cells in the immllni7.ing population
would simply represent x-irr~ t~17 auto}ogous fibroblasts that would constitute
no danger to the patient.
Transfectant Irradiation
Prior to return to the host, the transfected cells are preferably
irradiated. The transfectants are irradiated with a radiation dose sufficient torender them non-dividing, such as a dose of 25 By or 2500R. The cells are
then counted by trypan blue exclusion, and about 2 X 107 irradiated
. transfectants are resuspended in a volume of 0.2-0.4 ml of Hanks Balanced Salt
Solution.
~accination Procedure
The transfected cells are returned to the host to achieve
vaccination. The cells may be reimplanted at the same body site from which
they were originally harvested, or may be restored to a different site.
It is the object of the present invention to generate a systemic
tumor immllne response, so as to fight metastasis formation wherever any
metastases are found. Accordingly, there is no reason to inject the transfected
cells at the same body site from which they were taken. Intramuscular or
subcutaneous inoculation at a distal site would suffice to yield a systemic
response. Thus, patients are preferably vaccinated by subcutaneous inoculation
of the transfected cells.
CA 0224l9~2 l998-07-l4
W O 97/2S860 PCT~US97/00582.
- 42 -
For s-crc overexpression associated with colon carcinoma, partial
venous inoculation is preferred, as the liver is a frequent site of met~t~es. For
vaccinating against breast cancers and Iymphomas, systemic immlmi7~tion is
preferred.
S As a general rule, it is desirable to generate the strongest imml-ne
response consistent with clinical monitoring of no adverse side effects, i.e.
multiple rounds of inoculation with, for example 107 cells, at each round. The
number of rounds of inoculation is selected accordingly. The efficacy o~ the
inoculation schedule may be monitored by a delayed hypersensitivity reaction
~clmini~tf~red to the patient. A course of about up to 10 inoculations, at 2-3
week intervals, may be utilized. It may be appreciated that the inoculation
schedule may be modified in view of the immunologic response of the
individual patient, as determined with resort to the delayed-type hypersensitivity
(DTH) reaction.
Patient Response Monitorin~ bY Delayed-ty~e H~persensitivity Reaction
Patients are assessed for reactivity to the irradiated transfectants
~ by a test of skin reactivity in a DTH reaction. DTH has been used clinically
(Chang et al. (1993), Cancer Research 53:1043-10S0). To measure reactivity
to the autologous irradiated transfectants, 104- 106 cells in a volume of 0.1 mlHanks buffered saline solution (HBS~) are inoculated intradermally into the
host. Induration is measured 48 hours later, as an average of two perpendicular
diameters (responses of greater than 22 mm is considered positive).
One advantage to the DTH assay is that it can independently
assess the induction of T cell reactivity to (i) the transfectants used for
im m~mi7~tion (i. e. the set of 5 or more dCTGs chosen for imml~ni7~tion
purposes, each cont:~inin~ non-self determin~ntc) and (ii) transfectants, as
transfected with the human dCTG itself cont~ining only self determin~nt.c.
Thus, the induction of reactivity to the transfectants used for imm~lni7~tion
establishes that the immunizing transfectants are in fact immlln~genic, that is,the patient has not exhibiting a much ~veakened capacity for immlln~ response.
CA 02241952 1998-07-14
W O 97t2~860 PCT~US97100582
~ 43 -
If the patient is demonstrably capable of response to the immllni7ing
transfectants, then skin testing with the dCTG (human) transfectants would
establish whether or not reactivity to the human proto-oncogene encoded product
had been induced. According to the practice of the invention, inoculation of the5 immllni7.ing transfectants would continue for at least as long as the induction of
reactivity to the human proto-oncogene-encoded protein occurs.
The practice of the invention is illustrated by the following
nonlimitin~ examples.
Example 1
Imlnunization of Chickens A~ainst c-src(527)-Induced
Tumors By V~rcin~t;on with v-src DNA
A. Genes
The oncogene c-src(527) is an activated form of chicken c-src.
Its protein product pp60C-Src(5Z7) differs from the protein product of c-src, pp60C
src, by only a single amino acid substitution, phenyl~ nin.- ~or tyrosine at
residue 527 (Kmiecik and Shalloway, (1987~ ~ell 49, 65-73). This substitution
eliminz~tes the negative regulatory influence exerted on pp60C-SrC phosphokinaseactivity by the enzymatic phosphorylation of the position 527 tyrosine. The
protein product of v-src, pp60V-SrC, shows a number of sequence dirrel ellces with
pp60C-5rC (Takeya and Hanafusa, (1983) Cell 32, 881-890), including scattered
single amino acid substitutions within the first 514 residues and a novel C
terminus of 12 amino acids (residues 515-526), in place of the nineteen C
terminal amino acids of pp60C-SrC (residues 515-533). Both the v-src-positive
plasmid, pMvsrc. and the c-src(527)-positive plasmid, pcsrcS27, were originally
shown (Kmiecik and Shalloway, (1987) Cell 49, 65-73) to transform murine
NIH 3T3 cells in culture. However, the v-src-ind~ ed transformants exhibited
a more rapid or more extensive colony growth in soft agarose than the c-
src(527)-intl~lced transformants, as well as a usually shorter latency of tumor
formation in nude mice (id.).
CA 022419~2 1998-07-14
.
WO 97/25860 PCT/US97/00582
- 44 -
B. Plasmids
1. pvSRC-C 1
The pVSRC-C1 plasmid was prepared as described by Halpern
et al., (1991) Virology 180, 857-86. Essentially, the plasmid was derived from
the pRL'-src plasmid (~alpern et al., (1990) Virology 175, 328-331) by
subcloning the v-src(~) XhoI-EcoRI fragment of the latter into the multiple
cloning sequence of pSP65 (Melton et al., (1984) Nucleic Acids Res. 12, 7035-
7056) which had been cleaved with Sall and EcoRI; since ligation of the XhoI
overhang at the San site destroys both recognition sequences, subsequent
rcmoval of the v-src('r) insert from the vector was achieved by digestion with
EcoRI and with HindIII, which cleaves at a position in the multiple cloning
sequence adjacent to the Sall site. The pVSRC-C1 plasmid was restricted with
EcoRI and HindlII, so as to liberate the tumorigenic insert. This insert included
the v-src oncogene of the subgroup A strain of Prague RSV, as flanked
downstream by a portion of the long terminal repeat (LTR) of RSV (from the
5' start of the LTR, to the single EcoR~ site).
2. pMvsrc
The pMvsrc plasmid was generously provided by Dr. David
Shalloway, Cornell University, Ithaca, NY. The plasmid is prepared according
to Johnson et al., (1985) Mol. Cell. Biol. 5, 1073-1083. Briefly, the 3.1-kb
BamHI-Bg/II Schmidt Ruppin A v-src fragment from plasmid pN4 (Iba et al.,
(1984) Proc. Nat. Acad. Sci. USA 81, 4424-4428) is inserted into the pEVX
plasmid (Kriegler et al., (1984) Cell 38,483-491) at a Bg/II site Iying between
two Moloney murine leukemia virus (MoMLV) long terminal repeats (LTRs).
This fragment contains 276 bp of pBR322 DNA from the pBR322 BamHI to
Sall sites followed by 2.8 kb of Rous sarcoma virus (RSV) DNA from the SalJ
site that is about 750 bp upstream of the env termination codon down to the
NruI site that is about 90 bp downstream of the v-src termination codon. (The
CA 02241952 1998-07-14
WO 97/2S860 PCT~US97/00~82
- 45 -
NruI site is converted to a Bg/II site in the construction of pN4.) Ligation is
performed by using a 10:1 insert-vector DNA fragment molar ratio.
The pMvsrc plasmid was restricted with NheI, so as to liberate
a tumorigenic fragment. The fragment included the v-src oncogene of the
5 subgroup A strain of Schmidt-Ruppin RSV, as flanked upstream by most of the
Moloney murine le~lkemi~ virus (MoMLV) I,TR (from the NheI site near the
5' start of the LTR, to the 3' end of this LTR) and downstream by a small
portion of the MoMLV LTR (from the 5' start to the NheI site).
3. pcsrcS27
The pcsrc527 plasmid is prepared according to Kmiecik and
Shalloway, (1987) Cell 49, 65-73. Briefly, a plasmid is constructed by cleaving
expression vector pEVX (Kriegler et al., (1984) Cell 38,483-491 at its unique
BgIII site Iying between two MoMLV LTRs and inserting the 3.2 kilobase (kb)
pair BamEII-BgIII hybrid src fragment from plasmid pHB5 in the proper
15 orientation. This fragment contains sequences from pBR322, the SRA env 3'
region, SRA v-src, src from recovered ASV, and chicken c-src. The BgIII site
is generated by insertion of a linker at the SacI site about 20 bp downstream
from the c-src termination codon. The restriction map of pMHB5 contains the
MoMLV splice donor about 60 bp downstream from the 3 'end of the upstream
20 LTR and the v-src splice acceptor about 75 bp upstream from the src ATG.
Plasmid pMHB5527 is constructed by inserting the synthetic
double-stranded DNA oligomer
5' CCAGTTCCAGCCTGGAGAGAACCTATA (SEQ ID NO:l) 3'
3' TCGGGGTCAAGGTCGGACCTCTCTTGGATATCTAG (SEQ ID NO:2) 5'
25 into pMHB5 between the BanII site at c-src codon 524 and the downstream
unique BgIII site. This alters the TAC Tyr 527 codon to a TTC Phe codon
while preserving the rem~ining c-src coding region. Equimolar amounts of the
double-stranded oligomer and three gel-purified tandem restriction fragments
from pMHB5 are ligated in one reaction, which contains the following: the
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582
- 46 -
oligomer with BanII and BgIII complementary ends, the 3 kb BgIII-BgII (BgII
in the pEVX ampicillin resistance gene) partial digest fragment, the adjacent 6.1
kb BgII-BgII (downstream BgII in c-src) fragment, and the 0.38 kb BgII-BanII
(BanII at c-src codon 524) fragment.
S Plasmid pcsrcS27 is constructed by replacing the 2 kb SalI (in
e~lv)-MluI (in c-src) fragment in plasmid pM~B5527, with the homologous
fragment from plasmid p5II. This fragment contains the coding sequence for
the c-src amino region (codons 1 to 257) that have been isolated by molecular
cloning of a c-src provirus and previously shown by sequencing to contain
authentic c-src sequence without the mutation at codon 63 (Levy et al., (1986)
Proc. Natl. Acad. Sci. USA 83, 42284232). Equimolar amounts of
complementary gel-purified Sall-MluI fragments from p5H and the other
plasmids are ligated.
The pcsrc527 plasmid was restricted with NheI, so as to liberate
a tumorigenic fragment. The tumorigenic fragment included the c-src(527)
oncogene, as flanked by the same LTR complement as in pMvsrc.
~ C. Animals
Chickens of two closed lines, SC and TK, were utilized. These
lines differ at the major histocompatibility (B) complex (B~/B2 for the SC line,Bls/B2l for the TK line~. Embryonated eggs were obtained from Hylinc
International (Dallas Center, IA). All chickens were h~ltrh~d at the University
of New Hampshire Poultry ~esearch Farm and housed in isolation.
