Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL STRATEGY FOR CARBOHYDRATE-BASED
THERAPEUTIC VACCINES
FIELD OF THE INVENTION
This invention relates to the field of medical treatments and therapeutic
compositions for use therein. More specifically, it relates to methods and
compositions for treatment and prophylaxis of cancer in human patients.
BACKGROUND OF THE INVENTION
Despite the very extensive research efforts and expenditures over recent
years,
cancer remains one of the most life threatening diseases in the world. Cancer
therapy remains very difficult. Scientists have long been exploring the
possibility
of developing vaccines for the treatment and prevention of cancer in human
patients. Although this approach has been viewed as the therapy of the future,
progress has been modest and the incidence of clinical failure has been very
high.
Creating cancer vaccines is problematic, due largely to the fact that patients
fail
to mount an effective immune response to cancerous cells, because cancer cells
fail to produce immunogenic markers that sufficiently distinguish them from
normal
cells. Although the patterns of cell surface carbohydrate antigens of cancer
cells
differ from those of normal cells, the individual structures of their antigens
are
identical.
BRIEF REFERENCE TO THE PRIOR ART
Despite the structural identity of individual antigens found on normal cells
and
cancer cells, attempts have been made to exploit cancer cell carbohydrate
antigens as potential cancer vaccines. The observation that specific antigens
are
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overexpressed by certain tumor types has enabled the development of simple
monovalent antigen vaccines against various tumor types. It has also been
observed that, in animals and humans, provided that the carbohydrate antigens
are conjugated to a protein carrier, the resulting conjugate vaccines can be
used
to raise antibodies that are specific for each carbohydrate antigen.
However, the antibodies so induced are usually of low titer and poor endurance
(mostly IgM). Despite this drawback, they are used, on the basis that, after
surgical or chemical treatment of cancer, the antibody levels will remain
sufficiently
high, during a short convalescence period, to dispose of any remaining cancer
cells.
Clinical trials using some of these carbohydrate antigen-protein conjugate
vaccines have demonstrated that they can increase remission times in some
patients. However their use as therapeutic agents is far from satisfactory.
a2-8 Polysialic acid is expressed in a number of important human cancers,
including small cell lung cancer, neuroblastoma and Wilms' tumor. It is
strongly
expressed in neonatal tissue, but is not prevalently expressed in normal human
tissue following the neonatal period (1 month). Normal cells express a variety
of
different sialylated surface antigens.
It is known (U.S. Patent 5.811,102 Jenninas et al.) how to prepare vaccine
compositions based on chemically modified meningococcal polysaccharides, and
that they are useful for immunizing mammals against Neisseria mening~itidis
and
E. coli K1, microorganisms which are leading causes of meningitis in humans. A
modified B polysaccharide of N. menin itidis is prepared chemically, from the
polysaccharide isolated from N. meningitidis. The modified polysaccharide has
sialic acid residue N-acetyl groups (C2) replaced by a saturated or
unsaturated C3_s
acyl group. This modified polysaccharide is conjugated to an immunologically
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suitable protein to produce a conjugate of enhanced immunogenicity. A mammal
may be immunized with the vaccine composition, to induce a specific immune
response in the animal suitable to provide active protection from N. menin
itidis
infection. Alternatively, blood may be collected and the gamma globulin
fraction
may be separated from the immune serum, to provide a fraction for
administration
to a suitable subject to provide passive protection against or to treat on-
going
infection caused by these microorganisms.
Many different surface antigens containing modified sialic acid residues have
been
expressed in normal cells, using N-propionylated and N-levulinoyl-D-
mannosamine
as precursors. In one report, N-propionylated-D-mannose was introduced into a
cancer cell line (hepatoma).
It is an object of the present invention to provide novel compositions capable
of
being used as anti-cancer vaccines.
It is a further object of the invention to provide a process for enhancing the
specific
immunogenicity of mammalian cancer cells, and exploiting this enhanced
immunogenicity in a vaccination approach to the management of cancer in human
patients.
