Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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WO 94/06921 2 1 ~ 2 ~ 2 ~ Pcr/US93/09070
Description
RECOMBINANT RETROVIRAL VECTOR AGAINST FELV ANO/OR FIV.
Technical Field
The present invention relates generally to methods for treating
felines, and more specifically, to methods and compositions for treating feline
leukemia virus and feline immunodeficiency virus infections, as well as vaccinesfor preventing these infections.
Bachgl ound of the Invention
Feline lenkeTni~ virus ("FeLV") and feline immlmodeficiency virus
("FIV") are the two most common pathogenic retroviruses reported in feline
populations. In a major U.S. study of symptomatic and high-risk domestic cats, it
was found that 13~o were positive for FeLV antigens, 75'o were positive for FIV
antibodies, and only 2% were positive for both viruses (see O'Connor et al.,
JAVA~4 199:1348-1359, 1991). Although most studies indicate that FeLV and FIV
are acquired independently of each other, it has been reported that FeLV irlfected
cats are 1.5 to 4 times more susceptible to FIV infection than are FeLV-negativecats (see Cohen et al., JAVMA 197:220:225, 1990; Moraillon, Vet Rec. 126:68-69,
1990), and that dually infected cats have a more severe disease course than do cats
infected with either virus alone (see Hosie et al., Vet. Rec. 125:293-297, 1989).
Responses to FeLV infection can generally be classified into three
groups: acute infection, chronic viremia, and immnnity. The outcome for any
specific animal depends on a variety of viral, host, and environmental factors. In
acute infection, FeLV first replicates in the lymphocytes and macrophages of thetonsils, and within 2 to 12 days is carried throughout the cat to the bone marrow,
thymus, spleen, intestine, and lymph nodes. If the cat does not mount an adequate
immllne response, it will develop chronic viremia within 4 to 6 weel~s after initial
exposure.
Chronic viremia is confirmed when infective FeLV can be recovered
from the blood by use of a viral infectivity (VI) assay, by immlmofluorescent
antibody (IFA) detection of p27 in circlll~ting neutrophils and platelets, or bydetection of soluble p27 antigens through an enzyme-linked immlmosorbent assay
(ELISA) (see Fischinger et al., J. ~rol. 14:177-179, 1974; Hardy et al., "Detection
WO 94/06921 2 1 4 2 3 2 5 PCr/USs3/0907~
of the Feline Leukemia Virus and Other ~mm~ n Oncornaviruses by
Tmmlmofluorescence", Unifying Concepts of Leukemia, Dutcher, and Chieco-
Bianchi, (eds.) Karger (pub.), Basal, Switzerland: 778-799, 1973). In this stage, the
cat is highly contagious, and excretes large quantities of virus in its saliva and
S urine. It is also predisposed to death via a FeLV-related disease within months if
it is a kitten, or within 2 to 3 years if it is older. Examples of FeLV-related
~ e~cç~ include lyrnphomas, non-lymphoid leukemias, myeloproliferative
disorders, fibrosarcomas, cytosuppresive ~ e~ce~, and myelosuppresion, anemia
and leukopenia syndromes (see Hoover, JAVll~A 199:1287-97, 1991).
If the cat mounts an effective immnne response, it will curtail viral
replication and expression within 4 to 8 weeks after initial exposure. However,
many cats do not completely elimin~te the virus, and harbor a low-grade, latent,nonexpressed, FeLV infection of the bone marrow and lymph nodes for weeks to
years. If such a cat is severely stressed, the latent virus may subsequently be
reactivated, le~-ling to FeLV-related ~ e~es, and death of the cat.
A cat diagnosed with FeLV infection will generally die within 24 to
36 months. To prevent disease tr~n~mi~sir n, it has been strongly suggested thatFeLV-infected cats be isolated from other cats, regardless of whether or not theother cats have been v~crin~ted for FeLV.
In order to prevent FeLV, a number of v~ccines have been
developed. More specifically, eight FeLV v~ccines have been licensed by the
USDA, all of which are based upon an inactivated virus or purified subunit. Moststudies suggest that present commercial FeLV vaccines provide only a 60 to 90%
efflcacy in preventing cats from developing persistent infection (see Pollock et al.,
JA~A 799:1406-1409, 1991). All of these vaccines require a second dose and
annual boosts to m~int~in antibody persistance. In addition, one difficulty withpresent vaccines is, that if the diagnostic test which was ntili7ed to check forprevious infection was inaccurate, not available, or too expensive to be lltili7e~l,
v~cl in~tion of an infected animal may take place. Conventional v~ccinç~ are notexpected to be therapeutic, and therefore, infected ~nim~l~ which are
subsequently v~crin~ted may nevertheless develop FeLV. This has led to the
perception by some feline owners that v~ccines are either ineffective, or may
actually even lead to infection, and therefore are unnecessary.
Clinical management of feline leukemia virus infections has
generally been limited to treatment of FeLV-related diseases. In particular, a
number of researchers have experimented with therapeutic regimes, inrhl-ling forexample, ~lminictration of BCG, levamisole, mixed bacterial toxins (see Cotter,
2 ~ 2 ~
Wo 94/06921 Pcr/US93/09070
_~ 3
Proceedings, 7th Am. ColL Vet. Int. Med. For., 909-912, 1989, high doses of human
alpha interferon, and 3'-azido-3'-deoxythymidine ("AZT') (see Zeidner et al.,
An~imicrob. Ager~ Chemo. 34:1749-1756, 1990). These treatments, however, have
met ~vith limited success. Moreover, some of these therapeutics, such as AZT, are
5 additionally accompanied by undesirable side-effects, including for example AZT-
related hepatotoxicosis, and inability to elimin~te viremia after incorporation of
the virus into hematopoietic cells. Thus, there exists a need for effecatious
methods and compositions for both treating and preventing FeLV infections,
without undesirable side effects.
Similar to feline leukernia virus, feline immnnodeficiency virus is
found worldwide in both pet and stray cats (see Hoise et al., Vet. ReG 125:293-297,
1989; Ishida et al., Jpn. J. Vet. Sc~ 50:39-44, 1988; Pederson et al., "Prevalence of
infection with feline immllnodeficiency virus, feline lel~kemi~ virus and
toxoplasma gondii in feral cat population (Abstract), First Intern~ion~l
15 Conference of Feline Tmmlmodeficiency Virus Researchers, U111Ve1~ilY of
California, Davis, Calif., Sep. 4-7, 1991). The virus is believed to be l~ ille~l
primarily through infected saliva during fighting, (see Wasmien et al.,
"Tr~ncmicsion of feline immnnodeficiency virus from infected queens to kittens
(Abstract), First International Conference, supra), and therefore, cats which are
20 most~at risk are stray males over the age of 3 (see Y~m~moto et al., JAVMA
194:213-220, 1989). The incidence of feline immnnodeficiency virus in these high-
risk ~nim~lc ranges from 6 - 14~o in the United States (see August, JAVMA
199:1472-1477, 1991; Macy et al., "The clinical fin~lingc and prevalence of FIV and
FeLV in Colorado cats (Abstract), First International Conference, supra;
25 O'Connor et al., JA~A 199: 11348-1359, 1991), 18% in Denmark (see Peterson etal. supra), and 445~o in Japan (see Ishida et al, Jp~ J. Vet. Sci 50:3944, 1988). In
addition, vertical tr~ncmiccion from infected queens to kittens has been observed
(see ~ h~n et al, "Natural tr~ncmiccion of FIV in kittens. (Abstract), First
International Conference, supra; Wasmoen et al, supra), although the
30 epidemiological sigruficance of this route of infection has yet to be determined. In
the general population of low-risk, in-door cats, feline immnnodeficiency virus
infection is estimated to be 1 - 3 ~o (see Peterson et al., supra). Unlike the human
immunodeficiency virus ("HIV"), feline immlmndeficiency virus infection does notseem to be spread by sexual contact (see Gardner and Luciw, FASEB J. 3:2593-
35 2606, 1989).
Feline imununodeficiency virus induces an immnnodeficiencydisease in its host by the gradual and persistent depletion of CD4+ lymphocytes
W O 94/06921 214 2 3 2 5 PC~r/US93/0907 ~
with no apparent changes in the levels of CD8+, CrL, and T suppressor cells (seeAckley et al., J. ~rol. 64:5652-5655, 1990). This leads to an inversion of the
CD4+/CD8+ cell ratio which may be measured in order to determine the
irnmunological status of a feline.
The course of disease caused by feline immunodeficiency virus is
very sirnilar to that caused by HIV. More specifically, in the initial stage of
infection, generalized lymphoadenopathy, fever m~ e, and leukopenia may be
exhibited (see Jarret et al., AIDS 4:S163-S165, 1990). This is followed by a
relatively asymptomatic latent stage which does not present clinical signs. The
terminal stage, which can take months or years to appear, is usually characterized
by a number of chronic infections of a secondary or opportunistic nature.
Coinfection v~rith other viral or parasitic agents drastically shortens the second
stage latency period, and accelerates the progression of the terminal stage (seePedersen et al., Science 235:790-793, 1987). (~linic~l manifestations of this stage
inl~lntle oral cavity infections, chronic upper respiratory infecti~ns, chronic
enteritic, chronic conjunctivitis, and neurological abnorm~lities Feline
immlmodeficiency virus positive cats also present a high incidence of neoplasms
in~ tlin~ lymphoma, squamous cell carcinoma, and myelodysplastic disease (see
Hutson et al.,M~4 199:1357-1362, 1991).
Three types of tests are culle"Lly available to determine if cats are
infected with feline immllnodeficiency virus. They are the en_yrne-linked
immnnosorbent assay (ELISA), the immnnofluorescent antibody (IFA) test, and
the immlmoblot test, the latter of which is considered to be the standard for
methodology comparisons. Generally, as compared to an immlmoblot test, an
ELISA or IFA can produce results with a sensitivity of 93-95~o, and a specificity of
985'o (see Barr et al., JA~ 199:1377-1381, 1991).
After cats have been ~ gnosed with the feline immlmndeficiency
virus, the average survival period is about 24 months. Clinical management of
these ~nim~lc is minim~l, but inclllclss isolating them from other cats to help
prevent them from developing other life-shortening opportunistic infections (seeAugust et al., supra) as well as to prevent the spread of feline immlmQdeficiency
virus to non-infected cats.
Presently, for research purposes, the anti-retroviral human AIDS
drugs AZT and 9-(2-phosphonomethoxyethyl)-adenine (PMEA) have been
lltili7erl in cats with feline immllnodeficiency virus. These drugs i~ ove the
clinical condition of infected cats by increasing their CD4+/CD8+ ratio.
