Biochemical Engineering of Surface α2–8 Polysialic Acid for Immunotargeting Tumor Cells

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 theN-acetyl groups of surface α2–8 polysialic acid withN-propionyl groups. Expression of the altered α2–8N-propionylpolysialic acid on the surface of tumor cells induced their susceptibility to cell death mediated by monoclonal antibody 13D9 (mAb 13D9), which specifically recognizes α2–8N-propionylated polysialic acid. The expression of α2–8 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. 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 theN-acetyl groups of surface α2–8 polysialic acid withN-propionyl groups. Expression of the altered α2–8N-propionylpolysialic acid on the surface of tumor cells induced their susceptibility to cell death mediated by monoclonal antibody 13D9 (mAb 13D9), which specifically recognizes α2–8N-propionylated polysialic acid. The expression of α2–8 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. N-propionylated polysialic acid 3-(4,5-dimethulthiazol-2-yl)2,5-diphenyltetrazolium bromide N-propionyl-d-mannosamine fetal bovine serum phosphate-buffered saline 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 (2Shames S.L. Simon E.S. Christopher C.W. Schmid W. Whitesides G.M. Yang L.L. Glycobiology. 1991; 1: 187-191Crossref PubMed Scopus (47) Google Scholar, 3Lin C.-H. Sugai T. Halcomb R.L. Ichikawa Y. Wong C.-H. J. Am. Chem. Soc. 1992; 114: 10138-10145Crossref Scopus (111) Google Scholar, 4Kosa R.E. Brossmer R. Gross H.-J. Biochem. Biophys. Res. Commun. 1993; 190: 914-920Crossref PubMed Scopus (19) Google Scholar, 5Sparks M.A. Williams K.W. Lukacs C. Schrell A. Priebe G. Spaltenstein A. Whitesides G.M. Tertahedron. 1993; 49: 1-12Crossref Scopus (58) Google Scholar) have been exploited for the bioengineering of cell surface molecules. This strategy was first reported by Reutter and co-workers (6Kayser H. Zeitler R. Kannicht C. Grunow D. Nuck R. Reutter W. J. Biol. Chem. 1992; 267: 16934-16938Abstract Full Text PDF PubMed Google Scholar, 7Keppler O.T. Stehling P. Herrman M. Kayser H. Grunow D. Reutter W. Pawlita M. J. Biol. Chem. 1995; 270: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), who demonstrated that exposing mammalian cells in tissue culture andin vivo, to different N-acylmannosamine precursors, resulted in the expression of the unnaturalN-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 (7Keppler O.T. Stehling P. Herrman M. Kayser H. Grunow D. Reutter W. Pawlita M. J. Biol. Chem. 1995; 270: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 8Herrman M. von der Lieth C.W. Stehling P. Reutter W. Pawlita M. Virology. 1997; 71: 5922-5931Crossref Google Scholar). More recently, Bertozzi and co-workers (9Mahal L.K. Yarema K.J. Bertozzi C.R. Science. 1997; 216: 1125-1128Crossref Scopus (612) Google Scholar) have exploited this enzymatic permissiveness further by successfully usingN-levulinoylmannosamine as the precursor to introduceN-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 (10Slovan S.F. Scher H.I. Semin. Oncol. 1999; 26: 448-454PubMed Google Scholar) 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 α2–8 polysialic acid (polysialic acid) as our target antigen, because although not a universal cancer antigen, it is found on a number of important cancers (11Troy F.A. Glycobiology. 1992; 2: 5-23Crossref PubMed Scopus (322) Google Scholar, 12Roth J. Zuber C. Komminoth P. Scheidegger E.P. Warhol M.J. Bitter-Suermann D. Heitz P.U. Roth J. Rutishauser U. Troy F.A. Polysialic Acid. Birkhauser Verlag, Basel, Switzerland1993: 335-348Google Scholar, 13Martersteck C.M. Kedersha N.L. Drapp D.A. Tsui T.G. Colley K.J. Glycobiology. 1996; 6: 289-301Crossref PubMed Scopus (77) Google Scholar), and there is strong evidence that it is associated with metastasis (12Roth J. Zuber C. Komminoth P. Scheidegger E.P. Warhol M.J. Bitter-Suermann D. Heitz P.U. Roth J. Rutishauser U. Troy F.A. Polysialic Acid. Birkhauser Verlag, Basel, Switzerland1993: 335-348Google Scholar, 14Scheidegger E.P. Lackie P.M. Papay J. Roth J. Lab. Invest. 1994; 70: 95-105PubMed Google Scholar). In addition we have previously demonstrated that in its N-propionylated form (NPr polysialic acid)1 it is an excellent immunogen (15Jennings H.J. Roy R. Gamian A. J. Immunol. 1986; 137: 1708-1713PubMed Google Scholar, 16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar). 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 (15Jennings H.J. Roy R. Gamian A. J. Immunol. 1986; 137: 1708-1713PubMed Google Scholar, 16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar). 