SUMMARY OF THE INVENTION
In the present invention, from one aspect, the sialic acid component of a
sialic acid
unit-containing cell surface marker characteristic of cancerous mammalian
cells,
such as a2-8 polysialic acid, is modified, so that cells normally expressing
such
a marker express instead a modified sialic acid unit-containing cell surface
marker
which is strongly immunogenic. For example, the present invention enables, in
a
portion of patient cells which regularly express a2-8 polysialic acid (i.e.
various
types of cancer cells), the expression of a highly immunogenic surface antigen
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namely, modified a2-8 sialic acid. Antibodies specific for the modified
antigen,
which can be induced using a conjugate of a suitable portion of the modified
sialic
acid unit-containing marker (such as a2-8 polysialic acid) and a protein, can
then
be used to eliminate cells such as the aforementioned cancer cells which
express
a2-8 polysialic acid. Vaccines can be prepared utilizing conjugates of the
modified
sialic acid-containing marker (such as modified a2-8 polysialic acid), or
utilizing
antibodies produced in response to exposure of a suitable subject to the
modified
sialic acid-containing marker, for managing cancer conditions which involve
cancer
cells characterized, at least in part, by expression of modified sialic acid
unit
containing marker.
For instance, an appropriate a2-8 polysialic acid modification for use in the
present invention is N-acylation of a2-8 polysialic acid. This can be
accomplished
by feeding the cells an N-acylated precursor of a2-8 polysialic acid, for
example
N-propionylated-D-mannosamine, which in the natural intracellular biochemical
synthesis of a2-8 polysialic becomes chemically incorporated therein and thus
results in the production of N-propionylated a2-8 polysialic acid. Such N-
acylated
a2-8 polysialic acids are strongly immunogenic, in contrast to the naturally
expressed a2-8 polysialic acid.
Antibodies specific for the modified antigens can be induced by normal
antibody
raising techniques using a corresponding N-acylated a2-8 polysialic acid-
protein
conjugate. The N-acylated a2-8 polysialic acids for antibody production and
vaccine preparation may be produced from commercially available poly-2,8-N-
acetylneuraminic acid using methods disclosed herein. Alternatively, though
less
preferably, they may be purified by standard means from cells expressing them.
Examples of cells expressing N-acylated a2-8 polysialic acid include cells
such as
RMA tumor cells which normally produce 2-8 poly-N-acetylneuraminic acid, when
induced to use an N-propionated precursor such as N-propionated mannosamine.
Choice of immunologically suitable protein for conjugation, and techniques for
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binding the polysaccharide to the protein to form the conjugate for use as the
vaccine are matters within the skill of the art. Tetanus toxoid TT is a
specific,
suitable example of such a protein.
The binding of anti-N-acylated-a2-8 polysialic acid antibodies, prepared by
standard antibody raising techniques using the modified polysialic acids
described
herein, to cancer cells, in the presence of complement, will result in at
least partial
destruction of the cancer cells. The antibody can be previously induced in the
patient using an N-acylated-a2-8 polysialic acid-protein conjugate prior to
antigen
modification, or alternatively the antibody can be raised outside the
patient's body,
by known techniques such as hybridoma incubation, and administered passively
to the patient following antigen modification.
Thus according to one aspect of the present invention, there is provided a
process
of enhancing the specific immunogenicity of viable, proliferating mammalian
cancer cells to levels sufficient to allow the effective recognition and
destruction
of such cells by an immuno-response in vivo, which comprises providing to said
cells a chemically modified precursor of a suitable sialic acid unit-
containing cell
surface marker capable of rendering said cancer cells immunologically
distinctive
from related, normal cells; causing biochemical incorporation of said modified
precursor into the sialic acid unit-containing cell surface marker during
intracellular
synthetic processes; and eventual surface expression of said sialic acid unit-
containing surface marker incorporating said modified precursor in a form
capable
of eliciting said level of immune response.
Afurther aspect of the invention comprises immunogenic mammalian cancer cells,
said cells having surface markers incorporating modified sialic acid units
capable
of initiating an immune response. Such cells may elicit a strong enough immune
response in the mammalian system containing them, to effectively combat the
proliferation and even the viability of such cells.
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Another aspect of the invention provides use of chemically modified sialic
acid
precursors, such as N-propionylated mannosamine, in creating mammalian cancer
cells expressing sialic acid unit-containing surface markers capable of
eliciting an
immune response, and use of a vaccine comprising a conjugate of a polysialic
acid
compound incorporating said chemically modified precursor, and a protein, to
combat said mammalian cancer cells.