However, both cause decreased hematocrit and hemoglobin levels which
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Wo 94/06921 PCI /US93/09070
precludes their use as long term therapies (see Hartmann et al., "Use of two
virustatica (AZT, PMEA) in the treatment of FIV- and FeLV-seropositive cats
with clinical syrnptoms," (Abstract), First International Conference, supra). Inaddition, the discovery of ~ZT-resistant mutants of the feline immnnQdeficiency
S virus (see North et al., "Drug resistant mutants of feline imm11nodeficiency virus
isolated in vitro, (Abstract), First International Conference, supra) may further
restrict the utility of AZT in feline immllnodeficiency virus therapy.
The present invention provides compositions and methods for
treating FeLV and FIV, v~crines for preventing FeLV and FIV, and additionally
10 provides other related advantages.
Sumrnary of the Invention
As noted above, the present invention provides methods for
preventing or treating feline viral infections. Briefly, within one aspect of the
15 present invention methods of treating or preventing feline leul~emia virus
infections are provided, comprising, ~clministering to a feline a vector construct
which directs the expression of at least one imm11nogenic portion of a feline
lenk~mi~ virus antigen, such that a cellular immnne response is generated. Within
one embodiment of the invention, vector constructs are provided which direct the20 expression of an antigen selected from the group consisting of pl5gag, pl2gag,
p27gag, plOgag, pl4pol, p80pol, p46pol, gp70env, and plSenv. Within a particularly
preferred embotlimerlt vector constructs are provided which direct the expression
of gp85env.
Within another aspect of the present invention, methods of treating
25 or preventing feline imm1mode~lciency virus infections are provided, comprising,
lminictering to a feline a vector construct which directs the expression of at least
one imm11nogenic portion of a feline immnncldeficiency virus antigen, such that a
ce11n1~r immune response is generated. Within one embodiment, vector
constructs are provided which direct the expression of an antigen selected from
30 the group consisting of plSgag, p24gag, plOgag, pl3pol, p62pol, plSpol and p36pol.
Within a particularly preferred embodiment, vector constructs are provided with
direct the expression of gp6&nv, gp27env and rev.
Within another aspect of the present invention, methods of treating
or preventing feline leukemia virus and feline immnnodeficiency virus infections35 are provided, co~ hlg, ~lminictering to a feline a vector construct which directs
the co-expression of at least one imm1mogenic portion of a feline leukemia virus
WO 94/06921 2 1 4 ~ 3 ~ 5 PCI/US93/090~
antigen, and at least one immnnogenic portion of a feline immunodeficiency virusantigen, such that a cellular immnne response is to said viruses is generated.
Also provided within the present invention are vector constructs
which direct the expression of at least one immnnogenic portion of a feline
S lenkemi~ virus, vector constructs which direct the expression of at least one
immllnogenic portion of a feline immnnodeficiency virus, and vector constructs
which direct the co-e~res~ion of at least one immnnogenic portion of a feline
immnnodeficiency virus, and at least one immunogenic portion of a feline
leukemia virus.
Within various embodiments, the above-described vector constructs
are carried by recombinant retroviruses, or by a recombinant virus selected fromthe group con.~i~ting of poliovirus, rhinovirus, pox virus (e.g., the canary pox virus
or the vaccinia virus), inflllen7~ virus, adenovirus, parvovirus (e.g., the adeno-
associated virus B19 or MVN), herpes virus, SV40, HIV, measles, and alpha
15 viruses such as the Sindbis virus. Also provided are target cells infec~ed with the
above-described viruses.
Within yet another embodiment of the invention, pharmaceutical
compositions are provided, comprising the above described recombinant retroviralor recombinant viral constructs, in combination with a pharmaceutically
20 acce~table carrier or diluent.
These and other aspects of the present invention will become
evident upon reference to the following detailed description and ~tt~ched dra~ving.
In addition, all referellces which have been cited below are hereby incorporatedby reference in their entirety.
Briçf Description of the Drav~rings
Figure 1 is a schematic illustration of the construction of a
multivalent FIV - FeLV, retroviral vector.
30 Detailed Description of the Invention
Prior to setting forth the invention, it may be helpful to an
underst~nrlin~ thereof to first set forth definitions of certain terms that will be
used hereinafter.
"Immuno~enic portion" as utilized within the present invention,
refers to a portion of the respective antigen which is capable, under the
a~,o~liate conditions, of c~n~ing a cellular (~e., cell-mediated or humoral)
~ WO94/06921 ~ 232~ PCI/US93/09070
immnne response. "Portions" may be of variable size, but generally should be at
least 9 amino acids long, and may include the entire antigen. Representative
assays which may be utilized to determine immlmogenicity (e.g., cell-mediated
imm11ne response), are described in more detail below, as well as in Example 10~5 Cellular immnne responses may be mediated through Major Histocompatibility
("MHC") Class I presentation, MHC Class II presentation, or both.
"Vector construct" refers to an assembly which is capable of
directing the expression of the sequence(s) or gene(s) of interest. The vector
construct must include promoter element(s), as well as at least one sequence
10 which, when transcribed, is operably linked to the sequence(s) or gene(s) of
interest and acts as a tr~n~1~tion initiation sequence. Optionally, the vector
construct may also include a selectable marker such as Neo, SV2 Neo, TK,
hygromycin, phleomycin, histidinol, or DHFR, a signal which directs
polyadenylation, a translation termin~tion sequence, and one or more restriction15 sites. In ~AAition~ if the vector construct is placed into a retrovirus~ the vector
construct must include a p~k~ging signal and long terminal repeats (LTRs)
a~lu~liate to the retrovirus used (if these are not already present).
As noted above, the present invention is generally directed towards
compositions and methods for treating, as well as v~crines for preventing, various
20 feline, diseases, including for example feline leukemia virus and feline
immlln( deficiency virus infections. Briefly, the ability to recognize and defend
against foreign pathogens is central to the function of the imml1ne system. Thissystem, through imm11ne recognition, is capable of distinguishing "self' from
"nonself" (foreign), and is essential to ensure that defensive meçh~ni~ms are
25 directed towards invading entities rather than against host tissues. The
~mtl~mental features of the immnne system are highly polymorphic cell surface
recognition structures (receptors), and effector me~h~nicmc (antibodies and
cytolytic cells), which act to destroy invading pathogens.
One cell type of particular importance in immnne recogIution is the
30 Cytotoxic T Lymphocyte ("ClL"), which is primarily restricted in recognition to
antigens which have been processed in association with MHC class I products.
Briefly, CTLs are normally in-l~lcefl by the display of processed pathogen-specific
peptides in conjunction with MHC molecules along with molecules such as CD3,
ICAM-1, ICAM-2, LFA-1, LFA-3, ~-microglobulin, chaperones, and analogs
35 thereof (e.g., Altmann etal., Nature 338:512, 1989). Other genes coding for
proteins that enhance the stim1-l~tion or recognition of cell mediated responsesmay also be used in this context. Antigenic peptide presentation in association
WO 94/06921 2 1 ~ 2 3 2 5 PCr/US93/0907a~
with MHC Class I molecules leads to CD8+ CIL production. Peptides presented
in association with MHC Class II molecules leads to production of antibodies,
helper cells and B-cell memory, and may induce CD4+ CI Ls. The methods which
are described in greater detail below provide an effective means of inducing
S potent class I-restricted protective and therapeutic Cl'L responses, as well as
humoral responses.
As noted above, within one aspect of the present invention methods
for treating or preventing feline leukemia virus infections are provided,
comprising ~lminictering to a feline a vector construct which directs the
10 expression of at least one immnnogenic portion of a feline leukemia virus antigen,
such that a cellnl~r immlme response is generated.
Briefly, feline leuke~nia virus (FeLV) is a retrovirus of the
on~:o~ virus subfamily. FeLV is presently believed to exist in three subgroups
- A, B or C - which are differentiated by their envelope antigens gp70 and plSE.15 FeLV is also comprised of a number of core antigens, inrlll~in~ pl5, p~2, p27, and
plO, which are highly conserved for all subgroups of FeLV (see Geering et al., ~r.
36:678-680, 1968; Hardy et al., JA~lqA 158:1060-1069, 1971; Hardy et al., Science
166:1019-1021, 1969). As noted above, within one embodiment of the invention,
vector constructs are provided which direct the expression of at least one portion
20 of a f,eline leukemia virus antigen selected from the group consisting of plSgag,
pl2gag, p27gag, plOgag, pl4pol, p80pol, p46pol, gp70env, and pl5env. Within a
particularly preferred embodiment, vector constructs are provided which direct
the expression of gp85env. Sequences which encode these antigens may be readily
obtained given the disclosure provided herein (see Donahue et al., J. rr.
25 62(3):722-731, 1988; Stewart et al.,J. Yr. 58(3):825-834, 1986; Kumar et al.,J. ~r.
63(5):2379-2384, 1989; Elder et al., J. ~r. 46(3):871-880, 1983; Berry et al., J. ~r.
62(10):3631-3641, 1988; Laprevotte et al.,J. ~r. 50(3):884-894, 1984).
Within another aspect of the present invention, a method for
treating or preventing feline immnnodeficiency virus infections is provided,
30 comprising ~tlminictering to a feline a vector construct which directs the
expression of at least one immllnQgenic portion of a feline immlmodeficiency virus
antigen, such that a cellular immlme response is generated.
Briefly, feline immunodeficiency virus (FIV) has been classified as a
retrovirus of the lentivirus subfamily, based upon the m~necium requirement for
35 reverse transcriptase (RT) and the morphology of viral particles (see Pederesen et
al., Science 235:790-793, 1987). The feline immnnQdeficiency virus is
morphologically and antigenically distinct from other feline retroviruses, including
~4~
~ WO 94/06921 Pcr/us93/o9o7o
feline leukemia virus, type C oncorna virus (RD-114), and feline syncytium-
forrning virus (FeSFV) (see Y~m~moto et al., "Efficacy of experimental FIV
vaccines, (Abstract), First International Conference of Feline TmmllnodeficiencyVirus Researchers, University of California, Davis, CA, Sep. 4-7, 1991). As noted
5 above, within one embodiment of the invention, vector constructs are provided
which direct the expression of at least one immllnogenic portion of an feline
immllnodeficiency virus antigen selected from the group consisting of plSgag,
p24gag, plOgag, pl3pol, p62pol, plSpol and p36pol. Within a particularly preferred
embo(liment vector constructs are provided which direct the expression of
10 gp6&nv, gp27env and rev. Within the context of the present invention, "rev" is
understood to refer to the antigen corresponding to the rev open reading frame
(see, Phillips et al., First International Conference, supra). Sequences which
encode these antigens may be readily obtained by one of skill in the art given the
~1icclosllre provided herein (see Phillips et al., J. ~r. 64(10):4605-4613, 1990;
15 Olrnsted et al., PNAS 86:2448-2452, 1989; Talbott et al., PNAS 8~5743-5747,
1989).
