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J. Biol. Chem., Vol. 275, Issue 42, 32832-32836, October 20, 2000
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From the Institute for Biological Sciences, National Research
Council of Canada, Ottawa, Ontario K1A 0R6, Canada
Received for publication, August 23, 2000
To target tumor cells for immunotherapy, we
evaluated the feasibility of altering the epitopes on the surface
polysialic acid of tumor cells. A precursor
(N-propionylmannosamine), when incubated with leukemic
cells, RBL-2H3 and RMA, resulted in substitution of the
N-acetyl groups of surface Sialic acid is ubiquitous on the surface of eukaryotic cells,
where as a glycoconjugate substituent, it is involved in a number of
crucial biological processes (1). The permissiveness of the enzymes
involved in sialic acid biosynthesis and sialoside formation (2-5)
have been exploited for the bioengineering of cell surface molecules.
This strategy was first reported by Reutter and co-workers (6, 7), who
demonstrated that exposing mammalian cells in tissue culture and
in vivo, to different N-acylmannosamine precursors, resulted in the expression of the unnatural
N-acylated sialic acid residues on the cell surface
glycoconjugates. This technique was used by the authors to study the
effect of cell surface sialoside structural changes on viral receptors
(7, 8).
More recently, Bertozzi and co-workers (9) have exploited this
enzymatic permissiveness further by successfully using
N-levulinoylmannosamine as the precursor to introduce
N-levulinoylsialic residues on the surface of a
number of human cell lines. This procedure introduces unique active
keto groups on the surface of the cells, which via the use of
appropriate chemical reagents, can be used for the chemotargeting of drugs.
We now report the successful application of the enzymatic
permissiveness of sialic acid to the immunotargeting of cancer cells and the potential of our protocol to further the development of efficacious carbohydrate-based vaccines. Although some success has been
reported (10) in creating cancer vaccines based on cell surface
glycoconjugate antigens, the area remains problematic due to the fact
that cancer cells fail to produce markers that distinguish them from
normal cells. Population densities of cell surface carbohydrate
antigens of cancer cells do differ from those of normal cells, but
their individual structures are identical. Thus glycoconjugate vaccines
based on these antigens are poorly immunogenic. Therefore we propose to
introduce modified carbohydrate antigens on the surface of cancer cells
to which a strong immunogenic response can be induced. We chose Cell Lines--
The rat leukemic cell line (RBL-3H3) (13) was
obtained from the American Type Culture Collection (Manassas, VA), and
the mouse leukemic cell line (RMA) was the gift of H. G. Ljunggren (Karolinska Institute, Stockholm, Sweden).
Mice--
Female C57BL/6 mice were purchased from Charles Rivers
(Montreal, Quebec, Canada) and maintained in our Institutional Animal Facility.
Polysialic Acids--
NAc and NPr polysialic acids (11-kDa
fractions) were obtained from colominic acid as described previously
(16).
Monoclonal Antibodies--
mAb 13D9, specific for NPr polysialic
acid, has been described previously (16); mAb 735, specific for
polysialic acid (17), was the gift of D. Bitter-Suermann (Medizinishe
Hochschule, Hannover, Germany).
Flow Cytometry--
For flow cytometry, cells were incubated
with mAbs 13D9 or 735 in 50 µl of RPMI + 1% FBS on ice. After 30 min
the cells were washed and incubated with fluorescein
isothiocyanate anti-mouse IgG2a (obtained from Cedarlane
Laboratories, Ontario, Canada) in 50 µl of RPMI + 1% FBS on ice.
After another 30 min the cells were washed and fixed in 1%
formaldehyde and assayed on a flow cytometer (Coulter Incorporation,
Miami, FL). Fluorescence intensities are expressed in arbitrary units.
Antibody-dependent Cytotoxicity--
For
antibody-dependent cytotoxicity measurements, 1 × 106 cells were pretreated with ManNPr in 24-well plates.