D. Tumor Induction bv Plasmid DNA
Tumors were in~ cecl by subcutaneous inoculation in the wing
web of a src-positive plasmid according to the technique described by Fung et
al. (1983) Proc. Natl. Acad. Sci. USA 80, 353-357 and Halpern et al., (1990)
Virology 175, 328-331. Of the three tumorigenic plasmids utilized here, all
were adjusted, prior to inoculation, to a concentration of 100 ,ug of enzyme--
restricted DNA per 100 ,ul of phosphate-buffered saline. The conditions of
CA 02241952 1998-07-14
W O 97/2586~ PCTrUS97/00582
- 47 -
inoculation used for particular experiments (age of chicken at time of
inoculation, amount of plasmid, etc.) are indicated below.
E. Growth of Primarv (win~ web~ Tumors in TK or SC Chickens
Inocul~tçA with p V S R C-Cl, p M vsrc or pcsrcS27
Individual l-day-old chickens of line TK or of line SC were
inoculated with 100 ~g of either pVSRC-Cl, pMvsrc or pcsrc527. The mean
tumor fli~m~tPr (mm) at a particular time point and for any one group of TK or
SC line chirkP.n~ inoc~ t~l with an individual src-positive construct was
computed as the sum of the diameters of the primary tumors divided by the
10 number of chickens surviving to that po;nt. The results are shown in Fig. lA
(line TK) and Fig. lB (line SC). The ratios at each time point show, for a
particular group, the number of chickens bearing palpable tumors to the total
number of survivors to that point (standard typeface for pcsrc527, italics for
pVSRC-Cl, bold typeface for pMVsrc). Error bars (unless obscured by the
15 symbol) in~lir~te standard error.
F. Growth of Challenge (win~ web) Tumors in Test and Control
l_ine TK Chickens Under Conditions of Primin~ and Homolo~ous
Challen~e with pcsrcS27~ or Priming and Homolo~ous Challen~e
with pVSRC-Cl
Growth of challenge (wing web) tu~mors in test and control line
TK chickens was determined under conditions of (i) priming and homologous
challenge with pcsrcS27, or (ii) priming and homologous challenge with
pVSRC-Cl. Test chickens were primed at 1 day posthatch with 100 Ig of
construct; test and control chickens were challenged at five weeks posthatch
25 with 200 Ig of construct. The mean challenge tunnor diameter was computed
as described in the prec.eding section. At each time point the ratio of
chickens bearing palpable challenge tumors to total number of survivors to
that point is in-lic~t~cl for priming and homologous challenge with pcsrcS27
(Fig. 2A) and priming and homologous challenge with pVSRC-CI (Fig. 2B)
SlJ~5 ~ JTE SHEET (RULE 26)
-
CA 022419~2 1998-07-14
W O 97/25860 PCT~US97/00582
- 48 -
(standard typeface for control group, bold typeface for test group~. The
st~ti~tic~l comparison between the mean challenge tumor diameters of the test
versus the control group at a particular time point was made using a two-tailed
student's t test, *(p<0.05), **(p<0.01), ***(p<0.0~1). The st~ti.ctic~
5 comparison between the ratios of chickens bearing palpable challenge tumors tototal number of survivors of the test versus the control group at a particular
time point was made using a chi-squared test; the paired ratios are underlined
for orlly those time points where p<0.05. Error bars indicate standard error.
G. Growth of Challen~e (win~ web) Tumors in Test and Control
line TE~ ch}ckens under Conditions of Primin~ with pVSRC-C1
and Heterologous Challen~e with pcsrcS27~ or Primin~ with
pcsrcS27 and Heterolo,~ous Challen~e with pVSRC-Cl
Growth of challenge (wing web) tumors in test and control line
TK chickens, was determined under conditions of (i) priming with pVSRC-C1
and heterologous challenge with pcsrc527, or (ii) prirning with pcsrcS27 and
heterologous challenge with pVSRC-Cl. Test chickens were primed at 1 day
posthatch with 100 ,ug of construct; test and control chickens were challenged
at five weeks posthatch with 200 ~g of construct. The mean challenge tumor
m~ter was computed as described in Section E. At each time point the ratio
of chickens bearing palpable challenge tumors to total number of survivors to
that point is in-lic~t~rl for priming with pVSRC-C1 and heterologous challenge
with pcsrc527 (Fig. 3A) and priming with pcsrc527 and heterologous challenge
with pVSRC-CI (Fig. 3B) (standard typeface for control group, bold typeface
for test group). Statistical comparisons were made between test and control
groups at a particular time point as described in the prece-ling section
[*(p < 0.05), **(p < 0.013, ***(p < 0.001), for the student's t test~, and the
paired ratios are underlined for only those time points where, in the chi-squared
test, p < 0.05. Error bars inrli~te standard error.
SlJ~S 111 UTE SHEET (RULE 26)
CA 02241952 1998-07-14
W O 97/25860 PCT~US97/00582
- 49 -
H. Discussion
In a direct comparison of the growth of tumors in~ ed in line
TK by either pMvsrc or pVSRC-C1, a similar pattern of relatively rapid
~ regression was observed. This result established that the difference in LTR
S complement between these two v-src positive constructs did not exert a major
influence on the tumor growth pattern in the TK line (Fig. lA). By contrast,
much more extensive and persistent tumor growth resulted from inoculation of
TK chickens with the pcsrc527 construct (Fig. 1A). The relatively greater
growth capacity of tumors in-hlced by this construct indicated that in the TK
line, the c-src(527) oncogene is much more highly tumorigenic than the v-src
oncogene. This difference did not, however, generalize to the SC line (Fig.
lB). The SC line was chosen for comparison with the TK line on the basis of
earlier observations (Halpern et al., (1993) Virology 197, 480-484) that v-src
DNA-in-luce~l tumors engender a much weaker tumor immune response in line
SC than in line TK. Whereas the growth of pcsre527-in-luçe-1 primary tumors
was virtually in~ finguishable in the two lines, the growth of the v-src-inducedtumors was considerably greater in the SC than in the TK line (Figs. lA and
lB). Thus v-src, but not c-sre(527), gives rise to primary tumors whose growth
patterns differ in the two lines analyzed here.
Only minim~l protection against homologous challenge was
observed under conditions of priming to c-sre(527) DNA, indicative of the
induction of a relatively weak tumor immlln~ response (Fig. 2A; a statistically
significant lowering of eh~llenge tumor growth in the test versus the control
chickens was observed at only one time point). By contrast, the v-sre DNA-
primed chickens showed excellent protection against the homologous tumor
challenge (Fig. 2B).
Priming with v-sre DNA engenders a relatively greater degree
of protection against challenge with c-sre(527) DNA, than that a~forded by
priming with c-src(527) DNA itself (Pig. 3A). The degree of protection was
weaker than that determined (Fig. 2B) for the case of priming and
homologous challenge with v-src DNA. Only marginal protection was
SlL~ JTE SHEET (RULE 26)
CA 02241952 1998-07-14
W O 97/25860 PCT~US97/00582
- 50 -
observed, however, when the heterologous challenge protocol was carried out
in the reverse order (Fig. 3B). These results demonstrate that induction of
reactivity to an antigenicity specified in tumor cells by an overexpressed proto-
oncogene can confers tumor i.n.,.~ ily.
Example 2
V~(~rin~tion Protocol
The following is a representative vaccination protocol according
to the present invention.
A. Skin Punch Biopsy
A punch biopsy of skin is obtained by a trained physician
following standard m~ l practice.
B. P~ a,alion of Primary Fibroblast Culture
Under sterile conditions, the skin obtained by punch biopsy is put
in a tube with 10 ml of the following wash medium: Dulbecco's Modified
Eagle Medium (DMEM), cont:~ining sodium bicarbonate (30 ml/liter of a 5.6%
solution) and penicillin/streptomycin (2 ml/liter of a pen-strep stock solution
cont~ining 5000 units penicillin and 5000 ,ug of streptomycin/ml, pH 7.2-7.4.).
In a sterile hood, the skin biopsy is added to a Petri dish, and then transferred
several times to new Petri dishes cont:lining the same wash m~ m. The
biopsy is then finely minced with two scalpels, and 2-4 pieces ( < l mm3) of theminced biopsied are placed in the middle part of one or more T25 flasks. The
flask is placed in a tissue culture in~l~b~tor at 37~C for one half hour with the
cap firmly closed, then opened for 10 minutes. The following culture mt~ m
is prepared: DMEM cont~ining sodium bicarbonate; antibiotics; and 10% fetal
calf serum cont~ining 2.5 ~g/ml fungizone, 40 ,ug/ml gentamicin, and 1%
glllt~min~( 3 % W/V3. Two ml of the culture medium is then added to the flask,
and the flask is incubated at 37~C (5% CO2), with the cap lightly uns~ wed.
The flask is left for three days without moving so as to obtain adhesion of the
SUBSTITUTE SHEET (RULE 26~
CA 02241952 1998-07-14
W O 97/25860 PCT~US97/00582
- 51 -
separate pieces of skin to the plastic. Afterwards, the medium is changed two
times per week over a 3-4 week period always adding 2-3 ml of medium. To
trypsinize the skin cell culture, one needs zones of confluence. After aspirating
the culture medillm, 5 ml of the Puck's Saline A/EDTA solution (0.4 g EDTA
S to 1 liter of Puck's Solution A) is added and imm~di~tely aspirated. Then 1 mlof trypsin solution (0.05/0.02 % trypsin in PBS, without Ca~ + or Mg + ~) is
added and incubated for 5 min at 37~C, at which time 2 ml of culture fluid is
added to stop the action of the trypsin. The cells are then transferred to a larger
flask (T75) and incubated at 37~C in 15 ml of culture fluid, which is changed
10 every 2 days.
C. Fibroblast Transfection
The fibroblasts (2 X 105 cells) are washed twice in DMEM
without serum or antibiotics. A LipofectAMINETM-DNA solution is prepared
by mixing in tube #1 mix 400~1 DMEM and 10,ul of dCTG vector DNA
(l,ug/ul). In tube ~2, 400 ~ul DMEM and 25 Ml of LipofectAMINE Reagent
(Life Technologies, cat. no. 18324-012) are mixed. The contents of tube #1
and #2 are mixed together and are then left sitting at room temperature for 30
hours. Then, 3.2 ml of the LipofectAMINETM-DNA solution is added to the
cells. The cells are incubated for six hours at 37~C, washed once with Hank's
20 B~ n- ed Salt Solution, and then refed with growth medium and incubated for
an additional 24 hours at 37~C
D. Transfectant Irradiation
Transfectants are irradiated to a dose of 25 By or 2500R. the
cells are then counted by trypan blue exclusion. 2 X 107 irradiated transfectants
25 are resuspended in a volume of 0.2-0.4 ml of Hanks Balanced Salt Solution.
E. Vaccination
Patients are vaccinated by subcutaneous inoculation of 2 X 107
irradiated cells at 2-3 week intervals. A shorter or longer regimen is used,
CA 0224l9~2 l998-07-l4
.
W O 97/25860 PCT~JS97/00582
- 52 -
depending upon the results of delayed type hypersensitivity (DTH) reaction
monitoring (described below).
F. Patient Assessment by DTH Monitorin~
Patients are assessed for reactivity to the irradiated transfectants
by a test of skin reactivity in a DTH reaction, as described by Chang et al.
(1993), Cancer Research 53:1043-1050. To measure reactivity to the
autologous irradiated transfectants, 104 - 106 transfected irradiated cells in avolume of 0.1 ml HBSS are inoculated intradermally. Induration is measured
48 hours later, as an average of two perpendicular diameters. Responses of
10 greater than 2 mm are considered positive.
