Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO94/16735 ~l 3 2 G 3 2 PCT~S94/00858
TITLE: ANTI-ZONA PELLUCIDA ANTIBODIES FOR
DELIVERY OF THERAPEUTIC AGENTS TO THE
OVARY
FIELD OF THE INVENTION
This invention relates to antibodies which
specifically bind to zona pellucida and to immuno-
chemical agents made from those antibodies, and to
therapeutic methods utilizing such immunochemical
agents.
BACKGROUND OF THE INVENTION
Ovarian disease, and particularly ovarian cancer
is preferably treated by agents which are specifically
targeted to the ovary. In addition, agents for
fertility control may be more effective when such
agents are specifically directed and distributed to the
ovary and not diluted in other, non-ovarian tissues.
~; 15 The mammalian zona pellucida surrounds the oocytes
present within the ovary. lts unique structure
includes a novel glycoprotein network. Because of the
unique nature of the zona pellucida proteins,
` ~ antibodies raised against these proteins are generally
specific to the ovary. Thus, anti-zona pellucida
antibodies are ideally suited as carriers for targeting
therapeutic agents to the ovary.
Cytotoxic agents have been conjugated to ovarian
tumor cells and specific tumor cell antigens for the
purpose of targeting delivery of the cytotoxic agent to
tumor tissues (See, for example, U.S. Patent Nos.
4,956,453 to Bjorn et al; 4,806,494 to Pastan et al;
and 4,925,922 to Byers et al). Delivery of the agent
to the ovary requires that the ovarian tissue contain
the specific tumor antigen recognized by the antibody.
While such antibody recognition may be expected when
Wos4ll6735 PCT~S94/00858
the antibody i5 raised against the patient's own tumor
cells, tumor antigens vary from one patient to another
and a single antibody may not be expected to carry
cytotoxic agents to the ovaries of all patients. In
addition, non-carcinogenic conditions of the ovary
would benefit from targeted delivery of therapeutic
agents. Targeted, specific delivery of therapeutic
agents is desired to enable lower dosages of
therapeutic agents and specific ovarian action of non-
specific agents. Targeted, specific delivery oftherapeutic agents is desired to enable use of lower
dosages of therapeutic agents, especially those which
may be detrimental to normal tissues, and to permit
specific ovarian action of non-specific agents. It
would be of great utility to provide antibodies for use
in specifically targeting therapeutic agents to the
ovary, which antibodies did not rely on the recognition
of antigens which vary from patient to patient.
SUMMARY OF THE INVENTION
Monoclonal and polyclonal antibodies raised
against zona pellucida antigens have been found to
carry a desired therapeutic agent specifically to the
ovary without significant loss of the therapeutic agent
to non-ovarian tissues. Such antibodies are used to
specifically direct therapeutic agents to the ovary
without significant variation in recognition from ovary
to ovary. In a preferred embodiment, anti-zona
pellucida antibodies target agents to the ovary for
specific fertility and contraceptive effects.
DETAILED DESCRIPTION OF THE INVENTION
Anti-zona pellucida antibodies include those
antibodies raised against whole or $ractionated
mammalian zona pellucida and those raised against
wos4/1673s ? ~ 3 ~ PCT~S94/00858
specific zona pellucida proteins, native or
recombinant, or fractions thereof. The antibodies may
be raised against any species of mammalian zona
pellucida, although non-rodent species are preferred.
It has been reported that non-rodent zona pellucida
antibodies may not be cross-reactive with rodent zona
pellucida, but are cross-reactive among non-rodent
species.
Antibodies, monoclonal and polyclonal, may be
prepared by methods generally known in the field. In
general, polyclonal antibodies are raised in host
animals by the injection of zona pellucida antigen into
a host animal. The host animal's blood serum is
removed, and fractionated to obtain antisera. The
antisera is screened for the presence of anti-zona
pellucida antibodies by demonstrated reactivity with
zona pellucida and zona pellucida antigens, e.g., by
known immunoassay techniques. The antibodies are also
~ screened for specificity to ovarian tissue by methods
- 20 known in the art, e.g., immunocytochemistry or Western
Blot analysis.
Monoclonal antibodies are produced from hybridoma
cells by known methods in the art. In general, a host
animal is injected intraperitoneally with zona
pellucida antigen. The spleens of immunized animals
are collected and used to prepare hybridomas with a
murine tumor partner using the general zymotic cell
hybridization technique of Xohler and Milstein, Nature,
1975, 256:495-497. This technique involves fusing
tumor cells and splenocytes using a fusogen such as
polyethylene glycol. After fusion, the hybrid cells
are separated from the fusion medium and grown in a
selective growth medium, such as HAT medium, to
eliminate unhybridized parent cells. The hybridomas
are expanded, if desired, and conditional culture
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WO94/16735 PCT~S94100858
medium is assayed for anti-zona pellucida activity by
conventional immunoassay procedures including
radioimmunoassay, enzyme immunoassay or fluarescence
immunoassay.