Sequences which encode the above-described feline leukemia virus
and feline immllnodeficiency virus antigens may be prepared as described within
the references cited above, or obtained from a variety of sources. For example,
20 seque,nces which encode the envelope protein of FeLV may be readily obtained
from the American Type Culture Collection ("ATCC"; Rockville, Maryland) (see
for example, ATCC Nos. 39528, 39529, and 39530). Similarly, the AnDS
Repository (Division of AIDS, National Institute of Allergy and Infections
Disease, Bethesda, Maryland; see NIH Publication No. N2-1536) holds a deposit
25 of a plasmid clone which contains a sequence encoding a full-length, replication
competent FeLV (e.g., clone p61E-FeLV, Catalog No. 109), as well as a deposit ofa plasmid clone which cont~in~ a sequence encoding a feline imml-nodeficiency
virus (eg., clone pFIV-14-Pet~ m~, Catalog No. 851).
Alternatively, sequences which encode the above-described feline
30 viral antigens may be readily obtained from cells which express or contain
sequences which encode these viruses (e.g., from cats which have been infected
with FeLV or FIV). Briefly, within one embodiment, primers are prepared on
either side of the desired sequence, which is subsequently amplified by polymerase
chain reaction ("PCR") (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159)35 (see also, PCR Technology: Principles and Applications for DNA Amplification,Erlich (ed.), Stockton Press, 1989). In particular, a double stranded DNA is
denatured by heating in the presence of heat stable Taq polymerase, sequence
Wo 94/06921 2 1 4 ~ 3 2 ~ lo Pcr/US93/O9O~
specific DNA primers, ATP, CI P, GTP and TTP. Double-stranded DNA is
produced when synthesis is complete. This cycle may be repeated many times,
resulting in a factorial amplification of the desired DNA.
Sequences which encode the above-described feline viral antigens
5 may also be synthesized, for example, on an Applied Biosystems Inc. DNA
synth~si7~r (e.g, ABI DNA synthesizer model 392 (Foster City, California)). Suchsequences may also be linked together through complementary ends, followed by
PCR amplification, in order to forrn long double-stranded DNA molecules.
As noted above, at least one immnnogenic portion of a feline viral
10 antigen, inclll~ling for example, a feline leukemia virus antigen, a feline
immnnodeficiency virus antigen, any of the feline viral antigens which are
described in greater detail below, or any combination of these antigens, is
incorporated into a vector construct. The immllnogenic portion(s) which are
incorporated into the vector construct may be of varying length, although it is
15 generally preferred that the portions be at least 9 amino acids ~ long, and
yreferably, inrllltle the entire antigen. TmmlmQgenicity of a particular sequence is
often difficult to predict, although T cell epitopes may be predicted utilizing
coll~uLer algoliLhllls such as TSites (Me-lTmmlme~ Maryland), in order to scan
coding regions of FeLV gag, env, FIV gag, env and rev for potential T-helper sites
20 and C~TL sites. This analysis is primarily based upon 1) structural properties of the
proteins (principally alpha-helical periodicity and amphipathicity), and 2) motifs
found in sequences recognized by MHC Class I and Class II molecules. In general
however, it is preferable to determine irnmunogenicity in an assay.
Representative assays include an ELISA which detects the presence of antibodies
25 against a newly introduced vector, as well as assays which test for T helper cells,
such as g~mm~-interferon assays, IL-2 production assays, and proliferation assays.
A particularly preferred assay is described in more detail below in Example 10A.Tmmnnogenic proteins of the present invention may also be
manipulated by a variety of methods known in the art, in order to render them
30 more immnnogenic Representative examples of such methods include: adding
arnino acid sequences that correspond to T helper epitopes; promoting cellular
uptake by adding hydrophobic residues; by forrning particulate structures; or any
combination of these (see generally, Hart, op. cit., Milich et al., Proc. Natl. Acad.
Sci USA 85:1610-1614, 1988; Willis, Nature 340:323-324, 1989; Griffiths etal.,
35 J. rrO~ 65:450-456, 1991).
Particularly preferred immlmogenic portions for incorporation into
a vector construct include, for feline leukernia virus the gp85env antigen, and for
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WO 94/06921 ~ Pcr/US93/09070
11 '
feline immnnndeficiency virus the gp68env, gp27env, and rev antigens. Within a
particularly preferred embodiment of the invention, a vector construct is provided
which expresses both FeLV and FIV envelope antigens (gp85env; and gp68env,
gp27env, and rev, respectively) or both FeLV and FIV gag antigens (plSgag,
5 pl2gag, p27gag, plOgag, and pl4pol; and plSgag, p24gag, plOgag, and rev,
respectively).
In addition, as noted above more than one immlmogenic portion
may be incorporated into a vector construct. More specifically, within one
embodiment of the invention, multivalent vector constructs are provided which
10 may be lltili7ed for more than one rli~e~ce For example, a vector construct may
express (either separately or as one construct) all or portions of a feline leukemia
virus antigen, a feline immnnodeficiency virus antigen, as well as antigens which
are associated with other feline diseases. Representative examples of such
antigens inrlllcle VP1 and VP2 for feline panleukopenia virus (see Martyn et al.,
15 "Nucleotide sequence of feline panleukopenia virus: comparison with canine
parvovirus identiffes host-specific differences", J. Ge~ ~r. 71:2747-2753, 1990;Parrish, "Mapping Specific Functions in the Capsid Structure of Canine Parvovirus
- and Feline Panleukopenia Virus Using Infectious Plasmid Clones", ~r. 183:195-
205, 1991); the capsid protein of Feline Calicivirus (see Neill et al., "Nucleotide
20 Sequence and Expression of the Capsid Protein Gene of Feline Calicivirus", J. ~r.
65(10):5440-5447, 1991; Tohya et al., "Sequence Analysis of the 3' end of FelineCalicivirus Genome", ~r. 183:810-814, 1991; Carter et al., "Monoclonal antibodies
to Feline Calicivirus", J. Ge~ ~r. 70:2197-2200, 1989); the "N" or nucleoprotein of
the Rabies virus (see Ertl et al., "Induction of Rabies Virus-Specific T-Helper
25 Cells by Synthetic Peptides that carry domin~nt T-Helper Cell Epitopes of theViral Ribonucleoprotein", J. ~r. 63(7):2885-2892, 1989); and the surface
glycoprotein of Feline Herpesvirus (see Nunberg et al., "Identification of the
Thymidine Kinase Gene of Feline Herpesvirus: Use of Degenerate
Oligonucleotides in the Polymerase Chain Reaction to Isolate Herpesvirus Gene
30 Homologs",J. ~r. 63(8):3240-3249, 1989).
Within a further embodiment of the present invention, one or more
of the above-described immnnogenic portions may be co-expressed with an
immlmomodulatory cofactor. Briefly, as utilized within the context of the present
invention, an "i~ nmodulatory cofactor" refers to factors which, when
35 m~mlf~ctllred by one or more of the cells involved in an immune response, or,which when added exogenously to the cells, causes the immnne response to be
different in quality or potency from that which would have occurred in the absence
wO 94/06921 ' Pcr/US93/0907
of the cofactor. The quality or potency of a response may be measured by a
variety of assays known to one of skill in the art, including for example, in vitro
assays which measure cellular proliferation (e.g, 3H thymidine uptake), and in
vitro cytotoxic assays (e.g., which measure 51Cr release) (see Warner et al., AIDS
S Res. and Human Retrovin~ses 7:645-655, 1991). Tmmllnomodulatory cofactors may
be active both in vivo and ex vivo. Representative examples of such cofactors
in~ e alpha interferons (Finter et al., Drugs 42(5):749-765, 1991; U.S. Patent No.
4,892,743; U.S. Patent No. 4,966,843; WO 85/02862; Nagata et al., Nature 284:316-
320, 1980; Familletti et al., Methods in En~. 78:387-394, 1981; Twu et al., Proc.
Nat~ Acad. Sci USA 86:2046-2050, 1989; Faktor et al., Oncogene 5:867-872, 1990),beta interferons (Seif etal., J. ~rology 65:664-671, 1991), g~mm~ interferons
(Radford et al., The American Society of Hepatolog)t 20082015, 1991; Watanabe
et al., PNAS 86:9456-9460, 1989; Gansbacher et al., Cancer Research 50:7820-7825,
1990; Maio etal., Car~ ImmunoL Immunother. 30:34-42, 1989; U.S. Patent No.
4,762,791; U.S. Patent No. 4,727,138), GCSF (U.S. Patent Nos. 4,Q99,291 and
4,810,643), GMCSF (WO 85/04188), TNFs (Jayaraman etal., J. Immunology
144:942-951, 1990), Interleukin 2 ("IL-2") (Karupiah et al., J. Immunology 144:290-
298, 1990; Weber et al., J. ~xp. Me~ 166:1716-1733, 1987; Gansbacher et al., J.
Exp. Med. 172:1217-1224, 1990; U.S. Patent No. 4,738,927), Interleukin-4 ("IL-4")
(Tep~?er et al., Cell 57:503-512, 1989; Golumbek et al., Science 254:713-716, 1991;
U.S. Patent No. 5,017,691), Interleukin-6 ("IL-6") (Brakenhof et al., J. Immuno~139:4116-4121, 1987; WO 90/06370), ICAM-1 (Altman et al., Nature 338:512-514,
1989), ICAM-2, LFA-1, LFA-3, MHC class I molecules, MHC class II molecules,
~-microglobulin, chaperones, CD3, or analogs thereo
Sequences which encode the above-described immlmomodulatory
cofactors may be readily obtained from a variety of sources, inçlll~lin~ for example,
the American Type Culture Collection (ATCC, Rockville, Maryland), or from
cornrnercial sources such as British Bio-technology Lirnited (Cowley, Oxford
F.ngl~n~l). Representative examples include BBG 12 (cont~ining the GM-CSF
gene coding for the mature protein of 127 a~nino acids), BBG 6 (which contains
sequences encoding g~mm~ interferon), ATCC No. 39656 (which contains
sequences encoding TNF), ATCC No. 20663 (which contains sequences encoding
alpha interferon), ATCC Nos. 31902, 31902 and 39517 (which contains sequences
encoding beta interferon), ATCC Nos. 39405, 39452, 39516, 39626 and 39673
(which contains sequences encoding Interleukin-2), ATCC No. 57592 (which
cont~inc sequences encoding Interleukin-4), and ATCC 67153 (which contains
sequences encoding Interleukin-6).