Tumor cells (1-2 × 104), after treatment with
ManNPr, were harvested, washed with PBS, and incubated with antibodies
(735 or 13D9, 1 mg/ml) on ice for 1 h. Cells were washed and
incubated with 10% rabbit complement (Cedarlane Laboratories, Ontario,
Canada) at 37 °C for 2 h. The cytotoxic assay was performed as
described previously (18) in 96-well plates, and cell viability was
measured by the MTT colorimetric method. MTT was dissolved at a
concentration of 5 mg/ml in PBS, and the solution was sterilized by
filtration. After adding 10 µl of MTT solution into each well, cells
were incubated for 4 h. 150 µl of 1.5 M HCl and 500 µl of isopropyl alcohol were used to rupture the cells. A
standard curve was established by measuring MTT incorporation
(A570 nm) of a known number of tumor cells, and the percent cytotoxicity of the unknown samples was calculated using the formula: % cytotoxicity = (1 Inhibition of Antibody-dependent Toxicity--
For
inhibition of antibody-dependent cytoxicity, RMA
cells were preincubated with ManNPr (2 mg/ml) for 24 h, and the
washed cells (1-2 × 104 in 35µl of PBS) were
distributed into wells of a 96-well plate. 25 µl of mAb 13D9 (20 µg/ml) was then added to each well. This was followed by 40 µl of
NAc or NPr polysialic acids (1 mg/ml) into the first well with the
2-fold serial dilutions of the inhibitor solution in subsequent wells.
The cells were washed and incubated with rabbit complement at
37 oC for 2 h, and the cytotoxic assay was performed
as described above.
To examine the feasibility of our strategy for targeting cancer
cells, we first synthesized the required precursor ManNPr, essentially
using a previously described method (7). We then performed a series of
experiments to demonstrate that both a rat leukemic cell line (RBL-2H3)
(13) and a mouse leukemic cell line (RMA) (19) can incorporate ManNPr
into the cell surface polysialic acid (Fig.
1). RBL-2H3 cells were treated with
ManNPr at the same concentration for different times (Fig.
1A) and for the same time at difference concentrations (Fig.
1B). The pretreated cells were stained with mAb 13D9,
specific for NPr polysialic acid (16). Flow cytometric analysis
indicated that the uptake of ManNPr, as determined from the relative
surface expression of NPr polysialic acid, was both time- (Fig. 1A) and
dose (Fig. 1B)-dependent. The RBL-2H3 cells
above were, in addition to mAb 13D9, also stained with mAb 735, specific for polysialic acid (17). The predominant specificities of
these mAbs allowed for the successful monitoring of the transformation
of the cell surface polysialic acid to its N-propionylated
analog. Flow cytometric analysis showed that as the expression of
polysialic acid on the cell surface declined with exposure of the cells
to increasing amounts of ManNPr, the expression of NPr polysialic acid
on the cell surface increased (Fig. 1B). RMA cells gave
similar flow cytometric profiles when subjected to the above
experiments (data not shown), and from these data curves depicting the
time dependence of the transformation of the polysialic acid on the
surface this cell line to NPr polysialic acid were constructed. (Fig.
1C). The curves indicate that as the density of NPr
polysialic acid on the cell surface increases with time and eventually
plateaus, the density of polysialic acid decreases and plateaus
concomitantly.
To determine whether NPr polysialic acid is a useful marker to target
and kill tumor cells, assays of antibody-dependent
cytotoxicity were carried out, and the results are shown in Fig.
2, A and B. Following preculture with the precursor (ManNPr), RBL-2H3 cells were
further treated with mAb 13D9 and incubated with rabbit complement at
37 °C. The resultant cell counts demonstrated that lysis of tumor
cells was dependent only on the time and dose of their exposure to
ManNPr, because mAb 13D9 alone failed to lyse the cells. Thus, the more
NPr polysialic acid was expressed on the cell surface, the more cells
were killed (Fig. 2A). Previous studies (16) demonstrated
that although mAb 13D9 did not cross-react with polysialic acid, its
antigenic specificity has some similarities, being based on an epitope
located on an extended helical segment (n > 10) of NPr
polysialic acid (20). Thus our results show that ManNPr can be
incorporated into the cells in sufficient quantities to form this
complex epitope, which has a requirement for many contiguous N-propionylated sialic acid residues. To confirm this result
further, RMA cells were subjected to the same assay except that mAb 735 was used as the antibody. mAb 735 exhibited strong binding to the
native cell surface polysialic acid and also mediated strong killing of
the RMA cells. However, this killing was reduced in a
time-dependent manner as ManNPr was incorporated into the
cells (Fig. 2B). The killing of tumor cells by rabbit
complement alone was not significant, thus indicating that the
cytoxicity of the above cells is controlled by the specificity of the
antibody used.