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 (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar). The rat leukemic cell line (RBL-3H3) (13Martersteck C.M. Kedersha N.L. Drapp D.A. Tsui T.G. Colley K.J. Glycobiology. 1996; 6: 289-301Crossref PubMed Scopus (77) Google Scholar) 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). Female C57BL/6 mice were purchased from Charles Rivers (Montreal, Quebec, Canada) and maintained in our Institutional Animal Facility. NAc and NPr polysialic acids (11-kDa fractions) were obtained from colominic acid as described previously (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar). mAb 13D9, specific for NPr polysialic acid, has been described previously (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar); mAb 735, specific for polysialic acid (17Frosch M. Gorgen I. Boulnois G.T. Bitter-Suermann D. Proc. Natl. Acad, Sci. U. S. A. 1985; 82: 1194-1198Crossref PubMed Scopus (341) Google Scholar), was the gift of D. Bitter-Suermann (Medizinishe Hochschule, Hannover, Germany). 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. 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 (18Virag L. Kerekgyarto C. Fachet J. J. Immunol. Methods. 1995; 185: 199-208Crossref PubMed Scopus (29) Google Scholar) 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 (A 570 nm) of a known number of tumor cells, and the percent cytotoxicity of the unknown samples was calculated using the formula: % cytotoxicity = (1 − number of live cells/total number of cells) × 100%. 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 (7Keppler O.T. Stehling P. Herrman M. Kayser H. Grunow D. Reutter W. Pawlita M. J. Biol. Chem. 1995; 270: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). We then performed a series of experiments to demonstrate that both a rat leukemic cell line (RBL-2H3) (13Martersteck C.M. Kedersha N.L. Drapp D.A. Tsui T.G. Colley K.J. Glycobiology. 1996; 6: 289-301Crossref PubMed Scopus (77) Google Scholar) and a mouse leukemic cell line (RMA) (19Kärre K. Ljunggren H.G. Piontek G. Kiessling R. Nature. 1986; 319: 675-678Crossref PubMed Scopus (1706) Google Scholar) 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.1 A) and for the same time at difference concentrations (Fig.1 B). The pretreated cells were stained with mAb 13D9, specific for NPr polysialic acid (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar). 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. 1 B)-dependent. The RBL-2H3 cells above were, in addition to mAb 13D9, also stained with mAb 735, specific for polysialic acid (17Frosch M. Gorgen I. Boulnois G.T. Bitter-Suermann D. Proc. Natl. Acad, Sci. U. S. A. 1985; 82: 1194-1198Crossref PubMed Scopus (341) Google Scholar). 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. 1 B). 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.1 C). 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.Figure 1NPr 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 inA. 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 Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 2 A). Previous studies (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar) 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 (20Baumann H. Brisson J.-R. Michon F. Pon R. Jennings H.J. Biochemistry. 1993; 32: 4007-4013Crossref PubMed Scopus (55) Google Scholar). 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 contiguousN-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. 2 B). 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. 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 (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar), 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. 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 (106cells/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 monitored by of tumor The data showed that in with ManNPr, mAb 13D9 a effect on tumor mAb 13D9 although mAb 13D9 alone was also to tumor when with a control group of mice results indicate although this bioengineering procedure is to tumor it is not to tumor cells from the This can be by the fact that the was a of RMA cells, some of which were not and and were to the helical epitope of NPr polysialic acid on which the cytotoxicity of mAb 13D9, in the of ManNPr, (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar). of the solid tumor cells to polysialic acid was when mAb 735 failed to bind to tumor cells from the mice (data not our to solid we carried experiments to determine whether our bioengineering strategy could be to the of cancer cells. We have shown that leukemic cells and polysialic acid on their and it is on the basis of our results that in their they a high density of this surface (12Roth J. Zuber C. Komminoth P. Scheidegger E.P. Warhol M.J. Bitter-Suermann D. Heitz P.U. Roth J. Rutishauser U. Troy F.A. Polysialic Acid. Birkhauser Verlag, Basel, Switzerland1993: 335-348Google Scholar). This be to their because polysialic acid, in addition to its (15Jennings H.J. Roy R. Gamian A. J. Immunol. 