21~232~
W O 94/06921 - PC~r/US93/09070
13
The choice of which immnnomodulatory cofactor to include within a
vector construct may be based upon known therapeutic effects of the cofactor, ordetermined experimentally. For example, blood samples may be taken from a
feline with a particular ~lice~ce, for use in CTL assays. Briefly, peripheral blood
5 lymphocytes (PBLs) are separated from the blood, and stiml~l~ted in vi~ro withconcanavalin A, Interleukin-2, bovine T-cell growth factor, and autologous
irradiated cells, followed by tr~ncduction with the above-described recombinant
ret,oviluses which direct the expression of an immllnogenic portion of an antigen
which is associated with the above described diseases, and an imml-nomodulatory
10 cofactor. Stimlll~te~l PBLs are used as effectors in a CTL assay with the
autologous transduced cells as both restimlll~tors and targets. An increase in CTI
response over that seen in the same assay performed using autologous stim~ tor
and target cells tr~nc-lllred with a vector encoding the antigen alone, indicates a
useful immlmomodulatory cofactor.
Once the immllnngenic portion(s) discussed above ~ have been
selected, genes which encode these proteins are placed into a vector co,~sL,uct
which directs their expression. In general, such retroviral vectors encode only
these genes, and no selectable marker. Vectors encoding and le~rling to
expression of a specific antigen(s) may be readily constructed by those skilled in
20 the art. In particular, construction of vector col~llucts as well as ~lmini~tration of
retroviral constructs by direct injection is described in greater detail in an
application entitled "Recombinant Retroviruses" (U.S.S.N. 07/586,603, filed
September 21, 1990), which is herein incorporated by reference in its entirety.
These vector constructs may be used to generate transduction competent
25 retroviral vector particles by introducing them into a~rop,iate p~k~gjng cell lines (see U.S.S.N. 07/800,921).
Within a particularly preferred embodiment, vector constructs may
be col~llùcted to inrl~l(le a promoter such as SV40 (see Kriegler et al., Cell 38:483,
1984), cytomegalovirus ("CMV") (see Boshart et al., Cell 41:521-530, 1991), or an
30 Internal Ribosomal Binding Site ("IRBS"). Briefly, with respect to IRBS, the five
prime untr~ncl~ted region of the immlmoglobulin heavy chain binding protein has
been shown to support the internal engagement of a bicistronic m~sc~ge (see
Jacejak and Sarnow, Na~ure 353:90-94, 1991). This sequence is small (300 bp),
and may readily be incorporated into a retoviral vector in order to express
35 multiple genes from a multi-cistronic message whose cistrons begin with this
sequence. A representative vector construct lltili7ing IRBS is set forth in moredetail below in Example 4.
WO94/06921 2~2~2~ ;- Pcr/uss3/oso7a
In addition, vector constructs may also be developed and utilized
with other viral carriers incl-llling, for example, poliovirus (Evans et al., Nature
339:385-388, 1989, and Sabir~ J. of Biol. Standardization 1:115-118, 1973);
rhinovirus (Arnold, J. Cen Biochem. L401-405, 1990); pox viruses, such a canary
5 pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner
et al.,An~L N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990;
U.S. Patent Nos. 4,603,112 and 4,769,330; WO 89/01973); SV40 (~Illlig~n et al.,
Nature 277:108-114, 1979); influenza virus (Luytjes et al., Cell 59:1107-1113, 1989;
McMicheal et al., The New England Joumal of Medicine 309:13-17, 1983; and Yap
10 et al., Nature 273:238-239, 1978); adenovirus (Berkner, Biotechniques 6:616-627,
1988, and Rosenfeld et al., Science 252:431-434, 1991); parvovirus such as adeno-
associated virus (S~mlllcki etal., Joumal of ~rology 63:3822-3828, 1989, and
Mendelson et al., ~rology 166:154-165, 1988); herpes (Kit, Adv. Exp. Med. Bio~
215:219-236, 1989); SV40; HIV; measles (EP 0 440,219); and Sindbis virus (Xiong
15 et al., Science 234:1188-1191, 1989). Furthermore, viral carrie~ may be
homologous, non-pathogenic (defective), replication competent virus (e.g.,
Overbaugh et al., Science 239:906-910, 1988), and yet induce cellular immllne
responses, in~ lin~CTL.
Once the above-described vector constructs have been constructed,
20 they~may be tested for tumorigenicity prior to ~rlminictration to felines, for
example, by determining the extent of tumor formation in nude mice, or by
ev~ tin~ colony formation in soft agar. Briefly, tumor formation in nude mice isa particularly important and sensitive method for determining tumorigenicity.
Nude mice lack a functional cellular immllne system, do not possess mature T-
25 cells, and therefore provide a useful in vivo model in which to test the tumorigenicpotential of cells. Normal non-tumorigenic cells do not display uncontrolled
~rowth properties if injected into nude mice. However, transformed cells will
rapidly proliferate and generate tumors in nude mice. In one embodiment the
vector construct is ~rlminictered by injection into nude mice. The m-ice are visually
30 eY~mined for a period of 4 to 16 weeks after injection in order to determine tumor
growth. The mice may also be sacrificed and autopsied in order to determine
whether tumors are present. (Giovanella et al.,J. NatL CancerInst. 48:1531-1533,1972; Furesz et al., "Tumorigenicity testing of cell lines considered for production
of biological drugs," Abnormal Cells, New Prod~cts and Risk, Hopps and
35 Petricciani (eds.), Tissue Culture Association, 1985; and Levenbook et al., J. Biol.
Std. 13:135-141, 1985).
~ wo 94/06921 ~ 1 4 2 3 2 ~ PCr/US93/09070
Tumorigenicity may also be assessed by vicn~li7in~ colony formation
in soft agar (MacPherson and Montagnier, ~r. 23:291-294, 1964). Briefly, one
property of normal non-tumorigenic cells is anchorage-dependent growth. More
specifically, normal non-tumorigenic cells will stop proliferating when they areS plated in a semi-solid agar medium, whereas tumorigenic cells v~rill continue to
proliferate and form colonies in soft agar.
Once a vector construct has been prepared, it may also be
~rlminictered to a feline in order to treat the above-described feline ~lice~cecSimilarly, the vector construct may be ~lminictered prophylactically, in order to
~revel~L the above-described feline diseases. Methods for ~flminictering a vector
construct via a retroviral vector (such as by direct injection of the retroviralconstruct) are described in greater detail in an application entitled "Recombinant
Retloviluses" (U.S.S.N. 07/586,603).
Within another aspect of the present invention, methods are
provided for treating or preventing a feline ~lice~ce, comprising the ~teps of (a)
removing cells from a feline, (b) ~rlminictering to the removed cells a vector
construct which directs the e~lession of at least one ~ mogenic portion of a
feline leukemia virus antigen, a felirle immllnQdeficiency virus antigen, or both,
and (c) returning the cells to a feline, such that a cellular immnne response isgenerated. Within the context of the present invention, it should be understood
that the removed cells need not nececc~rily be returned to the same feline, but
may be ~ltili7erl within other cats as well. In such a case it is generally preferable
to have histocompatibility matched felines (although not always, see e.g.,
Y~m~rnoto etal., "Efficacy of Experiment~l FIV V~crines," First International
Conference of FIV Researchers, University of California at Davis, September
1991).
Cells may be removed from a variety of locations, inrlntling for
example from the skin (dermal fibroblasts) and the blood (peripheral blood
leukocytes). If desired, particular fractions of cells such as a T cell subset or stem
cells may also be removed from the blood for ~lminictration of the vector
construct (e.g, PCT WO 91/16116, an application entitled "Immnnoselection
Device and Method"). Vector constructs may then be ~minictered to the
removed cells ntili7ing any of the above-described techniques, followed by the
return of the cells to the feline.
Within another aspect of the present invention, a vector construct is
provided which directs the expression of an imm-~nQgeniC portion as described
above, and directs the expression of a prodrug activator. For example, within one
W O 94/06921 ~ ~ ~ 2 ~ 2 ~ PC~r/US93/0907
16
embodiment, genes for an immnnQgenic portion and a prodrug activator, such as
Herpes Simplex Virus Thymidine Kinase (HSVTK), are incorporated into a
vector construct. This vector construct is then ~minictered to cells, which can
then be elimin~te-l by ~tlminictration of an exogenous substance, such as acyclovir,
S which kills cells that express the HSVTK. As one of ordinary skill in the art will
readily appreciate, this vector construct may be utilized to ensure that even if the
delivered genes contribute to a tumorigenic event in cells which have taken up the
vector, these cells can be killed by, for example, exogenous application of
acyclovir.
In addition to the recombinant viral vectors discussed above, other
methods may also be utilized to ~ ler vector constructs of the present
invention, or nucleic acids which encode the immlmogenic portion(s) discussed
above, to felines, or to feline cells ex vivo. Such methods int~lnde, for example,
transfection by methods ~ltili7ing various physical methods, such as lipofection(Felgner etal., PrOG Nat~ Aca~ Sci USA 84:7413-7417, 1989), d~rect DNA
injection (Acsadi et al., Nature 352:815-818, 1991); microprojectile bombardment(Williams et al., PNAS 88:2726-2730, 1991); liposomes (Wang et al., PNAS
84:7851-7855, 1987); CaPO4 (Dubensky et al., PNAS 81:7529-7533, 1984); or DNA
ligand (Wu et al, J. of Bio~ Chem. 264:16985-16987, 1989).
Furthermore, a cellular response (inchltlin~ CTL) may also be
generated by ~tlminictration of a bacteria which expresses the immnnogenic
portion(s) discussed above. Represe~tive examples include BCG (Stover,
Nature 351:456-458, 1991), salmonella (Newton et al., Science 244:70-72, 1989),
and Listeria (Schafer et al., J. Imm. 149:53-59, 1992).
Cell mt~ te~l and humoral responses may also be induced again t
feline leukemia virus or feline immnnodeficiency virus, by ~lminictration of theimmllnogenic portion(s) ~liccllccefl above. Briefly, immllnogenic portions carrying
relevant epitopes can be produced in a number of known ways (Ellis and Gerety,
J. Med. ~ro~ 31:54-58, 1990), inclllflin~ chemical synthesis (Bergot et al., App~ied
Biosystems Peptide Synthesizer User Bulletin No. 16, 1986, Applied Biosystems,
Foster City California) and DNA expression in recombinant systems, such as the
insect-derived baculovirus system (Doerfler, Glrrent Topics in Immunology 131:51-
68, 1986), m~mm~ n-derived systems (such as CHO cells) (Berman et al.,
J. rroL 63:3489-3498, 1989), yeast-derived systems (McAleer etal., Nature
307:178-180), and prokaryotic systems (Burrel et al., Nanlre 279:43~7, 1979).