Hybridomas that produce monoclonal antibodies may
be grown in vitro or in vivo using known procedures.
Preferably the hybridomas are maintained as ascites
fluid in mice. The monoclonal antibodies may be
isolated from culture media or body fluids by
conventional immunoglobulin purification procedures
such ammonium sulfate precipitation, gel
electrophoresis, dialysis, chromatography, and
ultrafiltration if desired.
. Important characteriætics of the monoclonal and
polyclonal anti-zona pellucida antibodies of the
present invention include their reactivity with zona
pellucida antigen and their specificity to ovarian
tissues. The antibodies of the present invention may
be raised against a zona pellucida antigen of the same
species as the species to be treated (self-antigen), or
may be raised against a second species zona pellucida
(foreign antigen), where species show antigenic cross
reactivities. For example, polyclonal antisera
produced against a first species zona pellucida in a
second host species may recognize the first, second,
and additional species zona pellucida as tested by
Western blot, ELISA, or immunohistochemical techniques.
Antibodies may be labeled or conjugated to therapeutic
agents for use in the specific delivery of such agents
to the ovaries of the first, second or other species~
Similarly, recombinant zona pellucida proteins (foreign
` or self-antigens) and synthetically produced zona
pellucida peptides may be used as antigens to produce
antibodies useful in the method invention.
~ ~ ~ ? ~ 3 j
WO94/16735 PCT~S94100858
Anti-zona pellucida antibodies are conjugated to
chemical therapeutic agents by known methods in order
to form a desired immunochemical agent. The
conjugation may be made using a variety of bifunctional
protein coupling agents. Examples of such reagents are
N-succinimidyl-3-(2-pyridyldithio) propionate,
iminothiolane, bifunctional derivatives of imido
esters, aldehydes such as glutaraldehyde, bis-azido
compounds such as bis(P-diazonium benzoyl)-ethylene
diamine, diisocyanates such as tolylene 2,6-
diisocyanate, and bis-active fluorene compounds such as
l,5-difluoro-2,4-dinitrobenzene.
Examples of desired therapeutic agents for
coupling to the anti-zona pellucida antibodies include
radioisotopes, chemotherapy agents, cytotoxins, and the
; like. Radioisotopes are preferably high linear energy
emitting isotopes, e.g., Y, Pr, and the like. The
cytotoxin may be a cytotoxic drug or an enzymatically
active toxin of bacterial, fungal, or plant origin.
~;~ 20 Examples of such enzymatically active toxins are
diphtheria toxin A, exotoxin A, Ricin A, abrin A,
modeccin A, alpha sarcin, curcin, crotin, restrictocin,
phenomycin, and enomycin. The preferred cytotoxins are
ribosomal inhibiting proteins such as the A chain of
ricin or the A chain of abrin. The A chain of ricin
has been employed in cytotoxic conjugates as disclosed
in U.S. Patent No. 4,590,071.
The therapeutic immunochemical agents of the
present invention may be used in a variety of
therapeutic applications where specific, targeted
delivery of a therapeutic agent to the ovary is
desired. Examples of such therapeutic applications
include ovarian cancer, fertility control, and t~e
like. In a preferred embodiment, ricin con~ugated
anti-zona pellucida antibody is used to accelerate
~ ~ 5 ,~
WO94/1673S PCT~S94/00858
recruitment of primary follicles in the target ovary.
Accelerated recruitment is useful for example, in
contraceptive methods which effect later stage, e.g.,
tertiary, follicles. The anti-zona pellucida antibody
specifically delivers the cytotoxic agent, ricin to the
ovary. Ricin treatment results in loss of large
follicles from the ovary (eg, tertiary follicles) and
also results in recruitment of follicles (e.g.,
primordial follicles) into a growing pool where they
may be acted upon by fertility agents.
The immunochemical agents of the present invention
may be administered to a patient by known conventional
methods, e.g., injection, inhalation, or modified for
oral delivery. Preferably, the agent is administered
parenterally, i.e., intravenously, intraperitoneally,
or the like. The agent is prepared in a
pharmaceutically acceptable carrier, for example, in an
aqueous medium such as water, buffer, saline, glycine,
or an oil based carrier, as appropriate for the
specific antibody-agent couple. A therapeutically
effective dose, i.e., one that will eliminate, reduce,
or retard the growth of the patient's tumor or one that
will induce a desired fertility effect is administered.