Biochemical Engineering of Surface
2-8 Polysialic Acid for
Immunotargeting Tumor Cells*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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2-8 polysialic acid with
N-propionyl groups. Expression of the altered 2-8
N-propionylpolysialic acid on the surface of tumor
cells induced their susceptibility to cell death mediated by monoclonal
antibody 13D9 (mAb 13D9), which specifically recognizes 2-8
N-propionylated polysialic acid. The expression of
2-8 N-propionylated polysialic acid and the lysis of
tumor cells by antibody-dependent cytotoxicity depended on
the time and dose of incorporation of N-propionylated
mannosamine. In vivo, mAb 13D9 effectively controlled
metastasis of leukemic cells RMA when mice were administered the
precursor N-propionylated mannosamine.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-8
polysialic acid (polysialic acid) as our target antigen, because
although not a universal cancer antigen, it is found on a number of
important cancers (11-13), and there is strong evidence that it is
associated with metastasis (12, 14). In addition we have previously
demonstrated that in its N-propionylated form (NPr
polysialic acid)1 it is an
excellent immunogen (15, 16). In fact it is the basis of a potential
group B meningococcal vaccine and is able, when conjugated to a protein
carrier, to induce in mice high affinity NPr polysialic acid-specific
antibodies (15, 16). Although NPr polysialic acid protein conjugates do
induce some antibodies that cross-react with polysialic acid, the
protective antibody is predominantly based on a
length-dependent (helical) form of the NPr polysialic acid,
which mimics a unique capsular epitope on the surface of group B
meningococci (16).
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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number
of live cells/total number of cells) × 100%.
RESULTS AND DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
NPr polysialic acid expression on the surface
of tumor cells. A, rat leukemia cells (RBL-2H3) were
incubated with 4 mg/ml ManNPr in RPMI medium supplemented with 8% FBS
for 3 days. At daily intervals aliquots of the cells were harvested,
and the expression of NPr polysialic acid was monitored by flow
cytometry using mAb 13D9. B, RBL-2H3 cells were incubated
with different concentrations of ManNPr in the same medium described in
A. Following harvesting of the cells the expression of
polysialic acid and its NPr analog were measured by flow cytometry
using mAb 735 and mAb 13D9, respectively. C, mouse leukemic
cells (RMA) were incubated with 2 mg/ml ManNPr, and the expression of
polysialic and its NPr analog were measured by flow cytometry using mAb
735 and mAb 13D9, respectively.
View larger version (14K):
[in a new window]
Fig. 2.
Antibody-mediated cytotoxicity is dependent
on the expression of NPr polysialic on tumor cells. A,
RBL-2H3 cells were incubated with increasing concentrations of ManNPr
for 3 days. At daily intervals the cells were harvested, washed with
PBS, and incubated with mAb 13D9 as described previously. The cells
were then subjected to a cytotoxicity assay (18). B, RMA
cells were incubated with ManNPr (4 mg/ml), and aliquots were harvested
at different time intervals. They were then washed with PBS and
incubated without antibody, with mAb 735 and mAb 13D9, and subjected to
the cytotoxicity assay as described previously.
Confirmatory evidence that the cytotoxicity of RMA cells is mediated by
surface NPr polysialic acid was obtained by showing that cytotoxicity
could be inhibited by NPr polysialic acid (Fig. 3). Although we have also demonstrated
previously that mAb 13D9 does not bind to short NPr
sialooligosaccharides (16), we cannot, however, eliminate the
possibility that nonspecific binding to these antigens occurs when they
are situated on the surface of RMA cells. If this did occur it could
also possibly result in them making a contribution to the total
cytotoxic effect.
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To determine whether the above bioengineering procedure could control
tumor growth in vivo, we established a mouse solid tumor model. Mice were inoculated with RMA cells (106
cells/mouse), and 5 days after inoculation the mice were treated daily
with mAb 13D9 (200 µg/mouse) and precursor ManNPr (5 mg/mouse) for a
period of 8 days. Tumor growth was routinely monitored by measurement
of tumor size. The data showed that in combination with ManNPr, mAb
13D9 had a greater effect on tumor size than mAb 13D9 alone, although
mAb 13D9 alone was also able to reduce tumor size when compared with a
control group of mice (Figs. 4, A-C). These results indicate what although this
bioengineering procedure is able to curtail tumor growth, it is not
able to completely eradicate tumor cells from the mice. This can be
explained by the fact that the original inoculum was a mixture
of RMA cells, some of which were not polysialylated (Figs. 1,
A and B), and were therefore unable to express
the helical epitope of NPr polysialic acid on which the cytotoxicity of
mAb 13D9, in the presence of ManNPr, depends (16). Failure of the solid
tumor cells to express polysialic acid was confirmed when mAb 735 failed to bind to tumor cells extracted from the mice (data not
shown).