BRIEF REFERENCE TO THE DRAWINGS
l0 The accompanying drawings are graphical presentations of results obtained
according to various specific examples and experiments in accordance with the
invention, as described in more detail below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
It is preferred, according to the invention, to choose a polysialic acid as
the sialic
acid-unit containing cell surface marker for modification. This is desirable
as
polysialic acids, and particularly those those containing over 20 sialic acid
units,
are large enough to stimulate an effective immune response. One specific
example
of a suitable polysialic acid unit is a2-8 polysialic acid as the sialic acid
unit-
containing cell surface markerfor modification, since this marker is well
recognized
as a characteristic of certain cancer cells, and its biochemical synthesis is
quite
well understood. A variety of different N-acyl modifications can be made to
the a2-
8 polysialic acid for use in the present invention, by appropriate chemical
modification of the mannosamine precursor. One can introduce the appropriate
chemical modifying group by simple chemical reaction of the mannosamine with
the organic acid carrying the appropriate acyl group, i.e. an acid (or acid
equivalent such acid halide, anhydride, etc.) of formula R-COOH where R
represents a C~_s alkyl group, straight or branch chained, a C,_6 alkenyl
group,
straight or branch chained, a C~_6 ketonic group or the like. Specific
preferred
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examples include propionic acid and levulinic acid.
The invention is further described, for illustrative purposes, in the
following specific
examples.
Example 1- Synthesis of N-propionylated Group B meningiococcal polysaccharide
- tetanus toxoid (NPrGBMP-TT) conjugate vaccine (ref.):
1 ) Synthesis of NPRGBMP
Colominic acid (500 mg) was N-deacetylated in 2N NaOH (15ml) containing NaBH4
(50 mg) for 7 hours at 105°C. The solution was neutralized with 5N HCI
to ca. pH
8.0 and then dialyzed against distilled water overnight. The resulting product
was
sized by passing a Bio-Gel A.5 column to collect the fraction of MW ~ 10-11
kD.
After lyophilization, deacetylated GBMP was obtained (370 mg. 'H NMR of the
product indicated that the deacetylation was complete.
To the deacetylated GBMP (300 mg) solution in 0.1 N NaOH (10 ml) was added
propionyl anhydride (1 ml) in 5 portions while 2N NaOH was added to maintain
the
pH at 8-9. Four hours later, the reaction was adjusted to pH 11-12 and stirred
for
1 h. The reaction mixture was dialyzed against distilled water and then
lyophilized
to give NPrGBMP (310 mg). 'H NMR of the product indicated that the
propionylation was complete.
2) Synthesis of NPrGBMP-TT conjugate
NPrGBMP (150 mg) was activated by oxidation of the non-reducing terminus using
Na104 (100 mg) in 0.1 N NaOAc-HOAc solution at pH 6.5 overnight. The mixture
was dialyzed against distilled water and then lyophilized.
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Oxidized NPrGBMP (20 mg), freshly prepared TT monomer (8 mg)
and NaBCNH3 (10 mg) were dissolved in 0.1 N NaHC03 (1 ml) and the mixture was
incubated at room temperature for 3 days. The product was separated by gel
filtration chromatography on a Bio-Gel A.5 column (2x45 cm). The first peak
contained the expected glycoconjugate. It was then dialyzed and lyophilized to
give NPrGBMP-TT conjugate (8.1-8.2 mg). Sialic acid content of the conjugate
was determined using the resorcinol method and the protein content was
measured using BCA protein microanalysis. The final conjugate was found to
contain about 20% (wt/wt) of sialic acid, which is equivalent to 4 sialic acid
chains
per TT molecule.
In similar manner, GBMP-TT conjugate, as control conjugate vaccine, was also
prepared.
Ref. R.A. Pon, M. Lussier, Q.-L. Yang, H.J. Jennings, J. Exp. Med., 1997, 185,
1929.