The imm~mogenic proteins or peptides of the present invention may
also be purified by conventional means, and delivered by a number of methods in
21423~
~0 94/06921 - Pcr/US93/09070
17
order to induce cell-mediated responses, inrlll-ling class I and class II responses.
These methods include the use of adjuvants of various types, such as ISCOMS
(Morein, Immunology Letters 25:281-2~4, 1990; T~k~h~chi et al., Nature 344:873-
875, 1990), squalene/Tween-80/pluronic L121 (Morrow et al., Poster ~32,
- S "Advances in AIDS Vaccine Development, Proceedings of the Fifth Annual
Meeting of the National Cooperative Vaccine Dev. Group for AIDS", Aug, 1992),
saponin (Wu, Poster # 16, Advances, supra), proteoliposomes (Letvin,
"V~crin~tion of Rhesus monkeys with synthetic peptide in a fusogenic
proteoliposome elicits FIV specific CD8+ cytotoxic T-lymphocytes" "Procee-linEc,supra), liposomes (Gergoriadis et al., Vaccine 5:145-151, 1987), lipid conjugation
(Deres et al., Nature 342:561-564, 1989), coating of the peptide on autologous cells
(Staerz et al., Nature 329:449-451, 1987), pinosomes (Moore et al., Cell 54:777-785,
1988), alum, complete or incomplete Freund's adjuvants (Hart et al., PrOG Nat~
Acad. Sci USA 88:9448-9452, 1991), or various other useful adjuvants (e.g., Allison
and Byars, Vaccines 87:56-59, Cold Spring Harbor Laboratory, 1987)~that allow
effective parenteral ~llminictration.
Alternatively, the proteins or peptides corresponding to the
imm~mogenic portion(s) diccnssed above, can be enc~psitl~ted for oral or rectal
Llation to elicit an immlme response in enteric capsules (Ch~nnock et al.,
J. Ame,r. Med. Assoc 195:445452, 1966), or other suitable carriers, such as poly(DL-lactide-co-glycolate) spheres (Eldridge etal. in Proceedings of the
Internahonal Conference on Advances in AIDS Vaccine Development, DAIDS,
NIAID, U.S. Dept of Health & Human Services, 1991), for gastrointestinal
release.
Within a further aspect of the present invention, pharmaceutical
compositions are provided COlll~liSillg one of the above described recombinant
viruses, such as a recombinant retrovirus or recombinant virus selected from thegroup concictin~ of poliovirus, rhinovirus, pox virus, cana~y pox virus, v~rrini~
virus, influenza virus, adenovirus, palvovillls, adeno-associated virus herpes virus,
SV40, HIV, measles and Sindbis virus, in combination with a pharmaceutically
acceptable carrier or diluent. The composition may be prepared either as a liquid
solution, or as a solid form (e.g., lyophilized) which is suspended in a solution prior
to ~iminictration. In addition, the composition may be prepared with suitable
carriers or ~ entc for either injection, oral, or rectal ~lminictration. Generally,
the recombinant virus is utilized at a concentration ranging from 0.25% to 255~o,
and preferably about 5% to 20% before formulation. Subsequently, after
preparation of the composition, the recombinant virus will constitute about 1 ug of
WO 94/06921 ~ 2 5 Pcr/US93/0907
18
material per dose, with about 10 times this amount material (10 ug) as copurified
cont~min~ntc. Preferably, the composition is prepared in 0.1-1.0 ml of aqueous
solution formnl~ted as described below. In addition, the composition may containan adjuvant such as, for example, ~ min1lm hydroxide, saponin, and squalene.
Pharmaceutically acceptable carriers or diluents are nontoxic to
recipients at the dosages and concentrations employed. Representative examples
of carriers or ~li1nentc for injectable solutions include water, isotonic salinesolutions which are preferably buffered at a physiological pH (such as phosphate-
buffered saline or Tris-buffered saline), m~nnitol, dextrose, glycerol, and ethanol,
as well as polypeptides or proteins such as feline serum albumin. A particularlypreferred composition comprises a vector or recombinant virus in 10 mg/ml
m~nnitol, 1 mg/ml feline serum albumin, 20mM Tris pH=7.2 and 150mM NaCl.
In this case, since the recombinant vector represents approxim~tely 1 ~g of
material, it may be less than 1% of high molecular weight material, and less than
1/100,000 of the total material (in~-ln-ling water). This composition is~stable at -
70C for at least six months. The composition may be injected hlLlavellously (i.v.
or subcutaneously (s.c.), although it is generally preferable to inject it
intramuscularly (i.m.), or by aerosol ~rlminictration intranasally. These are
~lminictered at one to four week intervals for three or four doses initially
Subse~quent booster shots may be given as one or two doses after 6-12 months, and
thereafter ~nml~11y.
Oral formnl~tiQns may also be employed with carriers or diluents
such as cellulose, 1~rtose, mannitol, poly (DL-lactide-co-glycolate) spheres, and/or
carbohydrates such as starch. The composition may take the form of, for example,a tablet, gel capsule, pill, solution, or suspension, and additionally may be
formn1~te~1 for sustained release. For rectal ~llminictration, preparation of a
suppository may be ~ccomrlished with traditional carriers such as polyaLkalene
glucose, or a triglyceride.
The following examples are offered by way of illustration and not by
way of 1imit~fion.
23~23~
wo 94/06921 Pcr/US93/09070
19
EXAMPLES
EXAMPLE 1
5 A. Preparation of Retroviral Backbone KT-3
The Moloney murine lenkemi~ virus (MoMLV) 5' long terminal
repeat (LTR) EcoR I~EcoR I fr~gne~t in~llltlinE gag sequences, from the N2
vector (Armentano et al., J. ~r. 61:1647-1650, 1987; Eglitas et al., Science
230:1395-1398, 1985) is ligated into the plasmid SK+ (Stratagene, Calif.). The
resnltinE construct is decign~ted N2R5. The N2R5 construct is mnt~terl by site-
directed in vitro mutagenesis to change the ATG start codon to ATT preventing
gag e~ression. This mutagenized fragment is 200 base pairs (bp) in length and
fl~nkP~l by Pst I restriction sites. The Pst I-Pst I mllt~ted fr~Ement is purified from
the SK+ plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR~in plasmid
pUC31 to replace the non-mnt~te.~l 200 bp fragment. The plasmid pUC31 is
derived from pUC19 (Stratagene, Cali) in which additional restriction sites XhoI, Bgl II, BssH II and Nco I are inserted between the EcoR I and Sac I sites of the
polylinker. This construct is deciEn~te~l pUC31/N2R5gM.
A 1.0 Kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from
N2 is cloned into plasmid SK+ resnltin~ in a construct deci~n~ted N2R3-. A 1.0
Kb Cla I-Hind m fragment is purified from this construct.
The Cla I-Cla I dominant selectable mar}cer gene fragment from
pAFVXM retroviral vector (Kriegler et al., Cell 38:483, 1984; St. Louis et al.,
PNAS 85:3150-3154, 1988), comprising a SV40 early promoter driving expression
of the neomycin phosphotransferase gene, is cloned into the SK+ plasmid. A 1.3
Kb Cla I-BstB I gene fragment is purified from the SK+ pl~cmitl
The expression vector is constructed by a three part lig~tion in
which the Xho I-Cla I fragment cont~ining the gene of interest and the 1.0 Kb
MoMLV 3' LTR Cla I-Hind III fragment are inserted into the Xho I-Hind III site
of pUC31/N2R5gM plasmid. The 1.3 Kb Cla I-BstB I neo gene fragment from the
pAFVXM reLlovilal vector is then inserted into the Cla I site of this plasmid in the
sense orientation.
WO 94/06921 ~ ~ ~ 2 3 2 ~ PCI/US93/0907
B. Preparation of Retroviral Backbone KT-1
The KT-1 retroviral backbone vector is construc~ed essentially as
described for KT-3 in Example 1 A, with the exception that the dominant
5 selectable marker gene, neo, is not inserted into the expression vector.
EXAMPLE 2
PREPARATION OF SEQUENCES UTILIZING PCR
A. PCR of the FeLV ga~/prot gene
In order to obtain the FeLV gag/prot gene, a plasmid cont~inin~ a
15 FeLV sequence (p61E-FeLV) is obtained from the NIH Research and Reference
Reagent Program, Maryland. A reaction ~llLLLule is then prepared according to
procedures specified by Perkin Elmer Cetus (Emeryville, Calif.). More
specifically, a reaction mLxture is prepared cont~inin~ 1 ~Lg purified plasmid, 10 ~l
of 10X PCR reaction buffer, 2 ~Ll 2.5 mM of each dATP, dCTP, dGTP, and dTTP,
20 0.5 ~1 of 2.5 units/100 ~Ll Taq polymerase, 10 ~l of 10 mM MgC12, and 0.5-1.0 ug of
the primiers specificied below (Sequence ID No. 1 and Sequence ID No. 2).
The sense primer sequence is from the 5' region of the FeLV
gag/prot gene upstream from the ATG start codon at position 609 of the FGA
~JlOVilUS. The 5' end of the primer contains two consecutive Xho I restriction
25 sites.:
(Sequence ID No. 1)
5'-3': CC CTC GAG CTC GAG GGC GGT GGG ATC GAA GGA GCT GAC
G
The anti-sense primer sequence is complementary to a sequence at
30 position 2800 of the FGA provirus and contains two consecutive stop codons inframe with FeLV gag/prot gene. The 5' end of the primer contains two consecutiveCla I restriction sites.:
(Sequence ID No. 2)
5'-3': CC ATC GAT ATC GAT CTA TCA TGG CTC AAA TAG CCG ATA
35 CTC TTC TTC
3 2 ~
W O 94/06921 - PC~r/US93/09070
Z1
The reaction mixture is the brought to 100 ~l with DI H20, and
each tube is placed into a PCR m~chine (Gene Amp PCR System 9600, Perkin-
Elmer, Cetus, Calif.). The PCR program regulates the temperature of the
reaction vessel first at 94C for 2 minllte~ next at 56C for 30 seconds, 7ZC for 30
5 seconds, and finally, 94C for 30 seconds. This cycle is repeated 35 times. After
the 35th cycle, the re~ctionc are held at 4C.
