CA2279134A1

CA2279134A1 - Novel strategy for carbohydrate-based therapeutic vaccines

Info

Publication number: CA2279134A1
Application number: CA002279134A
Authority: CA
Inventors: Tianmin Liu; Qinling Yang; Subash Sad; Harold J. Jennings; Zhongnu Guo
Original assignee: National Research Council of Canada
Current assignee: National Research Council of Canada
Priority date: 1999-07-29
Filing date: 1999-07-29
Publication date: 2001-01-29
Also published as: WO2001009298A3; AU6420000A; EP1198244A2; IL147776A0; MXPA02000958A; WO2001009298A2; ZA200200731B; JP2003506034A

Abstract

The sialic acid component of a sialic acid unit-containing cell surface marker characteristic of cancerous mammalian cells, such as .alpha.2-8 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 .alpha.2-8 polysialic acid (i.e. various types of cancer cells), the expression of a highly immunogenic surface antigen namely, modified .alpha.2-8 polysialic acid. The modification is suitably N-acylation of a precursor of the sialic acid, so that the N-acylated precursor becomes chemically incorporated in the polysialic acid during its intracellular biochemical synthesis.
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 .alpha.2-8 polysialic acid) and a protein, can then be used to eliminate cells which express .alpha.2-8 polysialic acid. Vaccines can be prepared utilizing conjugates of the modified sialic acid-containing marker, 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.

Description

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 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 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 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 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.

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 _7_ 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.

_8_ 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), 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 - 1~ -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 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 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.

Claims

1. 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.

2. The process of claim 1 wherein the sialic acid unit-containing cell surface marker is a polysialic acid containing at least 20 sialic acid units.

3. The process of claim 2 wherein the polysialic acid is .alpha.2-8 polysialic acid

4. The process of claim 1 wherein the chemically modified precursor is an N-acylated precursor.

5. The process of claim 4 wherein the precursor is N-propionylated-D-mannosamine.

6. Immunogenic mammalian cancer cells, said cells having surface markers incorporating modified sialic acid units capable of initiating an immune response.
in a mammalian system containing them which is sufficiently strong, to effectively combat the proliferation of such cells.

7. A conjugate of a modified poly .alpha.2-8 sialic acid incorporating N-acylated D-mannosamine units and a protein.

8. Use of a conjugate of a modified .alpha.2-8 polysialic acid and a protein in the preparation of vaccine for managing cancer conditions in mammalian patients.