The therapeutically ef~ective dose and dose regimen
will vary according to specific characteristics of the
patient, the therapeutic indication, and the nature of
the therapeutic agent. The dose and regimen may be
calculated by the treating physician according to
acceptable procedures known in the therapeutic field.
In general, a therapeutically effective dose will
be less than the calculated LD50 f the therapeutic
agent as calculated for the weight of the patient. The
amount of the agent typically will be in the range of
about O.Ol to lO0 mg/kg body weight and preferably
between O~Ol and lO mg/kg.
:
~ ~ 3 ~ -, .3 ~
WO94tl6735 PCT~S94/00858
The invention may be better understood by
reference to the following examples:
EXAMPLE l
5PREPARATION OF ANTI-ZONA PELLUCIDA ANTIBODIES
Anti-zona pellucida antibodies against heat
solubilized dog zona pellucida (HSDZ) were prepared
generally following the procedures described by Dunbar
et al., (Biochemistry, 19:356-365, 1980), except that
ganged razor blades were used to mince the ovaries.
Rabbits were immunized with a mixture of HSDZ (250 ~g)
and MDP (250 ~g). Two additional boosts followed at
a~proximately three week intervals. The resultant
rabbit serum was next used for IgG fractionation.
Following the instructions provided with a Zymed
Protein A column, an IgG fraction was collected. The
collected anti-sera was incubated for one hour on the
Protein A column. The column was then washed four
times with PBS, IgG was eluted using 0.1 M acidic
acid, pH 2.8, and buffered immediately with l M tris,
pH 9.5. The collected samples were then dialyzed
extensively against PBS, pH 7.2. The ELISA titer of
the IgG fraction was greater than 64 K against HSDZ.
EXAMPLE 2
ANTI ZP ANTIBODY-RICIN A CONJUGATE
Antibodies produced as described for Example l
were conjugated to deglycosolated Ricin chain A by
Inland Laboratories, Dallas, Texas, following the
procedures described in R. Fulton et al., Cancer
RQsear$h, 4a: 2618-2625, 1989. The conjugated antibody
was subsequently used in animal studies.
~132G3~
WOg4/16735 PCT~S94/00858
Ricin-conjugated a~tibody (1 mg) prepared as
described above was placed in 0.5 ml PBS. The solution
was then injected intravenously into a seven month old
female dog. For the first 48 hours, the injected dog
exhibited elevated temperature, lethargy, and some
vomiting, but then returned to normal health. Seven
days post-injection, the dog was unilaterally
ovariectomized. The excised ovary was sectioned and
stained for histological analysis. Analysis of stained
sections of the ovary indicated accelerated atresia of
tertiary follicles within the treated ovary as compared
to control, untreated ovaries. Surprisingly,
recruitment of primary and secondary follicles was
a.ccelerated in the treated ovary as compared to the
controls. Control and treated histological sections
are shown in Figures 1-9. Figures 1-4 show sections of
control, untreated ovaries at 7.5, 10, 30, and ~OO
times magnification, respectively. Figures 5-8 show
sections of treated ovaries at 7.5, 10, 30, and lOO
times magnification, respectively.
EXAMPLE 3
l2sI-ANTI-ZONA PELLUCIDA ANTIBODIES
Antibodies were produced against heat solubilized
cat zona pellucida (HSCZ) following the procedures
described for Example 1. Affinity purified rabbit
anti-HSCZ IgG was iodinated by Dr. R. Kittoke,
~niversity of Nebraska, using the Iodogen method. A
molar ratio of 2:1 l2sI:protein was used. Reaction
products were separated on a Bio-Gel P-60 column and
the iodinated protein was eluted in the void volume.
Approximately 124 million DPM of radioactivity was
associated with 15 ~g of recovered protein.
The radiolabeled protein t2.95 ~g, 10.99
microcuries~ in 0.59 ml PBS containing 0.1% gelatin was
W094l16735 2 l .3 ~ S 3 ~ PCT~S94/00858
injected intravenously into a female cat. The animal
was also injected with 50 ~g of sodium Iodicle in
0.5 ml PBS. The amount of sodium Iodicle was lO,OO0
times the amount of 125I in the labeled protein, as
calculated on a molar basis.
Twenty-four hours post-injection the animal was
euthanized and various tissues were removed. Tissues
were homogenized and analyzed for radioactivity using a
Micro-Medic gamma counter. As shown in Table 1, within
24 hours the amount of radioactivity within the ovary
(CPM/g) was four times that of other reproductive
tissues.
. TABLE 1
TISSUE CPM/q
heart 2534
kidney 5034
liver 5537
~;~ brain 415
muscle 882
spleen 3202
uterus 7981
~; ovary 31348
~`