B. Isolation of PCR DNA
The PCR reaction is transferred into a 1.5 ml microfuge tube, and
50 ~Ll of 3 M sodium acetate is added. Next, 500 ~Ll of chloroform:isoamyl alcohol
(24:1) is added to the solution, which is vortexed and then spun for 5 minlltes.The upper aqueous phase is transferred to a fresh microfuge tube and 1 ml 100~Z
EtOH is added. The solution is incubated at -20C for 4.5 hours, and then spun
for 20 minntes The supernatant is de~nte~l, and the pellet rinsed ~ith 500 ~Ll
70~o EtOH. The pellet is dried by s~h~illg under a v~cmlm, and then
resuspended in 10 ~Ll H20.
E~AMPLE 3
,.
CONSrR~C~10N OF RETROVIRAL VECTORS
A. Construction of FeLV env Retroviral Vector
The 2.0 Kb Pst I fragment from FeLV-A-Gardner-Arnstein [FGA]
~l`OVilllS (Donahue et al., J. ~r. 62:722-731, 1988) is subcloned into the Pst I site of
the psp72 vector (Promega Biotech, Wisc.). Subclones cont~ininE FeLV env in the
sense orientation with respect to the 5' Xho I and 3' Cla I sites are selected by
30 restriction enzyme analysis. This co~Lluct is de~iEn~ted psp72 FeLV env. The
Xho I-Cla I fragment is then excised and inserted into the KT-3 backbone.
B. Construction of FeLV gag/prot Retroviral Vector
DNA encoding the FeLV gag/prot gene is prepared as described
above in Example 2, and placed into the Xho I and Cla I sites of the pBluescriptKS II+ plasmid (Stratagene, Calif.) and verified by DNA sequencing. This
W O 94/06921 ~ 3 2 ~ ` PC~r/US93/0907
construct is designated pBluescript KS II+ FeLV gag/prot. The Xho I-Cla I
fragrnent is then excised and inserted into the KT-3 backbone.
C. Construction of FIV env/rev/RR~ Retroviral Vector
Sequences encoding the FIV env/rev/RRE gene are amplified and
isolated from plasmid pFIV-14-Petaluma (NIH Research and Reference Reagent
Prograrn, Maryland), essentially as described in Example 2 above, using the
following primers:
The sense primer sequence has two consecutive Xho I restriction
sites that are placed at the S' end at position 6020 of clone 34F10 (Talbott et al.,
PNAS 86:5743-5747, 1989).:
(Sequence ID No. 3)
5'-3': CC CTC GAG CTC GAG GGG TCA CTG AGA AAC TAG AAA AAG
15 AAT TAG
The antisense primer sequence is complementary to a sequence at
position 9387 of clone 34F10. The 5' end of the primer has two consecutive Cla Isites:
20 (Sequence ID No. 4)
5'-3': CC ATC GAT ATC GAT GTA TCT GTG GGA GCC TCA AGG GAG
AAC
The PCR product is placed in the pBluescript KS II+ plasmid
25 (Stratagene, Calif.) and verified by DNA sequencing. This co~ uct is clesign~te~l
pBluescript KS II+ FIV env/rev/RRE. The Xho I-Cla I fragment is then excised
and inserted into the KT-3 backbone.
D. Construction of FIV ~aR/rev/RRE Retroviral Vector
The Cla I site in the sp72 (Promega, Wisc.) plasrnid is first killed by
1) Cla I digestion; 2) blunted by Klenow fragment; and 3) religated.
i. Construction of psp72 BIP-FIV gag
In order to construct the FIV gag open reading frame with Sph I and
35 Bgl II restriction sites fl~nking the open reading frame, a PCR reaction is
undertaken lltili7ing the following primers:
~ WO 94/06921 2 1 4 ~ 3 2 ~ PCr/US93/09070
The sense primer sequence is from position 612 of the clone 34F10.
Two consecutive Sph I restriction sites are placed at the 5' end of the primer:
(Sequence ID No. 5)
5'-3': CC GCA TGC GCA TGC GAG Al~ CTA CAG CAA CAT GGG GAA
5 TGG ACA G
The antisense primer sequence is complementary to a sequence at
position 1959 of the 34F10 clone. Two concecntive Bgl II sites are placed at the 5'
end of the primer. This oligonucleotide contains two consecutive in-frame stop
10 codons with the FIVgag open reading frame:
(Sequencing ID No. 6)
5'-3': CC AGA TCT AGA TCT CTA TCA CTC CAT TGG AGG TGC AGA
TGG CAT TTA CTG
The resnlt~nt PCR product is design~te~l Sph I-Bgl II/FIVgag.
In a three-part ligation, the Bgl II-Sph I BIP fragment (Peter
Sarnow, Univ. of Colo. Health Sciences Center, Denver, Human Tmmnnoglobulin
heavy chain binding protein), Sph I-Bgl II/FIV gag PCR product is ligated into the
CIP (calf intestinal phosphatase, New Fngl~ncl Biolabs, Mass.) treated Bgl II site
20 of the~ re-engineered psp72 vector, without the Cla I site. The insert is verified by
DNA sequencing. This construct is design~ted psp72 BIP-FIV gag. The Bgl II
fragment cont~inin~ BIP-FIVgag is excised and used in the ligation below.
ii. Construction of pBluescript KSII + /FIV rev/RRE
The FIV rev/~RE is constructed by PCR site directed mutagenesis
25 (Ho et al., Gene 77:51-59, 1989) with the sense primer sequence ID No. 3 and
antisense sequence ID No.4 used in generating the FIV env/~ev/RRE with two
lition~l oligonucleotides: the sense primer sequence
(Sequence ID No. 7)
5'-3': TGA TAG AGA (_ 1 l CCA CCATTA GTA GTC CCA G
30 and the antisense primer sequence
(Sequence ID No. 8)
5'-3': G TCT CTA TCA CCATACTAC CTG AGC GCC GGC TGT C
Embedded within primers Sequence ID Nos. 7 and 8 are two
co~cec~ltive stop codons in frame with the FIV env gene at position 6798 of the
35 34F10 clone. The primers Sequence ID Nos. 3 and 8 are used in the first PCR
reaction to generate the arruno terrninal region of FIV env with two in-frame stop
codons. This double stranded DNA is decign~ted FIV env/amino/stop. The
WO94/06921 21 ~32~ PCI`/US93/090
primers Sequence ID Nos. 7 and 4 are used in the second PCR reaction to
generate the carboxyl terrninal region of FIV env with a complementary region
encompassing both stop codons of FIV env/amino/stop. This double stranded
DNA is decign~ted FIV env/carboxyl/stop. The PCR products, FIV
S env/amino/stop and FIV env/carboxyl/stop are denatured, reannealed, and
allowed to undergo a third PCR reaction with primers Sequence ID Nos. 3 and 4.
This double stranded DNA is clesiEn~ted FIV rev/RRE. The FIV rev/RRE DNA
is digested with Xho I and Cla I, and subcloned into the Xho I and Cla I sites of
the pBluescript KS II + plasmid and verified by DNA sequencing. This
10 intermediate construct was clesiEn~ted pBluescript KSII + /FIV rev/RRE.
iii. Construction of KT-3 FIV ga~/rev/RRE
pBluescript KSII+/FIV rev/RRE is digested with Bcl I at position
7249 (Talbott et al., PNAS 86:5743-5747, 1989) and crP treated. The Bgl II BIP-
15 FIV gag fragment is eY~ice~ from psp72 BIP-FIV gag and inserted in the Bcl I site
in the sense orientation. This construct is ~lesiEn~te~l pBluescript KSII+/BIP-FIV
gag/rev/RRE. The construct is cleaved at the Apa I site blunted by Klenow
fr~Ement followed by cleavage with Cla I. The Xho I site of the KT-3 backbone iscleaved by Xho I and blunted with Klenow fragment followed by cleavage with Cla
20 I. The BIP-FIV gag/rev/RRE fragment from the blunted Apa I to the Cla I site is
inserted into the blunted Xho I site and Cla I sites of the KT-3 backbone.
EXAMPLE 4
CONS~UCrlON OF MULTIVALENT RETROVIRAL VECrORS
~ Construction of FIV env/rev/RRE. FeLV env Retroviral Vector
i. Multivalent env Rello~hal Vector with IRBS
The Cla I-Hind III fragrnent cont~ininE the IRBS (BIP) from psp72
is first inserted in the respective sites within the pBluescript KS II+ plasrnid. The
2.0 Kb FeLV env Pst I fragment is inserted at the Pst I site in the sense orientation
with respect to BIP. This construct is desiEn~ted pBluescript KSII+/ BIP-FeLV
env.
The FIV env/rev/RRE is first excised from pBluescript KS II+/FIV
env/rev/RR~ plasmid by Xho I and Cla I digestion and inserted into the Xho I-Cla
2~ 3~
WO 94/06921 ~ Pcr/uss3/o9o7o
I sites of the KT-1 backbone. This construct is cleaved at the Cla I site and
blunted by Klenow fragment. The Cla I-BamH I fragment from pBluescript
KSII+/BIP-FeLV env is then isolated, blunted by Klenow fragment, and inserted
in the sense orientation at the blunted Cla I sites of the KT-1 retroviral backbone.
ii. Multivalent env Retroviral Vector with CMV Promoter
The FIV env/rev/RRE is first excised from pBluescript KS II+ /FIV
env/rev/RRE plasmid by Xho I and Cla I digestion and inserted into the Xho I-Cla10 I sites of the KT-1 backbone. This construct is cleaved at the Cla I site andblunted by Klenow fragment. The Xho I-Cla I FeLV env fragment is isolated from
the cloning intermediate psp72-FeLV env vector and substituted for Xho I - Cla Iinsert into pUC 18 CMV gag/pol/CAR. The CMV FeLV env is then excised as a
Pst I fr~m~nt blunted by T4 DNA polymerase (New F.n~l~nrl Biolabs, Mass.)
15 and inserted in the sense orientation at the blunted Cla I sites of the KT-1
retroviral backbone.
The pUC 18 CMV gag/pol/CAR is constructed essentially as
follows. Briefly, from pAF/CMV/EnvR (U.S. Patent Application No.
07/395,932), the 4.7 Kb CMV EnvR Pst-RI fragment is isolated, and inserted into
20 pUC 18 (New Fn~l~n~l Biolabs, Mass.) at the Pst I and RI sites. This construct is
deci~h~te.l pUC 18 CMV EnvR. To generate CMV gag/pol/CAR, HIV-1 IIIB
CAR is subcloned as a Sau 3A fragment into the Bam HI site of pBluescript II
KS+/CAR. The CAR fragment is excised from pBluescript II KS+/CAR as a
Xba I-Cla I fragment. The Xho I- Xba I HIV-1 IIIB gag/pol fragment is excised
25 from SK+ gag/pol SD delta (U.S. Patent Application No. 07/395,932). The
plasmid backbone cont~ining the CMV promoter is excised from pUC18
CMV/EnvR with Xho I and Cla I. In a three part ligation, the Xho I-Xba I HIV
IIIB gag-pol fragment, the Xba I-Cla I CAR fragment is inserted into the Xho I-
Cla I sites of the pUC 18 CMV/EnvR backbone to generate pUC 18 CMV
30 gag/pol/CAR.