Example 3
v-mYc Transfection of Murhlc Fibroblasts
A. Vector Preparation
The v-myc retroviral oncogene of avian myelocytomatosis virus
M C29 (Land et al. (1983), Nature 304:596-602) was obtained from the
American Type Culture Collection, Rockville, MD, 20852, as the pSVv-myc
vector (ATCC No. 45014). The v-myc-positive EcoR~-Kpnl fragment of pSVv-
myc was ligated into the polylinker sites of the pBK-CMV plasmid (Stratagene
Cloning Systems, La Jolla, CA).
B. Cell Transfection
Stable transfection using the pBK-CMV-v-myc vector was carried
out on a line of A31 fibroblasts (Balb/c origin), obtained from the ATCC. 2
X 105 cells were seeded in a 100 mm/dish and allowed to grow for 18-20 h
(RPMI 1640 medium and 10% fetal bovine serum), at which time the cells
reached 50-705tc confluence. The cells were then washed twice in Dulbecco's
Modified Eagles Medium (without serum or antibiotics). A LipofectAMINETM-
DNA solution was prepared according to Example 2.C., with the pBK-CMV-v-
CA 02241952 1998-07-14
W O 97/25860 PCTAUS97/00582
-53 -
myc vector DNA, and 3.2 ml of the LipofectAMINETM-DNA solution added to
the cells. The cells were then incubated for 6 hours at 37~C~, washed once with
Hank's Balanced Salt Solution, and then refed with the growth medium and
incubated for an additional 24 hour at 37~C. Thereafter, the cells were fed
S once every two days with growth medium cont~ining 250 ,ug/ml geneticin
(G418; Gibco BRL cat. no. 11811) as the selective marker. Within two weeks,
colonies were picked and expanded into permanent cell lines. The cells were
then washed and collected by centrifugation.
It should be noted that the procedure for transient transfection is
10 the same, through the point of incubation with the Lipofect~min~T'I-DNA
solution. Thereafter, the cells are washed and incubate~ for 72 hours in growth
medium.
All references cited with respect to synthetic, preparative and analytical
procedures are incorporated herein by reference.
15The present invention may be embodied in other specific forms without
departing from the spirit or essential attributes thereof and, accordingly,
-- reference should be made to the appended claims, rather than to the foregoing
specification, as indication the scope of the invention.
CA 0224l9~2 l998-07-l4
WO 97/25860 PCTrUS97/00~82
- 54 -
SEQUENCE LISTING
(1) GENERA~ INFORMATION:
(i) APPLICANT: Allegheny University o~ the Health Sciences
Halpern, Michael S.
England, James M.
(ii) TITLE OF L~V~NllON: CANCER VACCINE
(iii) NUMBER OF SEQUENCES: 14
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Seidel, Gonda, Lavorgna & Monaco, P.C.
(B) STREET: Suite 1800, Two Penn Center Plaza
(C) CITY: Philadelphia
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19102
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/010,262
(B) FILING DATE: l9-JAN-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Monaco, Daniel A.
(B) REGISTRATION NUMBER: 30,480
(C) REFERENCE/DOCKET NUMBER- 7933-33 PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 568-8383
(B) TELEFAX: (215) 568-5549
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: ~ucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SÉQUENCE DESCRIPTION: SEQ ID NO:l:
CCAGTTCCAG CCTGGAGAGA ACCTATA 27
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
~'A) LENGTH: 35 base pairs
B) TYPE: nucleic acid
C) STRANDEDNESS: single
D) TOPOLOGY: linear
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/00582.
- 55 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GATCTATAGG TTCTCTCCAG GCTGGAACTG GGGCT 35
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1599 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAGACTGTGC CCTGTCCACG GTGCCTCCTG CATGTCCTGC TGCCCTGAGC TGTCCCGAGC 60
TAGGTGACAG CGTACCACGC TGCCACCATG AATGAGGTGT CTGTCATCAA AGAAGGCTGG 120
CTCCACAAGC GTGGTGAATA CATCAAGACC TGGAGGCCAC GGTACTTCCT GCTGAAGAGC 180
GACGGCTCCT TCATTGGGTA CAAGGAGAGG CCCGAGGCCC CTGATCAGAC TCTACCCCCC 240
TTAAACAACT TCTCCGTAGC AGAATGCCAG CTGATGAAGA CCGAGAGGCC GCGACCCAAC 300
ACCTTTGTCA TACGCTGCCT GCAGTGGACC ACAGTCATCG AGAGGACCTT CCACGTGGAT 360
TCTCCAGACG AGAGGGAGGA GTGGATGCGG GCCATCCAGA TGGTCGCCAA CAGCCTCAAG 420
CAGCGGGCCC CAGGCGAGGA CCCCATGGAC TACAAGTGTG GCTCCCCCAG TGACTCCTCC 480
ACGACTGAGG AGATGGAAGT GGCGGTCAGC AAGGCACGGG CTA~AGTGAC CATGAATGAC 540
TTCGACTATC TCAAACTCCT TGGCAAGGGA ACCTTTGGCA AAGTCATCCT GGTGCGGGAG 600
AAGGCCACTG GCCGCTACTA CGCCATGAAG ATCCTGCGAA AGGAAGTCAT CATTGCCAAG 660
GATGAAGTCG CTCACACAGT CACCGAGAGC CGGGTCCTCC AGAACACCAG GCACCCGTTC 720
CTCACTGCGC TGAAGTATGC CTTCCAGACC CACGACCGCC TGTGCTTTGT GATGGAGTAT 780
GCCAACGGGG GTGAGCTGTT CTTCCACCTG TCCCGGGAGC GTGTCTTCAC AGAGGAGCGG 840
GCCCGGTTTT ATGGTGCAGA GATTGTCTCG GCTCTTGAGT ACTTGCACTC GCGGGACGTG 9oo
GTATACCGCG ACATCAAGCT GGA~AACCTC ATGCTGGACA AAGATGGCCA CATCAAGATC 960
ACTGACTTTG GCCTCTGCAA AGAGGGCATC AGTGACGGGG CCACCATGAA AACCTTCTGT 1020
GGGACCCCGG AGTACCTGGC GCCTGAGGTG CTGGAGGACA ATGACTATGG CCGGGCCGTG 1080
GACTGGTGGG GGCTGGGTGT GGTCATGTAC GAGATGATGT GCGGCCGCCT GCCCTTCTAC 1140
AACCAGGACC ACGAGCGCCT CTTCGAGCTC ATCCTCATGG AAGAGATCCG CTTCCCGCGC 1200
ACGCTCAGCC CCGAGGCCAA GTCCCTGCTT GCTGGGCTGC TTAAGAAGGA CCCCAAGCAG 1260
AGGCTTGGTG GGGGGCCCAG CGATGCCAAG GAGGTCATGG AGCACAGGTT CTTCCTCAGC 1320
ATCAACTGGC AGGACGTGGT CCAGAAGAAG CTCCTGCCAC CCTTCAAACC TCAGGTCACG 1380
TCCGAGGTCG ACACAAGGTA CTTCGATGAT GAATTTACCG CCCAGTCCAT CACAATCACA 1440
CCCCCTGACC GCTATGACAG CCTGGGCTTA CTGGAGCTGG ACCAGCGGAC CCACTTCCCC 1500
CA 022419~2 1998-07-14
W 0 97/25860 PCTrUS97/00582
- 56 -
CAGTTCTCCT ACTCGGCCAG CATCCGCGAG TGAGCAGTCT GCCCACGCAG AGGACGCACG 1560
CTCGCTGCCA TCACCGCTGG GTG~~ ACCCCTGCC1599
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4530 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: ~inyle
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AATTCTCGAG CTCGTCGACC GGTCGACGAG CTCGAGGGTC GACGAGCTCG AGGGCGCGCG 60
CCCGGCCCCC ACCCCTCGCA GCACCCCGCG CCCCGCGCCC TCCCAGCCGG GTCCAGCCGG 120
AGCCATGGGG CCGGAGCCGC AGTGAGCACC ATGGAGCTGG CGGCCTTGTG CCGCTGGGGG 180
CTCCTCCTCG CCCTCTTGCC CCCCGGAGCC GCGAGCACCC AAGTGTGCAC CGGCACAGAC 240
ATGAAGCTGC GGCTCCCTGC CAGTCCCGAG ACCCACCTGG ACATGCTCCG CCACCTCTAC 300
CAGGGCTGCC AGGTGGTGCA GGGA~ACCTG GAACTCACCT ACCTGCCCAC CAATGCCAGC 360
CTGTCCTTCC TGCAGGATAT CCAGGAGGTG CAGGGCTACG TGCTCATCGC TCACAACCAA 420
GTGAGGCAGG TCCCACTGCA GAGGCTGCGG ATTGTGCGAG GCACCCAGCT CTTTGAGGAC 480
AACTATGCCC TGGCCGTGCT AGACAATGGA GACCCGCTGA ACAATACCAC CCCTGTCACA 540
GGGGCCTCCC CAGGAGGCCT GCGGGAGCTG CAGCTTCGAA GCCTCACAGA GATCTTGAAA 600
GGAGGGGTCT TGATCCAGCG GAACCCCCAG CTCTGCTACC AGGACACGAT TTTGTGGAAG 660
GACATCTTCC ACAAGAACAA CCAGCTGGCT CTCACACTGA TAGACACCAA CCGCTCTCGG 720
GCCTGCCACC CCTGTTCTCC GATGTGTAAG GGCTCCCGCT GCTGGGGAGA GAGTTCTGAG 780
GATTGTCAGA GCCTGACGCG CACTGTCTGT GCCGGTGGCT GTGCCCGCTG CAAGGGGCCA 840
CTGCCCACTG ACTGCTGCCA TGAGCAGTGT GCTGCCGGCT GCACGGGCCC CAAGCACTCT 900
GACTGCCTGG CCTGCCTCCA CTTCAACCAC AGTGGCATCT GTGAGCTGCA CTGCCCAGCC 960
CTGGTCACCT ACAACACAGA CACGTTTGAG TCCATGCCCA ATCCCGAGGG CCGGTATACA 1020
TTCGGCGCCA GCTGTGTGAC TGCCTGTCCC TACAACTACC TTTCTACGGA CGTGGGATCC 1080
TGCACCCTCG TCTGCCCCCT GCACAACCAA GAGGTGACAG CAGAGGATGG AACACAGCGG 1140
TGTGAGAAGT GCAGCAAGCC CTGTGCCCGA GTGTGCTATG GTCTGGGCAT GGAGCACTTG 1200
CGAGAGGTGA GGGCAGTTAC CAGTGCCAAT ATCCAGGAGT TTGCTGGCTG CAAGAAGATC 1260
TTTGGGAGCC TGGCATTTCT GCCGGAGAGC TTTGATGGGG ACCCAGCCTC CAACACTGCC 1320
CCGCTCCAGC CAGAGCAGCT CCAAGTGTTT GAGACTCTGG AAGAGATCAC AGGTTACCTA 1380
TACATCTCAG CATGGCCGGA CAGCCTGCCT GACCTCAGCG TCTTCCAGAA CCTGCAAGTA 1440
ATCCGGGGAC GAATTCTGCA CAATGGCGCC TACTCGCTGA CCCTGCAAGG GCTGGGCATC 1500
CA 0224l9~2 l998-07-l4
.