EXAMPLE 2 - Synthesis of the biosynthetic precursor of sialic acid - N-
propionyl
mannosamine (NPrMan)
To a solution of D-mannosamine hydrogen chloride (10 g) in 0.1 N NaOH (40 ml)
was added propionyl anhydride (5 ml) in portions while 2N NaOH was added to
the
solution to maintain the pH at 6.5-7Ø Four hours later, the reaction mixture
was
purified by chromatography on a Silica-Gel column (10x20 cm) using ethyl
acetate
and methanol (2:1 -1:1 ) as eluent. The first portion contained various
partially 0-
acetylated N-propionyl mannosamines and some N-propionyl mannosamine
(NPrMAN). The second pure portion was the expected product of NPrMan (6.4 g)
as a mixture of both a-(60%) and ~3-anomers (40%). 'H NMR(D20):5.09 (s, a H-1
),
5.01 (s, ~i H-1 ), 4.44 (d, J 3.5 Hz, (3 H-2), 4.30 (d, J 4.0 Hz, a H-2), 4.03
(dd, J 3.5,
9.8 Hz, a H-3), 3.88-3.76 (m), 3.60 (t, J 9.5 Hz, a H-4), 3.50 (t, J 9.8 Hz,
~3 H-4),
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3.39 (m, a H-5), 2.35-2.28 (m, CH3CHZC0), 1.12-1.04 (m, CH3CHZC0).
EXAMPLE 3 - Surface expression of N-Propionylated Polysialic acid;
Tumor cells (RMA) were incubated with the precursor N-propionylated
mannosamine for various time periods indicated in Figure 1 in RPMI plus 8%
FBS.
After each incubation, cells were harvested and incubated with either 13D9
(anti-
N-propionyl polysialic acid) or 735 (anti-N-acetyl polysialic acid) monoclonal
antibodies. Subsequently, cells were stained with FITC anti-mouse IgG2a
l0 antibody and fixed with 1 % formaldehyde before analysis by Flow cytometry.
Figure 1 clearly indicates that the binding of 13D9 antibody on tumor cells
increases with the duration of incubation of the precursor N-propionylated
mannosamine in the previous culture. Interestingly, the binding of 735
antibody
(which recognizes N-acetyl polysaccharide) is down-regulated with the
incorporation of N-propionylated mannosamine. Figure 2 shows the measurement
of the mean fluorescent intensity of tumor cells binding to 13D9 or 735
antibodies.
Clearly, the expression of N-propionyl groups on the surtace polysialic acid
of
tumor cells is associated with a reduction in their expression of N-acetyl
groups.
This data suggests that the precursor N-propionylated mannosamine is
metabolized efficiently by the tumor cells.
EXAMPLE 4- Susceptibility to cell death due to expression of N-Propionylated
Polysialic acid:
It was determined whether the expression of N-propionylated sialic acid makes
cells more susceptible to killing by the antibody 13D9 (anti-N-propionyl
polysialic
acid). After feeding tumor cells with the precursor for various time intervals
(indicated in Figure 3), cells were harvested and incubated with either 13D9
or 735
monoclonal antibodies for 4 hours in the presence of rabbit complement (low
toxicity grade). Cytotoxicity towards tumor cells was measured by MTT method
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that measures the viability of cells in culture (Figure 3). In the presence of
complement alone, low level cytotoxicity was noted. The addition of monoclonal
antibody 735 resulted in a strong cytotoxicity indicating that the expression
of sialic
acid makes the cells vulnerable to killing depending on the presence of
antibody.
This cytotoxicity, mediated by the antibody 735, persisted at various time
points
tested and only at late time point was a reduction in cytotoxicity mediated by
735
observed. Taken together with Figure 2, this indicates that even if the
expression
of N-acetyl groups is low on the surface of tumor cells, the level of
expression is
still sufficient to result in tumor cell killing. The addition of the antibody
13D9
during the incubation resulted in an increase in killing of tumor cells in a
time
dependent manner. This correlates with the increase in the expression of N-
propionyl groups on sialic acid on the tumor cells (Figure 2). Before
incubation of
cells with the precursor, a background killing of 25% was observed and this
killing
went up to about 80% with the incorporation of N-propionyl groups on the
surface
of tumor cells. Similar results have also obtained with polyclonal antisera
against
N-propionyl polysialic acid. Similar results were also obtained with another
tumor
cell line.
Taken together these results indicate that tumor cells can be made to
metabolize
and incorporate a precursor N-propionyl mannosamine which gets expressed on
the surface polysialic acid residues of the tumor cells. This expression of N-
propionyl polysialic acid makes the tumor cells vulnerable to immune attack
depending on the presence of antibody against N-propionyl polysialic acid.