B. Construction of FIV ga~/rev/RRE. FeLV gag Retroviral Vector
i. Multivalent gag Retroviral Vector with IRBS
In order to construct a FeLV gag/prot with fl~nkin~ Xho I
restriction sites, a PCR reaction is undertaken with the following primers.
WO 94/06921 2 1 4 2 3 2 5 26 PCr/US93/090~
The sense primer sequence is from the 5' region of the FeLV
gag/prot gene upstream from the ATG start codon at position 609 of the FGA
provirus. The 5' end of the primer contains two consecutive Xho I restriction
sites:
5 (Sequence ID No. 1)
5'-3': CC CTC GAG CTC GAG GGC GGT GGG ATC GAA GGA GCT GAC
G
The antisense primer sequence is complementary to a sequence at
10 position 2800 of the FGA ~rovi~lls and contains two consecutive stop codons in
frame with FeLVgag/prot gene. The 5' end of the primer contains two consecutive
Xho I restriction sites:
(Sequence ID No. 9)
5'-3': CC CTC GAG CTC GAG CTA TCA TGG CTC AAA TAG CCG ATA
15 CTC TTC TTC
The res111t~nt PCR product is inserted at the Xho I site in the sense
orient~tion with respect to BIP of the sp72 BIP pl~cmi(l, and is decign~t~d psp72
BrP-FeLV gag/prot.
The BIP-FIV gag/rev/RRE fragment is isolated from pBluescript
20 KSII+/BIP-FIV gag/rev/RRE, cleaved at the Apa I site, blunted by Klenow
fr~nent followed by cleavage with Cla I. This fragment is inserted into the Xho
I blunted-Cla I sites of the KT-1 backbone and is design~ted KT-1/BIP-FIV
gag/rev/hUZ~. KT-1/BIP-FIV gag/rev/h~ is then cleaved at the Cla I site and
blunted by Klenow fr~gment The Cla I-Nde I BIP-FeLV gag/prot fragrnent from
25 psp72 BIP-FeLV gag/prot is blunted by Klenow fragment, and inserted in the
sense orientation at the blunted Cla I sites of the KT- 1 retroviral backbone.
ii. Multivalent gag Retroviral Vector with CMV Promoter
The pBluescript KSII+ /BIP-FIV gag/rev/RRE is cleaved at the Apa
I site, blunted by Klenow fragment and cleaved with Cla I. This BIP-FIV
gag/rev/RRE fragment from the blunted Apa I to the Cla I site is inserted into the
Xho I blunted and Cla I sites of the KT-1 backbone. This construct is then
cleaved at the Cla I site and blunted by Klenow fr~nent The X~o I-Cla I
fragment of FeLV gag/prot is isolated from the pBluescript KS II+/FeLV
gag/prot and substituted for the Xho I Cla I insert in pUC 18 CMV gag/pol/CAIR.
The CMV-FeLV gag/prot fragment is excised as a Pst I fragment, blunted by T4
~1~23~
WO 94/06921 Pcr/US93/09070
27
DNA polymerase, and inserted in the sense orientation at the blunted Cla I sitesof the KT-1 retroviral backbone.
EXAMPLE 5
TRANSIE~T TRANSFECrlON AND TRANSDUCIION
OF PACKAGING CELL LINES Dx AND DA
10 ~ Plasmid DNA Transfection
DX cells (W092/05266) are seeded at 5 x 105 confluence on a 6 cm
tissue culture dish on Day 1. On Day 2, the media is replaced with a 4 ml fresh
media 4 hours prior to transfection. A standard calcium phosphate-DNA
15 coprecipitation is performed by mixing 25 ~l 2.0 M CaCl2, 10 ~g plasm~id DNA (in
10 mM TAs-Cl, pH 7.5) and water to make 200 ~Ll total. Precipitation bu~fer is
freshly prepared by II~ixing 100 ~l 500 mM HEPES-NaOH (pH 7.1), 125 ~l 2.0 M
NaCl, 10 ~l 150 mM Na2HP04-NaH2P04 (pH 7.0) and water to make 1 ml total.
DNA-CaC12 solution (200 ~Ll) is added dropwise with constant ~it~tion to 200 ~l
20 precipitation buffer. After 30 min. at room temperature the resultant fine
precipitate is added to a dish of cells. Cells are exposed to the DNA precipitate
until Day 3 when the medium is aspirated and fresh medium is added. On Day 4
the virus-cont~ining medium is removed, and passed through a 0.45 ~Lm filter.
B. Packaging cell line transduction
DA (W092/05266) cells are seeded at 1 x 105 cells/6 cm dish. Half
a ml of the freshly collected virus-cont~inin~ DX medium is added to the DA cells
with fresh medium cont~ining 4 ~g/ml Polybrene (Sigma, Missouri). The
following day, G418 (800 ~g/ml) is added to these cells and a drug resistant pool is
generated over the following week. The pool of cells is ~ ntion cloned by adding0.8-1.0 cells to each well of 96 well plates. Forty-eight clones are expanded to 24
well plates, then to 6 well plates, at which time cell supern~r~nts are collected for
titer.
From producer cells expressing vectors cont~ining the selectable
marker neo, 1.0 ml of producer cell line supernatant is diluted five fold to 10-9
dilution and each dilution is used to transduce 5 x 105 Crandell feline kidney
wo 94/06921 21 4 2 ~ 2 ~ 28 PCI/US93/090~
(CRFK, ATCC CCL 94) cells. The following day G418 is added to the cells and
14 days later G418 resistant colonies are scored at each dilution.
DA producer cell expressing neo- multivalent vector are dilution
cloned 3 days after transduction of 0.5 rnl of freshly collected virus-cont~ining DX
S medium with fresh medium cont~ining 4 ~Lg/ml Polybrene. Forty-eight clones are
expanded to 24 well plates and cell supern~t~ntc titered in Example 6.
EXAMPLE 6
TrrER~NG FOR MULTI-VALENr VECrORS
Since the multivalent vectors do not contain a selectable marker
such as the neomycin gene, another way of titering the vector is described. Moresperific~lly~ 1.0 ml of vector supernatant is diluted five fold until 10-9 dilution, and
each rlillltion is then used to tr~n~ -ce S x 105 CRFK cells. One week later, DNA
is extracted from each dish (Willis et al., J. Biol. Chem. 259:7842-7849, 1984). The
FTV gag or FIV env is amplified by PCR using the following PCR primers.
For FIV gag, the sense primer sequence:
(Sequ~ence ID No. 10)
5'-3': GAG ATT CTA CAG CAA CAT GGG GAA TGG ACA G
is from position 612 of the clone 34F10.
The antisense primer sequence:
(Sequence ID No. 11)
5'-3': CTC CAT TGG AGG TGC AGA TGG CAT TAC TG
is complementary to position 1959 of the 34F10 clone.
For FIV env, the sense primer sequence:
(Sequence ID No. 12)
5'-3': GGG TCA CTG AGA AAC TAG AAA AAG AAT TAG
is from position 6020 of clone 34F10.
The antisense primer sequence:
(Sequence ID No. 13)
5'-3': GTA TCT GTG GGA GCC TCA AGG GAG AAC
is from position 9387 of clone 34F10.
The PCR products are analyzed by Southern blot analysis with the
aypropliate probes (Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Signal is
21~232!~
O 94/06921 - Pcr/uss3/o9o7o
29
expected to be seen in all the lower dilutions and fall off at a certain dilution with
all higher dilutions not having signal. The last dilution where a signal is visible
yields the infectious U/ml of the vector.
EXAMPLE 7
~ Transduction of Murine Cells with Vector Construct
The murine fibroblast cell line BClOME (Patek et al, Cell Imm.
72:113-121, 1992) (BC, H-2d) is grown in Dulbecco's modified eagle medium
(D~vIEM), 4500 mg/l glucose with L-glllt~mine (Irvine Scientific, Santa Ana,
California), cont~ining 10% fetal bovine serum (FBS) (Gemini, Calabasas,
California).
The BClOME cell line is transduced with the rellovila~vector and
clones are selected using 800 ~Lgm/ml G418 for 14 days as described in Ex~mple
SB. Cells are lysed to assay for protein expression using Western blots When theBClOME cell line is tr~nc~ ced with a multivalent vector, these cells are
transduced at a multiplicity of infection of 20 U/cell. One week after
tr~nc-lllction, cells are lysed and assayed for protein expression using Western blot
analysis.
B. Transduction of Feline Cells with Vector Constructs
The feline kidney cell line (CRFK) is grown in DMEM cont~ining
10% FBS. CRFK cells are transduced with the vector construct as described in
Example 5B, above and used to show vector expression in feline cells using
Western blot analysis.
EstabLished autologous feline T-cell (as described in Example 10B,
below) lines are transduced by cocultivation with the vector producer cell line.1 x 106 DA (vector producer cell line) is irradiated (10,000 rads, room
temperature) and plated for three days with 1 x 106 T-cells from the establishedautologous T-cell line. These cells are then cocultivated two more times with the
vector producer line in the same method as above. After the third cocultivation,these cells are placed under G418 selection. These cells are then assayed for
expression of the desired protein by Western blot analysis.
WO 94/06921 PCI/US93/090~
2~3~ 30
EXAMPLE 8
EXPRESSION OF TRANSDUCED GENES BY
S WESTERN BLOT ANALYSIS
Proteins are separated according to their molecular weight (MW) by
means of SDS polyacrylamide gel electrophoresis. Proteins are then transferred
from the gel to a IPVH Immobilon-P membrane (Millipore Corp., Bedford,
10 Mass.). The Hoefer HSI l l~ transfer apparatus (Hoefer Scientific Instruments,
Cali) is used to transfer proteins from the gel to the membrane. The membrane
is then probed with polyclonal antibodies that react specifically with the expressed
protein. The bound antibody is detected using 125I-labeled protein A, which
allows vic~ tion of the transduced protein by autoradiography.