W O 97/25860 PCT~US97/00~82
- 57 -
AGCTGGCTGG GGCTGCGCTC ACTGAGGGAA CTGGGCAGTG GACTGGCCCT CATCCACCAT 1560
AACACCCACC TCTGCTTCGT GCACACGGTG CCCTGGGACC AGCTCTTTCG GAACCCGCAC 1620
CAAGCTCTGC TCCACACTGC CAACCGGCCA GAGGACGAGT GTGTGGGCGA GGGCCTGGCC 1680
TGCCACCAGC TGTGCGCCCG AGGGCACTGC TGGGGTCCAG GGCCCACCCA GTGTGTCAAC 1740
TGCAGCCAGT TCCTTCGGGG CCAGGAGTGC GTGGAGGAAT GCCGAGTACT GCAGGGGCTC 1800
CCCAGGGAGT ATGTGAATGC CAGGCACTGT TTGCCGTGCC ACCCTGAGTG TCAGCCCCAG 1860
AATGGCTCAG TGACCTGTTT TGGACCGGAG GCTGACCAGT GTGTGGCCTG TGCCCACTAT 1920
AAGGACCCTC CCTTCTGCGT GGCCCGCTGC CCCAGCGGTG TGAAACCTGA CCTCTCCTAC 1980
ATGCCCATCT GGAAGTTTCC AGATGAGGAG GGCGCATGCC AGCCTTGCCC CATCAACTGC 2040
ACCCACTCCT GTGTGGACCT GGATGACAAG GGCTGCCCCG CCGAGCAGAG AGCCAGCCCT 2100
CTGACGTCCA TCGTCTCTGC GGTGGTTGGC ATTCTGCTGG TCGTGGTCTT GGGGGTGGTC 2160
TTTGGGATCC TCATCAAGCG ACGGCAGCAG AAGATCCGGA AGTACACGAT GCGGAGACTG 2220
CTGCAGGAAA CGGAGCTGGT GGAGCCGCTG ACACCTAGCG GAGCGATGCC CAACCAGGCG 2280
CAGATGCGGA TCCTGAAAGA GACGGAGCTG AGGAAGGTGA AGGTGCTTGG ATCTGGCGCT 2340
TTTGGCACAG TCTACAAGGG CATCTGGATC CCTGATGGGG AGAATGTGAA AATTCCAGTG 2400
GCCATCAAAG TGTTGAGGGA AAACACATCC CCCAAAGCCA ACAAAGAAAT CTTAGACGAA 2460
GCATACGTGA TGGCTGGTGT GGGCTCCCCA TATGTCTCCC GCCTTCTGGG CATCTGCCTG 2520
ACATCCACGG TGCAGCTGGT GACACAGCTT ATGCCCTATG GCTGCCTCTT AGACCATGTC 2580
~ CGGGAAAACC GCGGACGCCT GGGCTCCCAG GACCTGCTGA ACTGGTGTAT GCAGATTGCC 2640AAGGGGATGA GCTACCTGGA GGATGTGCGG CTCGTACACA GGGACTTGGC CGCTCGGAAC 2700
GTGCTGGTCA AGAGTCCCAA CCATGTCAAA ATTACAGACT TCGGGCTGGC TCGGCTGCTG 2760
GACATTGACG AGACAGAGTA CCATGCAGAT GGGGGCAAGG TGCCCATCAA GTGGATGGCG 2820
CTGGAGTCCA TTCTCCGCCG GCGGTTCACC CACCAGAGTG ATGTGTGGAG TTATGGTGTG 2880
ACTGTGTGGG AGCTGATGAC TTTTGGGGCC AAACCTTACG ATGGGATCCC AGCCCGGGAG 2940
ATCCCTGACC TGCTGGAAAA GGGGGAGCGG CTGCCCCAGC CCCCCATCTG CACCATTGAT 3000
GTCTACATGA TCATGGTCAA ATGTTGGATG ATTGACTCTG AATGTCGGCC AAGATTCCGG 3060
GAGTTGGTGT CTGAATTCTC CCGCATGGCC AGGGACCCCC AGCGCTTTGT GGTCATCCAG 3120
AATGAGGACT TGGGCCCAGC CAGTCCCTTG GACAGCACCT TCTACCGCTC ACTGCTGGAG 3180
GACGATGACA TGGGGGACCT GGTGGATGCT GAGGAGTATC TGGTACCCCA GCAGGGCTTC 3240
TTCTGTCCAG ACCCTGCCCC GGGCGCTGGG GGCATGGTCC ACCACAGGCA CCGCAGCTCA 3300
TCTACCAGGA GTGGCGGTGG GGACCTGACA CTAGGGCTGG AGCCCTCTGA AGAGGAGGCC 3360
CCCAGGTCTC CACTGGCACC CTCCGAAGGG GCTGGCTCCG ATGTATTTGA TGGTGACCTG 3420
CA 022419~2 1998-07-14
W O 97/25860 PCTAJS97/00582
- 58 -
GGAATGGGGG CAGCCAAGGG GCTGCAAAGC CTCCCCACAC ATGACCCCAG CCCTCTACAG 3480
CGGTACAGTG AGGACCCCAC AGTACCCCTG CCCTCTGAGA CTGATGGCTA CGTTGCCCCC .3540
CTGACCTGCA GCCCCCAGCC TGAATATGTG AACCAGCCAG ATGTTCGGCC CCAGCCCCCT .3600
TCGCCCCGAG AGGGCCCTCT GCCTGCTGCC CGACCTGCTG GTGCCACTCT GGAAAGGGCC 3660
AAGACTCTCT CCCCAGGGAA GAATGGGGTC GTCAAAGACG TTTTTGCCTT TGGGGGTGCC 3720
GTGGAGAACC CCGAGTACTT GACACCCCAG GGAGGAGCTG CCCCTCAGCC CCACCCTCCT 3780
CCTGCCTTCA GCCCAGCCTT CGACAACCTC TATTACTGGG ACCAGGACCC ACCAGAGCGG 3840
GGGGCTCCAC CCAGCACCTT CAAAGGGACA CCTACGGCAG AGAACCCAGA GTACCTGGGT 3900
CTGGACGTGC CAGTGTGAAC CAGAAGGCCA AGTCCGCAGA AGCCCTGATG TGTCCTCAGG 3960
GAGCAGGGAA GGCCTGACTT CTGCTGGCAT CAAGAGGTGG GAGGGCCCTC CGACCACTTC _ 4020
CAGGGGAACC TGCCATGCCA GGAACCTGTC CTAAGGAACC TTCCTTCCTG CTTGAGTTCC 4080
CAGATGGCTG GAAGGGGTCC AGCCTCGTTG GAAGAGGAAC AGCACTGGGG AGTCTTTGTG 4140
GATTCTGAGG CCCTGCCCAA TGAGACTCTA GGGTCCAGTG GATGCCACAG CCCAGCTTGG 4200
CCCTTTCCTT CCAGATCCTG GGTACTGAAA GCCTTAGGGA AGCTGGCCTG AGAGGGGAAG 4260
CGGCCCTAAG GGAGTGTCTA AGAACA~AAG CGACCCATTC AGAGACTGTC CCTGA~ACCT 4320
AGTACTGCCC CCCATGAGGA AGGAACAGCA ATGGTGTCAG TATCCAGGCT TTGTACAGAG 4380
TGCTTTTCTG TTTAGTTTTT A~ G TTTTGTTTTT TTAAAGACGA AATAAAGACC 4440
CAGGGGAGAA TGGGTGTTGT ATGGGGAGGC AAGTGTGGGG GGTCCTTCTC CACACCCACT ~4500
TTGTCCATTT GCAAATATAT TTTGGAAAAC ' 4530
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 891 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC ACAGATTCCA 60
GCTTCGGAAC AAGAGACCCT GGATCTTGAT GCTGGTGTAA GTGAACATTC AGGTGATTGG 120
TTGGATCAGG ATTCAGTTTC AGATCAGTTT AGTGTAGAAT TTGAAGTTGA ATCTCTCGAC 180
TCAGAAGATT ATAGCCTTAG TGAAGAAGGA CAAGAACTCT CAGATGAAGA TGATGAGGTA 240
TATCAAGTTA CTGTGTATCA GGCAGGGGAG AGTGATACAG ATTCATTTGA AGAAGATCCT 300
GAAATTTCCT TAGCTGACTA TTGGAAATGC ACTTCATGCA ATGAAATGAA TCCCCCCCTT 360
CCATCACATT GCAACAGATG TTGGGCCCTT CGTGAGAATT GGCTTCCTGA AGATAAAGGG 420
AAAGATAAAG GGGAAATCTC TGAGAAAGCC AAACTGGAAA ACTCAACACA AGCTGAAGAG 480
CA 022419~2 1998-07-14
~ W O 97/2~86~ PCTAUS97/00582
. = - 5g -
GGCTTTGATG TTCCTGATTG TAAAAAAACT ATAGTGAATG ATTCCAGAGA GTCATGTGTT 540
GAGGAAAATG ATGATAAAAT TACACAAGCT TCACAATCAC AAGAAAGTGA AGACTATTCT 600
CAGCCATCAA CTTCTAGTAG CATTATTTAT AGCAGCCAAG AAGATGTGAA AGAGTTTGAA 660
AGGGAAGAAA CCCAAGACAA AGAAGAGAGT GTGGAATCTA GTTTGCCCCT TAATGCCATT 720
GAACCTTGTG TGATTTGTCA AGGTCGACCT AAAAATGGTT GCATTGTCCA TGGCA~AACA 780
GGACATCTTA TGGCCTGCTT TACATGTGCA AAGAAGCTAA AGAAAAGGAA TAAGCCCTGC 840
CCAGTATGTA GACAACCAAT TCAAATGATT GTGCTAACTT ATTTCCCCTA G 89l
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 657 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) S~U~ DESCRIPTION: SEQ ID NO:6:
ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC ACAGATTCCA 60
GCTTCGGAAC AAGAGACCCT GGACTATTGG AAATGCACTT CATGCAATGA AATGAATCCC l20
CCCCTTCCAT CACATTGCAA CAGATGTTGG GCC~11~C~1'G AGAATTGGCT TCCTGAAGAT l80
AAAGGGAAAG ATAAAGGGGA AATCTCTGAG AAAGCCAAAC TGGAAAACTC AACACAAGCT 2g0
GAAGAGGGCT TTGATGTTCC TGATTGTAAA AAAACTATAG TGAATGATTC CAGAGAGTCA 300
TGTGTTGAGG AAAATGATGA TAAAATTACA CAAGCTTCAC AATCACAAGA AAGTGAAGAC 360
... TATTCTCAGC CATCAACTTC TAGTAGCATT ATTTATAGCA GCCAAGAAGA TGTGAAAGAG 420
TTTGAAAGGG AAGAAACCCA AGACAAAGAA GAGAGTGTGG AATCTAGTTT GCCCCTTAAT 480
GCCATTGAAC CTTGTGTGAT TTGTCAAGGT CGACCTAAAA ATGGTTGCAT TGTCCATGGC 540
AAAACAGGAC ATCTTATGGC CTGCTTTACA TGTGCAAAGA AGCTAAAGAA AAGGAATAAG 600
CCCTGCCCAG TATGTAGACA ACCAATTCAA ATGATTGTGC TAACTTATTT CCCCTAG 657
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 966 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC ACAGATTCCA 60