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Despite our failure to eradicate solid tumors, we carried out experiments to determine whether our bioengineering strategy could be applied to the elimination of metastatic cancer cells. We have shown that leukemic cells (RMA and RBL-2H3) already express polysialic acid on their surfaces, and it is likely, on the basis of our results (see later), that in their metastatic forms they still express a high density of this surface antigen (12). This would be to their advantage, because polysialic acid, in addition to its poor immunogenicity (15), is also a powerful inhibitor of alternative complement pathway activation (21). This accounts for the fact that polysialic acid is the major virulence factor in both pathogenic group B meningococci and Escherichia coli K1 (22). The experiments in mice were carried out as described for the solid tumor using RMA cells, except that in this case the spleens of the mice were analyzed for the presence of metastatic cells. One-fifth of a cell suspension of the whole spleen of the mice was used to initiate the tumor cell limiting dilution experiment. Following cell cultures of the spleen cells the metastasized tumor cells were easily distinguished from the normal spleen cells by microscopic examination. Our data in Tables I and II show that there were no tumor cells in the spleen of the mice treated with a combination of mAb 13D9 and ManNPr, indicating that all transported metastasized tumor cells were polysialylated and therefore were completely eliminated from the mice.
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The data also revealed that mAb 13D9 alone could also partially reduce
the metastasis of tumor cells to a certain extent in comparison with a
control group of mice (Tables I and II). One plausible explanation for
this phenomenon is that the cytotoxicity of mAb 13D9 can be attributed
to its ability to recognize a unique polysialic acid-associated epitope
found only on the surface of in vivo RMA cells. This
hypothesis has some credence, because a similar cytotoxic epitope is
expressed on group B meningococci and E. coli K1. The
epitope is composite in nature and is thought to be formed on the
surface of the bacteria by the interaction of extended helical segments
of their 2-8 polysialic acid capsules with another, probably lipid,
surface component (16, 23). Why the expression of this type of epitope
did not result in the complete cytotoxicity of all the metastatic cells
is not known.
In summation we have demonstrated in mice that the metastasis of tumor cells can be controlled by bioengineering their surface polysialic acid glycoconjugates to their N-propionylated analogs and then by applying immunotherapy based on antibodies specific for the modified antigen. These antibodies could be either passively administered as described herein or induced in situ by direct immunization using an appropriate NPr polysialic acid-protein conjugate vaccine. Although this new immunotherapeutic strategy was only partially able to inhibit the growth of tumor cells, its significance cannot be underestimated because of the importance of being able to successfully control metastasis in the treatment of cancer.
A serious problem with the implementation of this strategy for the
immunotargeting of cancer cells, which applies equally to their
chemotargeting (9), is that in all likelihood any precursor, including
ManNPr, will be taken up by both normal and cancer cells alike.
Therefore, the successful application of both the above protocols will
depend on a means of achieving specificity. By using polysialic acid as
our target antigen we can achieve specificity mediated by the immune
response, because although polysialic acid is ubiquitous on fetal
tissue, it is only found in a few discrete adult tissues (12, 24, 25).
In addition NPr polysialic acid conjugates have been successfully used
as experimental human vaccines against group B meningococcal in a number of animal species without deleterious consequences (15, 26).
Although the application of the above strategy to other sialylated
glycoconjugates on cancer cells is also theoretically possible, it will
be more difficult, because the former are also found on adult
tissues. Therefore, it will require the introduction of different
methods of achieving specificity to preferentially target cancer cells.
Perhaps specificity could be generated in these cases by
exploiting the differing densities of some of these glycoconjugates on
normal and cancer of cells or by the introduction of new technologies
whereby the precursor can be preferentially delivered to cancer cells.
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ACKNOWLEDGEMENTS |
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We thank D. Bitter-Suermann for providing mAb 735 and H. G. Ljunggren for providing the mouse leukemic cell line (RMA).
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FOOTNOTES |
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* This is National Research Council of Canada publication number 42421.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 613-990-0821;
Fax: 613-941-1327; E-mail: harry.jennings@nrc.ca.
Published, JBC Papers in Press, September 6, 2000, DOI 10.1074/jbc.C000573200
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ABBREVIATIONS |
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The abbreviations used are: NPr polysialic acid, N-propionylated polysialic acid; MTT, 3-(4,5-dimethulthiazol-2-yl)2,5-diphenyltetrazolium bromide; ManNPr, N-propionyl-D-mannosamine; FBS, fetal bovine serum; PBS, phosphate-buffered saline.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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