EXAMPLE 9
TUMORIGENICrrY AND TRANSFORMATION
A. ~ Tumori~enicity Assay
Tumor formation in nude rnice is a particularly important and
sensitive method for determining tumorigenicity. Nude mice do not possess
25 mature T-cells, and therefore lack a functional cellular imml-ne system, providing
a useful in vivo model in which to test the tumorigenic potential of cells. Normal
non-tumorigenic cells do not display uncontrolled growth properties if injected
into nude mice. However, transformed cells will rapidly proliferate and generatetumors in nude mice. Briefly, the vector construct is ~lmini~tered by injection into
30 nude mice. The rnice are visually eY~mined for a period of 4 to 16 weeks after
injection in order to determine tumor growth. The mice may also be sacrificed
and autopsied in order to determine whether tumors are present. (Giovanella et
al., J. NatL Cancer Inst. 48:1531-1533, 1972; Furesz et al., '~umorigenicity testing
of cell lines considered for production of biological drugs," Abnormal Cells, New
35 Products and Risk, Hopps and Petricciani (eds.), Tissue Culture Association, 1985;
and Levenbook et al., J. B~o~ Std. 13:13S-141, 1985). This test is performed by
Quality Biotech Inc., NJ.
2~4~32~
Wo 94/06921 Pcr/US93/09070
31
B. Transformation Assav
Tumorigenicity may also be assessed by vicll~li7ing colony formation
- 5 in soft agar (MacPherson etal., ~r. 23:291-294, 1964). Briefly, one property of
norrnal non-tumorigenic cells is anchorage dependent growth. Normal non-
turnorigenic cells will stop proliferating when they are in serni-solid agar support
medium, whereas tumorigenic cells will continue to proliferate and form coloniesin soft agar.
HT1080, (ATCC CCL 121) a neoplastic cell line derived from
human fibrosarcoma and known to cause tumors in 100% of nude mice is used as
the assay positive control. WI-38, (ATCC CCL 75) a diploid embryonic human
lung cell line which is not tumorigenic in nude mice, is used as the assay negative
control.
Primary feline fibroblasts or WI-38 cell lines are tr~nd~ with the
vector col~lluct as described in Example 6B. Duplicate samples of each of the
tr~nctl~lçe~l cell lines, HT1080, and WI-38, are cultured in agar. Briefly, a lower
layer of 5.0 ml 0.8% R~l to~g~r (Difco, Michigan) in DMEM 17% FBS is set on 60
mm tissue culture plates. This is overlaid with 2.0 rnl 0.3% Bactoagar in the same
m~ m with the cells suspended at a concentration of 5 x 105 cells per ml. To
reduce background clumps, each cell line is strained through a 70 ~Lm nylon meshbefore suspending in the agar solution. The plates are incubated at 37C in a
hllmi(lified atmosphere of 5% C02 for 14 days. Within 24 hours of plating,
represent~tive plates of each cell line are examined for cell clumps present at the
time of plating. On day 13, the plates are stained with 1.0 ml INT viral stain
(Sigrna, Missouri) and on day 14, they are scanned for colonies of 2150 ~m in
diaIneter using a 1 rnm eyepiece reticle.
Only colonies spanning 150 ,um in any orientation are scored,
because colonies of this size can be readily observed in all planes under the
rnicroscope and non-transformed cells rarely form colonies of this size. At the end
of the assay, the plating efficiencies for each cell line are calculated as b/a x 100,
where b = the sum of colonies on all plates, and a = the total number of cells
plates. A non-transforrned cell line is one which has a plating efficiency of lower
than or e~ual to 0.001%. Therefore, a transformed cell line will have a plating
3S efflciency of greater than 0.001% (see, Risser et al., ~r. 59:477-489, 1974).
2 1 ~ ~ 3 ~ 5 32 PCI/US93/090~
EXAMPLE 10
C'frOTOXIC~ ASSAY
5 ~ Mice
Six- to eight-week- old female BALB/c mice (Harlan Sprague-
Dawley, Tn~ n~polis, ~ntli~n~) are injected twice intraperitoneally (i.p.) with 1 x107 irradiated (10,000 rads at room temperature) vector transduced cells.
10 ~nim~lc are sacrificed 7 days later and the splenocytes (3 x 106/ml) cultured in
vitro with irradiated syngeneic transduced cells (6x 104/ml) in flasks (T-25,
Corning, Corning, New York). Culture medium consists of RPMI 1640 (Irvine
Scientific, Santa Ana, Calif., heat-inactivated fetal bovine serum (5~o, Hyclone,
Logan, Utah), sodium pyruvate (1 mM), gentamicin (50 ug/ml) and 2-
15 merc~loethanol (10-5 M, Sigma Chemical, St. Louis, Missouri.). Effector cellsare harvested 4-7 days later and tested using various Effector:Target cell ratios in
96 well microtiter plates (Corning, Corning, New York) in a standard 4-6 hour
assay. The assay employs Na251CrO4-labeled (Amersham, Arlington Heights,
Illinois) (100 uCi, 1 hr at 37C) target cells (1 x 104 cellstwell) in a final volume of
20 200 uL Following incubation, 100 ul of culture me~ m is removed and analyzed
in a Be~km~n g~mm~ spectrometer. Spontaneous release (SR) is determined as
CPM from targets plus medium and m~ximnm release (MR) is determined as
CPM from targets plus lM HCl. Percent target cell lysis is calcnl~ted as:
[(Effector cell + target CPM) - (SR)/(MR) - (SR)] x 100. Spontaneous release
25 values of targets are typically 10%-20% of the MR
B. Felines
Since the vectors which are described above are to be utilized for
30 treating felines, an assay demonstrating immllnological efficacy in felines is
needed. The following is a description of the generation of the autologous T-cell
lines needed for re~ tQrs and target cells for the st~n~rd 51Cr release assay
(Brown et al., J. rr. 65:3359-3364, 1991). Peripheral blood mononuclear cells
(PBMC) are obtained following venipuncture and Ficoll-sodium diatrizoate
35 (Histopaque-1077; Sigma, St. Louis, Mo.) density gradient centrifugation. These
PBMCs are stim~ ted by 5 ,ug/ml concanavalin A (Con A, Sigma) for three days,
and maintenance in medium cont~ining 25 U/rnl human recombinant interleukin-
214~.~2~
Wo 94/06921 Pcr/uss3/o9o7
33
2 (IL-2) (Boehringer Mannheim Biocherr~icals, Tn~ n~polis, Ind.) and 10~o bovineT-cell growth factor (TCGF). Cells are seeded into round-bottom 96-well
microtiter plates at an average of 1 or 0.3 cells per well with 5 x 104 irr~ te~(3,000 rads) autologous PBMC, 10~o bovine TCGF, and 25 U/rnl of IL-2 in a final
- 5 volume of 200 ~Ll of complete RPMI. Complete RPMI consisted of RPMI 1640
medium cont~ining 10~o FBS, 2 mM L-glllt~nnine, 5 x 10-5 M 2-mercaptoethanol,
and 50 ~g of gentamicin per ml. Clones are expanded sequentially to 48-well and
24-well plates. After several weeks, cells are tr~ncd-lcerl with retroviral vectors
e,-~res~hlg either FeLV or FIV gag or env genes as in Example 7B. Expression of
these cell lines are monitored by Western blot analysis as in Example 8. Cell lines
expressing high levels of the desired protein function as restimlll~tQrs and targets
in a st~n~3~rd 51Cr release assay as in Example 10A.
From the foregoing, it will be appreci~te~l that, although specific
emboriim~ntc of the invention have been described herein for purposes of
illustration, various modifications may be made without deviating from the spirit
and scope of the invention. Accordillgly, the invention is not limited except as by
the appended claims.
WO 94/06921 PCr/US93/09Ol~
2~2325
34
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
NAME: Viagene, Inc.
STREET: 11075 Roselle Street
CITY: San Diego, California
COUNTRY: USA
POSTAL CODE: 92121
TELEPHONE: (619) 452-1288
TELEFAX: (619) 453-0095
INVENTORS: Lee, William T.L.
Serbin, John J.
Jolly, Douglas J.
Barber, Jack R.
Chada, Sunil
Chang, Stephen M.W.
(ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR TREATING
FELINE LEUKEMIA VIRUS AND FELINE IMMUNODEFICIENCY VIRUS
(iii) NUMBER OF SEQUENCES: 13
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Seed and Berry
(B) STREET: 6300 Columbia Center, 701 Fifth Avenue
(C) CITY: Seattle
(D~ STATE: Washington
(E) COUNTRY: U.S.A.
(F) ZIP: 98104
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
2~4;~3~
W 0 94/06921 PCT/US93/09~70
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: N/A
(B) FILING DATE: N/A
(C) CLASSIFICATION: N/A
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McMasters, David D.
(B) REGISTRATION NUMBER: 33,963
(C) REFERENCE/DOCKET NUMBER: 930049.415PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 206-622-4900
(B) TELEFAX: 206-682-6031
(C) TELEX: 3723836
(2) INFORMATIaN FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCCTCGAGCT CGAGGGCGGT GGGATCG M G GAGCTGACG 39
(2) INFORMATION FOR SEQ ID NO:2:
W 0 94/06921 2 ~ ~ 2 ~ 2 ~ P ~ /US93/090 ~
36
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCATCGATAT CGATCTATCA TGGCTCAAAT AGCCGATACT CTTCTTC 47
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCCTCGAGCT CGAGGGGTCA CTGAGAAACT AG M AAAGAA TTAG 44
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~4~32~
W o 94/06921 P ~ /US93/09070
37
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCATCGATAT CGATGTATCT GTGGGAGCCT CAAGGGAGAA C 41
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCGCATGCGC ATGCGAGATT CTACAGC M C ATGGGG M TG GACAG 45
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
WO 94/06921 PCI/US93/09Oj~
21~2~5
38
CCAGATCTAG ATCTCTATCA CTCCATTGGA GGTGCAGATG GCATTTACTG 50
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TGATAGAGAC TTCCACCATT AGTAGTCCCA G 31
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GTCTCTATCA CCATACTACC TGAGCGCCGG CTGTC 35
(2) INFORMATION FOR SEQ ID NO:9:
2 ~
W 0 94tO6921 P ~ /US93/09070
39
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCCTCGAGCT CGAGCTATCA TGGCTCA M T AGCCGATACT CTTCTTC 47
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C)~STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAGATTCTAC AGC M CATGG GG M TGGACA G 31
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
W O 94/06921 2 1 ~ 2 3 2 5 ! PCT/US93/090 ~
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTCCATTGGA GGTGCAGATG GCATTACTG 29
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOT~HETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGGTCACTGA GA M CTAGAA AAAG M TTAG 30
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
~ W 0 94/06921 214232~
P ~ /US93/09070
41
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GTATCTGTGG GAGCCTCAAG GGAG M C 27