GCTTCGGAAC AAGAGACCCT GGTTAGACCA AAGCCATTGC TTTTGAAGTT ATTAAAGTCT 120
GTTGGTGCAC AAAAAGACAC TTATACTATG AAAGAGGATC TTGATGCTGG TGTAAGTGAA l80
CATTCAGGTG ATTGGTTGGA TCAGGATTCA GTTTCAGATC AGTTTAGTGT AGAATTTGAA 240
CA 0224l9~2 l998-07-l4
WO 97/25860 PCTrus97/00582
- 60 -
GTTGAATCTC TCGACTCAGA AGATTATAGC CTTAGTGAAG AAGGACAAGA ACTCTCAGAT 300
GAAGATGATG AGGTATATCA AGTTACTGTG TATCAGGCAG GGGAGAGTGA TACAGATTCA 360
TTTGAAGAAG ATCCTGA~AT TTCCTTAGCT GACTATTGGA AATGCACTTC ATGCAATGAA 420
ATGAATCCCC CCCTTCCATC ACATTGCAAC AGATGTTGGG CCCTTCGTGA GAATTGGCTT 480
CCTGAAGATA AAGGGAAAGA TAAAGGGGAA ATCTCTGAGA AAGCCA~ACT GGAAAACTCA 540
ACACAAGCTG AAGAGGGCTT TGATGTTCCT GATTGTAAAA AAACTATAGT GAATGATTCC 600
AGAGAGTCAT GTGTTGAGGA A~ATGATGAT AAAATTACAC AAGCTTCACA ATCACAAGAA 660
AGTGAAGACT ATTCTCAGCC ATCAACTTCT AGTAGCATTA TTTATAGCAG CCAAGAAGAT 720
GTGAAAGAGT TTGA~AGGGA AGA~ACCCAA GACAAAGAAG AGAGTGTGGA ATCTAGTTTG 780
CCCCTTAATG CCATTGAACC TTGTGTGATT TGTCAAGGTC GACCTAAAAA TGGTTGCATT 840
GTCCATGGCA A~ACAGGACA TCTTATGGCC TGCTTTACAT GTGCAAAGAA GCTA~AGA~A 900
AGGAATAAGC CCTGCCCAGT ATGTAGACAA CCAATTCA~A TGATTGTGCT AACTTATTTC 960
CCCTAG 966
(2) INFORMATION FOR SEQ ID NO:8:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 399 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC ACAGATTCCA 60
GCTTCGGAAC AAGAGACCCT GGTTAGACAA GA~AGTGAAG ACTATTCTCA GCCATCAACT 120
TCTAGTAGCA TTATTTATAG CAGCCAAGAA GATGTGAAAG AGTTTGAAAG GGAAGAAACC 180
CAAGACA~AG AAGAGAGTGT GGAATCTAGT TTGCCCCTTA ATGCCATTGA ACCTTGTGTG 240
Alll~l~AG GTCGACCTAA AAATGGTTGC ATTGTCCATG GCAAAACAGG ACATCTTATG 300
GCCTGCTTTA CATGTGCAAA GAAGCTAAAG A~AAGGAATA AGCCCTGCCC AGTATGTAGA 360
CAACCAATTC AAATGATTGT GCTAACTTAT TTCCCCTAG 399
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~SS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGTGCA~TA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC ACAGATTCCA 60
GCTTCGGAAC AAGAGACCCT GGTTAGACCA AAGCCATTGC TTTTGAAGTT ATTA~AGTCT 120
CA 0224l9~2 l998-07-l4
W O 97/2S860 PC~rUSg7/00582
- 61 -
GTTGGTGCAC AAAAAGACAC TTATACTATG AAAGAGGTTC 'll-l"ll"lATCT TGGCCAGTAT 180
ATTATGACTA AACGATTATA TGATGAGAAG CAACAACATA TTGTAAATGA TTGTGCTAAC 240
TTATTTCCCC TAGTTGACCT GTCTATAAGA GAATTATATA TTTCTAACTA TATAACCCTA 300
GGAATTTAG 309
(2) INFORMATION FOR SEQ ID NO:10:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1897 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 3ingle
(D) TOPOBOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CACAGATAAG GTTATTTGGG TACCCTCTCG A~AAGTTA~A CCGGACATCG CCCAaAAGGA 60
TGAGGTGACT AAGAAAGATG AGGCGAGCCC lClllll~GCA GGCTGGAGGC ACATAGATAA 120
GAGAATTATC ACTCTACATT CATCTTTCTC AAAGATTAAT CTACTTGTGT GTTTTATATT 180
TCATTAGAAT CGGACAGATG TTCAGTGCCA GCACCGGTGG CAGA~AGTAT TAAACCCAGA 240
ACTTAACA~A GGTCCATGGA CTA~AGAGGA GGATCAAAGG GTAATAGAAC ACGTGCAGAA 300
ATACGGTCCA AAGCGCTGGT CGGACATTGC TAAGCATTTG AAGGGAAGGA TTGGAAAACA 360
GTGCAGGGAG AGGTGGCACA ACCATCTGAA TCCAGAAGTG A~GAAAACCT CCTGGACAGA 420
AGAGGAAGAT AGAATTATTT ACCAGGCACA CAAGAGACTG GGA~ACAGAT GGGCAGA~AT 480
TGCA~AGTTG CTGCCTGGAC GGACTGATAA CGCTGTCAAG AACCACTGGA ATTCCACCAT 540
GCGCCGGAAG GTCGAGCAGG AGGGTTACCC GCAGGAGTCC TCCA~AGCCG GCCCGCCCTC 600
GGCAACCACC GGCTTCCAGA AGAGCAGCCA TCTGATGGCC TTTGCCCACA ACCCACCTGC 660
AGGCCCGCTC CCGGGGGCCG GCCAGGCCCC TCTGGGCAGT GACTACCCCT ACTACCACAT 720
TGCTGAGCCA CAAAATGTCC CTGGTCAGAT CCCATATCCA GTAGCACTGC ATATA~ATAT 780
TATCAATGTT CCTCAGCCAG CTGCTGCAGC TATTCAGAGA CACTATACTG ATGAAGACCC 840
TGAGAAAGAA AAACGAATAA AGGAATTAGA GTTGCTACTT ATGTCGACTG AGAATGAACT 90O
GAAAGGGCAG CAGGCATTAC CAACACAGAA CCACACAGCA AACTACCCCG GCTGGCACAG 960
CACCACGGTT GCTGACAATA CCAGGACCAG TGGTGACAAT GCGCCTGTTT C~ GGG 1020
GGAACATCAC CACTGTACTC CATCTCCACC AGTGGATCAT GGTTGCTTAC CTGAGGA~AG 1080
TGCGTCCCCC GCACGGTGCA TGATTGTTCA CCAGAGCAAC ATCCTGGATA ATGTTAAGAA 1140
TCTCTTAGAA TTTGCAGAAA CACTCCAGTT AATAGACTCC TTCTTA~ACA CATCGTCCAA 1200
TCACGAGAAT CTGAACCTGG ACAACCCTGC ACTAACCTCC ACGCCAGTGT GTGGCCACAA 1260
GATGTCTGTT ACCACCCCAT TCCACAAGGA CCAGACTTTC ACTGAATACA GGAAGATGCA 1320
CGGCGGAGCA GTCTAGAGCT CAATTATAAT AATCTTGCGA ATCGGGCTGT AACGGGGCAA 1380
CA 0224l9~2 l998-07-l4
W 097/25860 PCTrUS97/00582.
- 62 -
GGCTTGACCG AGGGGACTAT AACATGTATA GGCGAAAAGC GGGGTCTCGG TTGTAACGCG 1440
CTTAGGAAGT CCCCTCGAGG TATGGCAGAT ATGCTTTTGC ATAGGGAGGG GGAAATGTAG 1500
TCTTAATCGT AGGTTAACAT GTATATTACC AAATAAGGGA ATCGCCTGAT GCACCAAATA 1560
AGGTATTATA TGATCCCATT GGTGGTGAAG GAGCGACCTG AGGGCATATG GGCGTTAACA 1620
GAACTGTCTG TCCTTGCGTC ATTCCTCATC GGATCATGTA CGCGGCAGAG TATGATTGGA 1680
TAACAGGATG GCACCATTCA TCGTGGCGCA TGCTGATTGG TGCGACTAAG GAGTTGTGTA 1740
ACCCACGAAT GTACTTAAGC TTGTAGTTGC TAACAATAAA GTGCCATTCT ACCTCTCACC 1800
ACATTGGTGT GCACCTGGGT TGATGGCCGG ACCGTCGATT CCCTGACGAC TGCGAACACC 1860
TGAATGAAGC TGAAGGCTTC AGGTACCCTT ACTTGAT1897
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 8082 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AG~ll~lll~G GCCGTTTTAG G~lll~llGG AA'l'l~ll'lTll' TCGTCTATGT ACTTGTGAAT 60
TATTTCACGT TTGCCATTAC CG~ CCA TAGGGTGATG TTCATTAGCA GTGGTGATAG 120
GTTAATTTTC ACCATCTCTT ATGCGGTTGA ATAGTCACCT CTGAACCACT TTTTCCTCCA 180
GTAACTCCTC Tll~llCGGA CCTTCTGCAG CCAACCTGAA AGAATAACAA GGAGGTGGCT 240
GGAAACTTGT TTTAAGGAAC CGCCTGTCCT TCCCCCGCTG GA~ACCTTGC ACCTCGGACG 300
CTCCTGCTCC TGCCCCCACC TGACCCCCGC CCTCGTTGAC ATCCAGGCGC GATGATCTCT 360
GCTGCCAGTA GAGGGCACAC TTACTTTACT TTCGCAAACC TGAACGCGGG TGCTGCCCAG 420
AGAGGGGGCG GAGGGAAAGA CGCTTTGCAG CAAAATCCAG CATAGCGATT GGTTGCTCCC 480
CGCGTTTGCG GCAAAGGCCT GGAGGCAGGA GTAATTTGCA ATCCTTAAAG CTGAATTGTG 540
CAGTGCATCG GATTTGGAAG CTACTATATT CACTTAACAC TTGAACGCTG AGCTGCAAAC 600
TCAACGGGTA ATAACCCATC TTGAACAGCG TACATGCTAT ACACACACCC CTTTCCCCCG 660
AA~ lC TCTTTTGGAG GTGGTGGAGG GAGAGAAAAG TTTACTTAAA ATGCCTTTGG 720
GTGAGGGACC AAGGATGAGA AGAATGTTTT ll~ CA TGCCGTGGAA TAACACAAAA 780
TAAAAAATCC CGAGGGAATA TACATTATAT ATTAAATATA GATCATTTCA GGGAGCAAAC 840