1986; 137: 1708-1713PubMed Google Scholar), is also a inhibitor of complement PubMed Google Scholar). This for the fact that polysialic acid is the in both group B meningococci F. I. J. Med. PubMed Scopus Google Scholar). The experiments in mice were carried as described for the solid tumor using RMA cells, except that in this the of the mice were for the of cells. of a cell of the of the mice was used to the tumor cell Following cell of the cells the tumor cells were from the normal cells by data in and that there were tumor cells in the of the mice treated with a of mAb 13D9 and ManNPr, indicating that tumor cells were and were from the NPr polysialic acid control tumor metastasis in in + of tumor cells, mAb 13D9, and ManNPr into mice was carried as described in the to were and cell in medium RPMI = 8% of the aliquots from the individual mice were used to serial 2-fold in 24-well in 1 of 8% were and monitored a period of 1 to wells samples that tumor cells were and the samples that tumor cells at dilutions were in a of metastasis by of cells from individual cells treated + mAb of of total cell was used to a 2-fold serial The samples that tumor cells were The samples that tumor cells were and were subjected to by serial in a of tumor cells, mAb 13D9, and ManNPr into mice was carried as described in the to were and cell in medium RPMI = 8% of the aliquots from the individual mice were used to serial 2-fold in 24-well in 1 of 8% were and monitored a period of 1 to wells samples that tumor cells were and the samples that tumor cells at dilutions were of total cell was used to a 2-fold serial The samples that tumor cells were The samples that tumor cells were and were subjected to by serial The data also that mAb 13D9 alone could also the metastasis of tumor cells to a in with a control group of mice and for this is that the cytotoxicity of mAb 13D9 can be to its to a unique polysialic epitope found only on the surface of in RMA cells. This has some because a similar cytotoxic epitope is expressed on group B meningococci and The epitope is in and is to be on the surface of the by the of extended helical of their α2–8 polysialic acid with surface (16Pon R.A. Lussier M. Yang Q.-L. Jennings H.J. J. Exp. Med. 1997; 185: 1929-1938Crossref PubMed Scopus (96) Google Scholar, H.J. Gamian A. Michon F. J. Immunol. Google Scholar). the expression of this of epitope did not result in the cytotoxicity of the cells is not In we have demonstrated in mice that the metastasis of tumor cells can be controlled by bioengineering their surface polysialic acid to their N-propionylated and then by based on antibodies specific for the modified antibodies could be administered as described or induced in by using an appropriate NPr polysialic Although this strategy was only to the growth of tumor cells, its be because of the of being to successfully control metastasis in the treatment of A with the of this strategy for the immunotargeting of cancer cells, which to their chemotargeting (9Mahal L.K. Yarema K.J. Bertozzi C.R. Science. 1997; 216: 1125-1128Crossref Scopus (612) Google Scholar), is that in ManNPr, be by both normal and cancer cells the successful application of both the above on a of using polysialic acid as our target we can specificity mediated by the because although polysialic acid is ubiquitous on fetal it is only found in a (12Roth J. Zuber C. Komminoth P. Scheidegger E.P. Warhol M.J. Bitter-Suermann D. Heitz P.U. Roth J. Rutishauser U. Troy F.A. Polysialic Acid. Birkhauser Verlag, Basel, Switzerland1993: 335-348Google Scholar, J. Bitter-Suermann D. C. J. Immunol. Google Scholar, J. Zuber C. P. C. Heitz P.U. C. Bitter-Suermann D. Proc. Natl. Sci. U. S. A. PubMed Scopus Google Scholar). In addition NPr polysialic acid conjugates have been successfully used as human vaccines group B meningococcal in a number of (15Jennings H.J. Roy R. Gamian A. J. Immunol. 1986; 137: 1708-1713PubMed Google Scholar, Michon F. J. 1997; PubMed Scopus Google Scholar). Although the application of the above strategy to on cancer cells is also it be more because the are also found on it the of different of specificity to target cancer cells. specificity could be in these by the densities of some of these on normal and cancer of cells or by the of the precursor can be to cancer cells. We D. Bitter-Suermann for mAb 735 and H. G. Ljunggren for the mouse leukemic cell line

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