A~ATCATGTG TGGGGCTGGG CAACTAGCTG AGTCGAAGCG TAAATAAAAT GTGAATACAC 900
GTTTGCGGGT TACATACAGT GCACTTTCAC TAGTATTCAG AAAAAATTGT GAGTCAGTGA 960
ACTAGGAAAT TAATGCCTGG AAGGCAGCCA AATTTTAATT AGCTCAAGAC TCCCCCCCCC 1020
CCCCA~AAAA AGGCACGGAA GTAATACTCC TCTCCTCTTC TTTGATCAGA ATCGATGCAT 1080
CA 0224l9~2 l998-07-l4
W 097/25860 PCTrUS97100582
- 63 -
-llll~LGCA TGACCGCATT TCCAATAATA AAAGGGGA~A GAGGACCTGG A~AGGAATTA 1140
AACGTCCGGT TTGTCCGGGG AGGAAAGAGT TAACGGTTTT TTTCACAAGG GTCTCTGCTG 1200
ACTCCCCCGG CTCGGTCCAC AAGCTCTCCA CTTGCCCCTT TTAGGAAGTC CGGTCCCGCG 1260
GTTCGGGTAC CCCCTGCCCC TCCCATATTC TCCCGTCTAG CACCTTTGAT TTCTCCCAAA 1320
CCCGGCAGCC CGAGACTGTT GCAAACCGGC GCCACAGGGC GCAAAGGGGA TTTGTCTCTT 1380
CTGAAACCTG GCTGAGAAAT TGGGAACTCC GTGTGGGAGG CGTGGGGGTG GGACGGTGGG 1440
GTACAGACTG GCAGAGAGCA GGCAACCTCC CTCTCGCCCT AGCCCAGCTC TGGAACAGGC 1500
AGACACATCT CAGGGCTAAA CAGACGCCTC CCGCACGGGG CCCCACGGAA GCCTGAGCAG 1560
GCGGGGCAGG AGGGGCGGTA TCTGCTGCTT TGGCAGCAA,A TTGGGGGACT CAGTCTGGGT 1620
GGAAGGTATC CA~TCCAGAT'AGCTGTGCAT ACATAATGCA TAATACATGA CTCCCCCCAA 1680
CAAATGCAAT GGGAGTTTAT TCATAACGCG CTCTCCAAGT ATACGTGGCA ATGCGTTGCT 1740
GGGTTATTTT AATCATTCTA GGCATCGTTT TCCTCCTTAT GCCTCTATCA TTCCTCCCTA 1800
TCTACACTAA CATCCCACGC TCTGAACGCG CGCCCATTAA TACCCTTCTT TCCTCCACTC 1860
TCCCTGGGAC TCTTGATCAA AGCGCGGCCC TTTCCCCAGC CTTAGCGAGG CGCCCTGCAG 1920
CCTGGTACGC GCGTGGCGTG GCGGTGGGCG CGCAGTGCGT TCTCTGTGTG GAGGGCAGCT 1980
GTTCCGCCTG CGATGATTTA TACTCACAGG ACAAGGATGC G~l"l"l~lCAA ACAGTACTGC 2040
TACGGAGGAG CAGCAGAGAA AGGGAGAGGG TTTGAGAGGG AGCA,A~AAGAA AATGGTAGGC 2100
GCGCGTAGTT AATTCATGCG GCTCTCTTAC TCTGTTTACA TCCTAGAGCT AGAGTGCTCG 2160
GCTGCCCGGC TGAGTCTCCT CCCCACCTTC CCCACCCTCC CCACCCTCCC CATAAGCGCC 2220
CCTCCCGGGT TCCCA~AGCA GAGGGCGTGG GGGAAAAGAA A~AAGATCCT CTCTCGCTAA 2280
TCTCCGCCCA CCGGCCCTTT ATAATGCGAG GGTCTGGACG GCTGAGGACC CCCGAGCTGT 2340
GCTGCTCGCG GCCGCCACCG CCGGGCCCCG GCCGTCCCTG GCTCCCCTCC TGCCTCGAGA 2400
AGGGCAGGGC TTCTCAGAGG CTTGGCGGGA AAAAGAACGG AGGGAGGGAT CGCGCTGAGT 2460
ATAAAAGCCG GTTTTCGGGG CTTTATCTAA CTCGCTGTAG TAATTCCAGC GAGAGGCAGA 2520
GGGAGCGAGC GGGCGGCCGG CTAGGGTGGA AGAGCCGGGC GAGCAGAGCT GCGCTGCGGG 2580
CGTCCTGGGA AGGGAGATCC GGAGCGAATA GGGGGCTTCG CCTCTGGCCC AGCCCTCCCG 2640
CTGATCCCCC AGCCAGCGGT CCGCAACCCT TGCCGCATCC ACGAP~ACTTT GCCCATAGCA 2700
GCGGGCGGGC ACTTTGCACT GGAACTTACA ACACCCGAGC AAGGACGCGA CTCTCCCGAC 2760
GCGGGGAGGC TATTCTGCCC ATTTGGGGAC ACTTCCCCGC CGCTGCCAGG ACCCGCTTCT 2820
CTGAAAGGCT CTCCTTGCAG CTGCTTAGAC GCTGGATTTT TTTCGGGTAG TGGAP~ACCA 2880
GGTAAGCACC GAAGTCCACT TGCCTTTTAA TTTATTTTTT TATCACTTTA ATGCTGAGAT 2940
GAGTCGAATG CCTAAATAGG GT~L~Ll~'l"l'C TCCCATTCCT GCGCTATTGA CACTTTTCTC 3000
CA 0224l9~2 l998-07-l4
W 097/25860 PCTrUs97/00582
- 64 -
AGAGTAGTTA TGGTAACTGG GGCTGGGGTG GGGGGTAATC CAGAACTGGA TCGGGGTAAA 3060
GTGACTTGTC AAGATGGGAG AGGAGAAGGC AGAGGGAAAA CGGGAATGGT TTTTAAGACT 3120
ACCCTTTCGA GATTTCTGCC TTATGAATAT ATTCACGCTG ACTCCCGGCC GGTCGGACAT 3180
TCCTGCTTTA 'l"l'~'l'~'l"l'AAT TG~l~ GG GTTTTGGGGG GCTGGGGGTT GCTTTGCGGT 3240
GGGCAGAAAG CCCCTTGCAT CCTGAGCTCC TTGGAGTAGG GACCGCATAT CGCCTGTGTG 3300
AGCCAGATCG CTCCGCAGCC GCTGACTTGT CCCCGTCTCC GGGAGGGCAT TTA~ATTTCG 3360
GCTCACCGCA TTTCTGACAG CCGGAGACGG ACACTGCGGC GCGTCCCGCC CGCCTGTCCC = 3420
CGCGGCGATT CCAACCCGCC CTGATCCTTT TAAGAAGTTG GCATTTGGCT TTTTAAAAAG 3480
CAATAATACA ATTTA~AACC TGG~L~ A GAG~l~ AG GACGTGGTGT TGGGTAGGCG 3540
CAGGCAGGGG A~AAGGGAGG CGAGGATGTG TCCGATTCTC CTGGAATCGT TGACTTGGAA 3600
AAACCAGGGC GAATCTCCGC ACCCAGCCCT GACTCCCCTG CCGCGGCCGC CCTCGGGTGT 3660
CCTCGCGCCC GAGATGCGGA GGAACTGCGA GGAGCGGGGC TCTGGGCGGT TCCAGAACAG 3720
CTGCTACCCT TGGTGGGGTG GCTCCGGGGG AGGTATCGCA GCGGGGTCTC TGGCGCAGTT 3780
GCATCTCCGT ATTGAGTGCG AAGGGAGGTG CCCCTATTAT TATTTGACAC CCCCCTTGTA 3840
TTTATGGAGG GGTGTTAAAG CCCGCGGCTG AGCTCGCCAC TCCAGCCGGC GAGAGAAAGA 3900
AGAAAAGCTG GCA~AAGGAG TGTTGGACGG GGGCGGTACT GGGGGTGGGG ACGGGGGCGG 3960
TGGAGAGGGA AGGTTGGGAG GGGCTGCGGT GCCGGCGGGG GTAGGAGAGC GGCTAGGGCG 4020
CGAGTGGGAA CAGCCGCAGC GGAGGGGCCC CGGCGCGGAG CGGGGTTCAC GCAGCCGCTA 4080
GCGCCCAGGC GCCTCTCGCC ~L~l~llCA GGTGGCGCAA AACTTTGTGC CTTGGATTTT 4140
GGCAAATTGT TTTCCTCACC GCCACCTCCC GCGGCTTCTT AAGGGCGCCA GGGCCGATTT 4200
CGATTCCTCT GCCGCTGCGG GGCCGACTCC CGGGCTTTGC GCTCCGGGCT CCCGGGGGAG 4260
CGGGGGCTCG GCGGGCACCA AGCCGCTGGT TCACTAAGTG CGTCTCCGAG ATAGCAGGGG 4320
ACTGTCCA~A GGGGGTGAAA GGGTGCTCCC TTTATTCCCC CACCAAGACC ACCCAGCCGC 4380
TTTAGGGGAT AGCTCTGCAA GGGGAGAGGT TCGGGACTGT GGCGCGCACT GCGCGCTGCG 4440
CCAGGTTTCC GCACCAAGAC CCCTTTAACT CAAGACTGCC TCCCGCTTTG TGTGCCCCGC ~500
TCCAGCAGCC TCCCGCGACG ATGCCCCTCA ACGTTAGCTT CACCAACAGG AACTATGACC 4560
TCGACTACGA CTCGGTGCAG CCGTATTTCT ACTGCGACGA GGAGGAGAAC TTCTACCAGC 4620
AGCAGCAGCA GAGCGAGCTG CAGCCCCCGG CGCCCAGCGA GGATATCTGG AAGAAATTCG 4680
AGCTGCTGCC CACCCCGCCC CTGTCCCCTA GCCGCCGCTC CGGGCTCTGC TcGcccTccT 4740
ACGTTGCGGT CACACCCTTC TCCCTTCGGG GAGACAACGA CGGCGGTGGC GGGAGCTTCT 4800
CCACGGCCGA CCAGCTGGAG ATGGTGACCG AGCTGCTGGG AGGAGACATG GTGAACCAGA 4860
GTTTCATCTG CGACCCGGAC GACGAGACCT TCATCAAAAA CATCATCATC CAGGACTGTA 4920
CA 022419~2 1998-07-14
W O 97/25860 PCTnUS97/OOS82
- 65 -
TGTGGAGCGG CTTCTCGGCC GCCGCCAAGC TCGTCTCAGA GAAGCTGGCC TCCTACCAGG 4980
CTGCGCGCAA AGACAGCGGC AGCCCGAACC CCGCCCGCGG CCACAGCGTC TGCTCCACCT 5040
CCAGCTTGTA CCTGCAGGAT CTGAGCGCCG CCGCCTCAGA GTGCATCGAC CCCTCGGTGG 5100
TCTTCCCCTA CCCTCTCAAC GACAGCAGCT CGCCCAAGTC CTGCGCCTCG CAAGACTCCA 5160
GCGCCTTCTC TCCGTCCTCG GATTCTCTGC TCTCCTCGAC GGAGTCCTCC CCGCAGGGCA 5220
GCCCCGAGCC CCTGGTGCTC CATGAGGAGA CACCGCCCAC CACCAGCAGC GACTCTGGTA 5280
AGCGAAGCCC GCCCAGGCCT GTCAAAAGTG GGCGGCTGGA TACCTTTCCC ATTTTCATTG 5340
GCAGCTTATT TAACGGGCCA CTCTTATTAG GAAGGAGAGA TAGCAGATCT GGAGAGATTT 5400
GGGAGCTCAT CACCTCTGAA ACCTTGGGCT TTAGCGTTTC CTCCCATCCC TTCCCCTTAG 5460
ACTGCCCATG TTTGCAGCCC CCCTCCCCGT TTGTCTCCCA CCCCTCAGGA ATTTCATTTA 5520
G~TTTlTA~A CCTTCTGGCT TATCTTACAA CTCAATCCAC TTCTTCTTAC CTCCCGTTAA 5580
CATTTTAATT GCCCTGGGGC GGGGTGGCAG GGAGTGTATG AATGAGGATA AGAGAGGATT 5640
GATCTCTGAG AGTGAATGAA TTGCTTCCCT CTTAACTTCC GAGAAGTGGT GGGATTTAAT 5700
GAACTATCTA CA~AAATGAG GGGCTGTGTT TAGAGGCTAG GCAGGGCCTG CCTGAGTGCG 5760
GGAGCCAGTG AACTGCCTCA AGAGTGGGTG GGCTGAGGAG CTGGGATCTT CTCAGCCTAT 5820
TTTGAACACT GA~AAGCAAA TCCTTGCCAA AGTTGGACTT ll"l"lllll~CT TTTATTCCTT 5880
CCCCCGCCCT CTTGGACTTT TGGCAAAACT GCAATTTTTT TTTTTTTATT TTTCATTTCC 5940
AGTA~AATAG GGAGTTGCTA AAGTCATACC AAGCAATTTG CAGCTATCAT TTGCAACACC 6000
TGAAGTGTTC TTGGTAAAGT CCCTCAAAAA TAGGAGGTGC TTGGGAATGT GCTTTGCTTT 6060
GG~l~l~ C A~AGCCTCAT TAAGTCTTAG GTAAGAATTG GCATCAATGT CCTATCCTGG 6120
GAAGTTGCAC TTTTCTTGTC CATGCCATAA CCCAGCTGTC TTTCCCTTTA TGAGACTCTT 6180
ACCTTCATGG TGAGAGGAGT AAGGGTGGCT GGCTAGATTG GTT~L~l~lll~l~ l"l"l"l"l"l"l"l"l~C 6240
~'l"l"l"l"l''l'AAG ACGGAGTCTC A~l~l~l~AC TAGGCTGGAG TGCAGTGGCG CAATCAACCT 6300
CCAACCCCCT GGTTCAAGAG ATTCTCCTGC CTCAGCCTCC CAAGTAGCTG GGACTACAGG 6360
TGCACACCAC CATGCCAGGC TAATTTTTGT AATTTTAGTA GAGATGGGGT TTCATCGTGT 6420
TGGCCAGGAT GGTCTCTCCT GACCTCACGA TCCGCCCACC T,CGGCCTCCC AAAGTGCTGG 6480
GATTACAGGT GTGAGCCAGG GCACCAGGCT TAGATGTGGC TCTTTGGGGA GATAATTTTG 6540
TCCAGAGACC TTTCTAACGT ATTCATGCCT TGTATTTGTA CAGCATTAAT CTGGTAATTG 6600
ATTATTTTAA TGTAACCTTG CTAAAGGAGT GATTTCTATT TC~lTl~l~L~A AAGAGGAGGA 6660
ACAAGAAGAT GAGGAAGAAA TCGATGTTGT TTCTGTGGAA AAGAGGCAGG CTCCTGGCAA 6720
AAGGTCAGAG TCTGGATCAC CTTCTGCTGG AGGCCACAGC AAACCTCCTC ACAGCCCACT 6780
GGTCCTCAAG AGGTGCCACG TCTCCACACA TCAGCACAAC TACGCAGCGC CTCCCTCCAC 6840
CA 022419~2 1998-07-14
W O 97/25860 PCTrUS97/0~582
- 66 -
TCGGAAGGAC TATCCTGCTG CCAAGAGGGT CAAGTTGGAC AGTGTCAGAG TCCTGAGACA 6900
GATCAGCAAC AACCGAAAAT GCACCAGCCC CAGGTCCTCG GACACCGAGG AGAATGTCAA 6960
GAGGCGAACA CACAACGTCT TGGAGCGCCA GAGGAGGAAC GAGCTA~AAC GGAGCTTTTT 7020
TGCCCTGCGT GACCAGATCC CGGAGTTGGA AAACAATGAA AAGGCCCCCA AGGTAGTTAT 7080
CCTTAAAAAA GCCACAGCAT A QTCCTGTC CGTCCAAGCA GAGGAGCA~A AGCTCATTTC 7140
TGAAGAGGAC TTGTTGCGGA AACGACGAGA ACAGTTGAAA CACAAACTTG AACAGCTACG 7200
GAA~ GCGTAAGGAA AAGTAAGGAA AACGATTCCT TCTAACAGAA A~ C~l~AG 7260
CAATCACCTA TGAACTTGTT TCA~ATGCAT GATCAAATGC AACCTCACAA CCTTGGCTGA 7320
GTCTTGAGAC TGAAAGATTT AGCCATAATG TAAACTGCCT CAAATTGGAC TTTGGGCATA 7380
AAAGAACTTT TTTATGCTTA CCA~ TTTTTCTTTA ACAGATTTGT ATTTAAGAAT 7440
T~~ AAA AAATTTTAAG ATTTACACAA TGTTTCTCTG TA~ATATTGC CATTA~ATGT 7500
AAATAACTTT AATAAAACGT TTATAGCAGT TACACAGAAT TTCAATCCTA GTATATAGTA 7560
CCTAGTATTA TAGGTACTAT AAACCCTAAT lllllllATT TAAGTACATT TTGW l~ A 7620
AAGTTGATTT TTTTCTATTG TTTTTAGAAA A~ATAAAATA ACTGGCAAAT ATATCATTGA 1680
GCCAAATCTT AAGTTGTGAA ~1~'1111~111' CGTTTCTTCC CCCTCCCAAC CACCACCATC 7740
C~l~lll~ll~ TTCATCAATT GCCCCTTCAG AGGGCGGTCT TAAGAAAGGC AAGAGTTTTC 7800
CTCTGTTGAA ATGGGTCTGG GGGCCTTAAG GTCTTTAAGT TCTTGGAGGT TCTAAGATGC 7860
TTCCTGGAGA CTATGATAAC AGCCAGAGTT GACAGTTAGA AGGAATGGCA GAAGGCAGGT 7920
GAGAAGGTGA GAGGTAGGCA AAGGAGATAC AAGAGGTCAA AGGTAGCAGT TAAGTACACA 7980
AAGAGGCATA AGGACTGGGG AGTTGGGAGG AAGGTGAGGA AGAAACTCCT GTTACTTTAG 8040
TTAACCAGTG CCAGTCCCCT GCTCACTCCA AACCCAGGAA TT 8082
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4480 base pairR
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
AGGGTTACAC GTCTTAACTC AGAGTTGCAA CAGGCTTGAA CAAGCCCAGG CACGCCCAGA 60
TACCTAGGGC CGAGTCACCG TTAAAACTAA CAGACCATAA AAGGAAAGGA ATACAGAACA 120
GACTAGGAGT ACCGGATCTG ACTCACAGGC CACCTGGCAG GAAGAGATAA GCCCCAGCCC 180
CCGACATTCA GGACGTCCCA GCCCGCACGT ACTCTTACCA TGTTACAACC TCATTCGAAT 240
ATGATTCA~A CCTGCCAATG TGTGTAGCTA TACCTTATCA CCTCATCTTG TGA~ATAACC 300
AATCATATGT GAACATGTCT ATATGCTTCG TTTA~ATCCA CCAATCCCCG TAACTATGCA 360
CA 0224l9~2 l998-07-l4
W 097/25860 PCT~U$97/00582
- 67 -
TCTGCTTCTG TACGCCCGCT TCTGCTTCCC CAAACCCTAT AAAAGCCCCA TGCTAGAGCT 420
GTTGGGCGCG CAAGTCCTCC GAAGAGACTG TGTGCCCGCA GGTACCTGTG TTTTCCAATA 480
AACCCTCTTG CTGATTGCAT CCGAGTGGCC TCGGCTCGGT CATTGGGCGC TTGGGGGTCT 540
CCTCCTGAGG GAAAGGTCCT CTCCGGAGGT CTTTTCATTT TGGGGGCTCG TCCGGGATCT 600
GGAGATCCTC CGCCCAGAGA TCACCGACCA CCCACCGGGA GGTAAGCCGG CCGGCATCTG 660
TCGTGTCTTG CCCTGTCTTG TCTTGTCTTG TCCTGTGCGC GTGTTCAGTT CGTCTCAGTT 720
TTGGACTCAG ATCTGGGTTT TGGTCGAAGG AGAAGGCCCA GGGCTTCGGT TTCTCAGGGT 780
TCAGGACCCT CAGCGCCTCC.GTTTGGGCGG GTCAGAGAAG GAGCTGACGA GCTCGGACTT 840
CTCCCCCCGC AGCCCTGGAA GACGTTCCAA GGGTGTCTGG AGCCCGGTTC TTTGGGGCTC 900
AGCCCGTATC GGAGGGATAC GTGGTTTTGG TTGGAGGAGA GGGTCCAGGA CCCTCGGCAC 960
CTCCATCTGA CTCTTTGTTT TGGGTTTTAC GTCGAAGCCG CGCGGCGCGT CTGTCTGTTA 1020
TTTGTCTGAT CGTTGGATTT GTCTGTCTAA TCTGTGCCCT AATTTTCTTT GAAGCTACCA 1080
TGGGACAATC GCTAACAACC CCCTTGAGTC TCACTCTAGA CCATTGGAAG GACGTCCGAG 1140
ACCGAGCACG TGATCAGTCG GTCGAGATCA AGAAAGGTCC TCTCCGGAGG TCGGGGACAG 1200
TCGCGCCAGC AAGCGGTGGG GCAGGAGCTC CTGGTTTGGC AGCCCCTGTA GAAGCGATGA 1260
CAGAATACAA GCTTGTGGTG GTGGGCGCTA GAGGCGTGGG AAAGAGTGCC CTGACCATCC 1320
AGCTGATCCA GAACCATTTT GTGGACGAGT ATGATCCCAC TATAGAGGAC TCCTACCGGA 1380
AACAGGTAGT CATTGATGGG GAGACGTGTT TACTGGACAT CTTAGACACA GCAGGTCAAG 1440
AAGAGTATAG TGCCATGCGG GACCAGTACA TGCGCACAGG GGAGGGCTTC CTCTGTGTAT 1500
TTGCCATCAA CAACACCAAG ~ ll~AAG ACATCCATCA GTACAGGGAG CAGATCAAGC 1560
GGGTGAAAGA TTCAGATGAT GTGCC~ATGG TGCTGGTGGG CAACAAGTGT GACCTGGCCG 1620
CTCACACTGT TGAGTCTCGG CAGGCCCAGG ACCTTGCTCG CAGCTATGGC ATCCCCT~r~ 1680
TTGAAACATC AGCCAAGACC CGACCAGGTG TGGAGGATGC CTTCTACACA CTAGTACGTG 1740
AGATTCGGCA GCATAAACTG CGGAAACTGA ACCCGCCTGA TGAGAGTGGC CCTGGCTGCA 1800
TGAGCTGCAA GTGTGTGCTG TCCTGACACC AGGTTAAGGA CCTGATTTTC CGCCAGAAGC 1860
CGTACGGACA CCCTGACCAG GTGGCCTACA TTGTCACCTG GGAGAGCTTG GCATTTAGCC 1920
CTCCTCCTTG GGCAGAACCC TTTGTGGACC CGAATTGGCT TCCTGTTTCC CCTAAACCTG 1980
TTTCCCCGAG CCCACCTGAC CCTTTGGTTG CTTCTTCCTC TCTCTATCCT GCTCTAACTA 2040
AGGAAGAATC TCCCAAAGTC CCTCCCCCGA AACCTGTCCT CCCAGAGGAC CCAAATTCCC 2100
CCCTTATAGA TCTCCTGTTG GAAGAACCTC CTCCGTACCC TGTACCTACA GCCCCGCCAA 2160
GAGAAGAGGA AGTGGAGCCG CCTGCTAGAC CTCGACTCGA GGCGGCCCCT TCCCCTGTGG 2220
CTGGAAGACT TCGGGGACGA CGCGAGGTGG CGCCAGACTC CACCTCCCAG GCCTTTCCGC 2280
CA 022419~2 1998-07-14
W 097/25860 PCTrus97/00582
- 68 -
TTAGACAAGG GGCTGGCGGC CAGATACAAT ACTGGCCATT CTCAGCGGCC GACATATATA 2340
ACTGGAAACA ACACAACCCC CCCTTTTCTA AGGATCCGGT GGCTCTCACC AACCAGATAG 2400
AATCTGTCTT GCTTACCCAT CAGCCCACTT GGGATGATAT ACAGCAACTT TTACAGGCCC 2460
TCCTGACCTC TGAAGAGAAG CAGAGAGTGC TCTTAGAGGC CAGGAAACAT GTTTTGGGGG 2520
ACAATGGACG CCCCACCTTG CTCCCGAAAG AGATCGATGA TGCATTCCCA CTTACAAGAC 2580
CTGATTGGGA TTTCACCACG GCTAAAGGTA GGAGACACCT ACGCCTTTAT CGCCAGTTGC 2640
TCCTAGCGGG TCTCCGAGGG GCGGCACGAC GCCCCACCAA TTTGGCTCAG GTAAA~CAAG 2700
TGGTACAAGA GGCTGCGGAG ACTCCCTCAG CCTTCCTAGA GAGACTTAAG GAAGCTTATC 2760
GCATGTATAC CCCTTATGAT CCAGATGATC CAGGACAAAT GACA~ATGTC TCCATGTCCT 2820
TCATCTGGCA GGCAGCACCA GATATCAGGG CCAAGCTACA GAGAATAGAA AATTTACAAG 2880
GGTATACACT GCAGGATTTA CTTAAGGAGG CAGA~AGAAT TTATAACAAG AGAGAGACAC 2940
AAGAAGAAAA GA~AGATAAA ATACGTAGAG AAAAAGATGA GAGAGACCGA AAAAGA~ACA 3000
GAGAGTTGAG TCGAATCTTG GCCGCCGTAG TTCAGGGT Q AGAGAAAAGG GGAGAGAGGG 3060
TGGGAGTTCG AAAGGGGCCA AAGCTAGATA AGGATCAATG TGCGTATTGC A~AGA~AGAG 3120
GACACTGGGC CAGAGATTGC CCTAAGAAAC CCAGCGGCTC CGAAGACCCC GCCCACAGAC 3180
CTCCCTCTTG GCCCTAGATA AAGATTAGGG AGGTCAGGGC CAGGAGCCCC CCCCTGAGCC 3240
CAGGATAACT CTTGAAGTTG GGGGGCAGCC AGTCACCTTT CTGGTGGACA CAGGAGCCCA 33~0
GCACTCAGTC CTCACCCAGG CCCCTGGACA ACTCAGCGAC CGGACGGCCT GGGTACAAGG 3360
AGCCACTGGC AGCAAGAGAT ACCGTTGGAC TACAGATCGA CGGGTTCAGC TGGCTACTGG 3420
TAAGGTGACC CATTCCTTCT TACATGTTCC GGACTGCCCA TACCCTCTGC TGGGCCGTGA 3480
CTTGCTTACC A~ATTAAAAG CTCAGATCCA TTTTGAAGAA GGAGGGACCC GAGTAACCGG 3540
GCCCCGCGGT ATTCCTCTTC AGATTTTAAC CCTTCAGTTA GAAGATGAAT ATAGATTATA 3600
TGAACCAGAA CAGGACAAGC CA~AATCTCC AGAAATAGAC TCTTGGGTCA CGAAATTCCC 3660
ACTGGCCTGG GCAGAGACTG GCGGGATGGG GTTGGCGCTC CAACAGCCTC CCCTAATTAT 3720
CCAGTTAAAG GCCACCGCGA CTCCTGTCTC CATTAAACAG TACCCCATGT CATGGGAAGC 3780
TTATCAGGGC ATAAAGCCAC ATATCAGGAG GCTCTTAGAC CAAGGCATCC TAGTCCCTTG 3840
CCGGTCACCC TGGAATACGC CTCTGCTACC TGTTAAGAAG CCCGGCACTG GAGACTATAG 3900
GCCAGTACAA GATTTGAGAG AGGTCAACAA AAGAGTAGAA GATATTCATC CAACTGTCCC 3960
AAACCCTTAT AACCTACTCA GCACCCTGCC TCCCACCCAT ACTTGGTATA CGGTCTTAGA 4020
TCTGAAGGAT GCTTTCTTCT GCCTCCGGCT GAGCCCAGAA AGCCAGCCCT TATTTGCTTT 4080
TGAGTGGAAA GACTCTGA~A TGGGGCTTTC GGGACAGTTG ACTTGGACAA GGTTACCACA 4140
GGGTTTCAAA AACAGCCCAA CGCTCTTTGA TGAGGCCTTA CACCGGGACT TGGCTGACTT 4200
CA 0224l9~2 l998-07-l4
W O 97/25860 PCTrUS97/00582
- 69 -
TCGAGTCCAG CATCCCACTC TTATACTTCT TCAGTTTGTT GATGACCTTC TTCTAGGGGC 4260
CACTTCTGAG ACAGCATGCC ACCAGGGAAC AGAATCCCTC TTGCAGACTT TGGGGCGATT 4320
GGGCTATCGA GCTTCTGCCA GAAAGGCTCA AATTTGCCAG ACCCAGGTTA CTTATTTAGG 4380
CTATCAACTA AGGGATGGAC AGCGATGGCT GACTCCGGCT AGGAAACAGA CCGTGGCCAA 4440
CATCCCAGCC CCAAGAAATG GCCGACAGCT ACGGGAATTC 4480
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~65 base pairs
(B) TYPE: nucleic acid
(C) sTRpNn~nN~s single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GCTGAGTAGT GCGCGAGCAA AATTTAAGCT ACAACAAGGC AAGGCTTGGC CGACAATTGC 60
ATGAAGAATC TGCTTAGGGT TAGGCGTTTT GCG~LG~llC GCGATGTACG GGCCAGATAT 120
ACGCGTATCT GAGGGGACTA GGGTGTGTTT AGGCGAAAAG CGGGGCTTCG GTTGTACGCG 180
GTTAGGAGTC CCCTCAGGAT ATAGTAGTTT CGCTTTTGCA TAGGGAAGGG GA~ATGTAGT 240
CTTATGCAAT ACTCTTGTAG TCTTGCAACA TGCTTATGTA ACGATGAGTT AGCAACATGC 300
CTTACAAGGA GAGAAAAAGC ACCGTGCATG CCGATTGGTG GAAGTAAGGT GGTACGATCG 360
TGCCTTATTA GGAAGGCAAC AGACGGGTCT GACATGGATT GGACGAACCA CCGAATTCCG 420
CATTGCAGAG ATATTGTATT TAAGTGCCTA GCTCGATACA ATAAACGCCA TTTGACCATT 480
CACCACATTG GTGTGCACCT GGGTTGATGG CCGGACCGTT GATTCCCTGA CGACTACGAG 540
CACCTGCATG AAGCAGAAGG CTTCA 565
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 1804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GGATCCTCAG GGGTAACACC TTTTGGAGGT GGGCATCTTC CTCATTCTCA GTGGTGCCAA 60
GTTCATATCC TGCTGGCTTA ACACGTGGTG TTACTATATT TGTGGCCTTA TATGATTATG 120
AAGCTAGAAC TACAGAAGAC CTTTCATTTA AGAAGGGTGA AAAATTTCAA ATAATTAACA 180
ATACAGAAGG AGACTGGTGG GAAGCAAGAT CAATCACTAC AGGAAAGAAT GGTTATATCC 240
TGAGCAGTTA TGTAGCGCCT GCAGATTCCA TTCAGGCAGA AGAATGGTAT TTTGGCAAAA 300
TGGGGAGAAA AGATGCTGAA AGATTACTTC TGAATCCTGG AAATTAATGA GGTATTTTCT 360
TAGGAAGAGA GAGTGAAATG GCTGGGTGCA GTGGCTCATG CCTGTAATCC CAGCACTTTG 420
CA 022419~2 1998-07-14
.
W 097/25860 PCTtUS97tO0582
- 70 -
GGAGGCCGAG TTGGGCGGAT CACCTGAGGT CAGGAGTTCG AGACTAGCCT GGCCAACATG 480
GTGAAACCCC ATCTCTACTA APlU~AG TACAAAATTA GCTGGACGTG GTGGTGAGTG 540
CCTGTAATCC CAGCTACTCA GGAGGCTGAG GCAGCAGAAT CACTTGAACC TGGGAGGCGG 600
AGGTTGCAGT GAGCTGAGAT CGCGCCACTG CACTCCAGCC TCGGCGACAA GAGCAAAAAC 660
TCCGTCTAAA AAACAAATAA GCAAACAGAA CAAAACA~AA CAAAAACGAG AGAGCGA~AC 720
TACTAAAGGT GCTTATTCCC TCTCTATTCG TGATTGGGAT GAGGTAAGGG GTGACAATGT 780
GAAACACCAC AAAATTAGGA AACTTGACAA TGGTAGATAC TATATCACAA CCAGAGAACA 840
ACTTGATACT CTGCAGAAAT TGGCAAAACA CTACACAGAA CATGCTGATG GTTTATGCCA 900
CAAGTTAACA ACTGTGTGTC CAACTGTGAA ACCTCAGATT CAAGGTCTAG CAAAAGATGC 960
TTGGGAAATC CCTTGATAAT CTTTGCGACT AGAGGTTAAA CTAGGACAAG GATGTTTTGG 1020
CAAAGTGTGG ATGGGAATAT GGAATGGAAC CACAAAAGTA GCAATCAAAA CACTAAAACC 1080
AGGTACAATG ATGCCAGAAG CTTTTCTTCA AGAAGCTCAG GTAATGAAAA AAATAAGACA 1140
TGGTAAACTT GTTCCACTAT ATG~"l'~l"l'~'l' TTCTGAAGAG CCAATTTACA TTGTCACTGA 1200
ATTGATGTCA AAAGGAAGCT TATTCAATTT CCTTAAGGAA GGAGATGGAA AGTATTTGAA 1260
GCTTCCACAA ATGGTTGATA TGCCTGCTCA GATTGCTGAT GGTATGGCAT ATATTAAAAG 1320
AATGAACTAT ATTCACCGAG ATCTCTGGGC TGCTAATATT CTTGTAGGAG AAAATCTTCT 1380
GTGCAAAATA GCAGATTTTG GTTTAGCAAG GTTAATTGAA GACAATGAAT ACACATCAAG 1440
ACAAGGTGCA GAATTTCCAA TCAAATGGAC AGCTCCTGAA GTTGCACTGT ATGGTGGGTT 1500
TACAATAAAG TCTGGTGTCT GCTCATTTGG AATTCTACAG ACAGAACTGG TAACAAAGGG 1560
CAGAGTGCCA TATCCAGGTA TGGTGAACCA TGAAATACTG GAACAGGTGG AGCGAGGATA 1620
CAGGATGCCT TGCCCTCAGG GCTGTCCAGA ATCCCTCCAT GAATTGATGA ATCTGTGTTG 1680
GAAGAAGGAC CCTGATGAAA GACCAACATT TGAATATGTT CAGTCCTTCT TGGGAGACTA 1740
CTTCACTGCT ACAGAGCCAT AGTACCAGCC AGGAGAAAAC TTCTAATTCA AGTAGCCTAT 1800
TTTA 1804
