Purification of immunoprotective tumor antigens by preparative isotachophoresis (original) (raw)

Abstract

Tumor-specific transplantation antigens (TSTA) were purified from 3 M KCl and butanol extracts of C3H/HeJ 3-methylcholanthrene-induced fibrosarcomas by preparative isotachophoresis (pITP). Fractions from pITP which reacted with antisera to TSTA preparations in an enzyme-linked immunospecific assay were tested in vivo for induction of resistance to the growth of transplanted tumor cells. Isotachophoresis of crude 3 M KCl extracts yielded TSTA that was immunogenic at doses between 17 and 124 μg. Isotachophoresis of TSTA partially purified from crude 3 M KCl or butanol extracts by preparative isoelectric focusing (pIEF) of 3 M KCl and butanol extracts yielded TSTA that was immunogenic over a two-fold log dose range. As little as 10 ng purified TSTA reduced tumor growth by 50%. Tumor growth reduction was specific, and immunized animals survived longer than non-immunized controls. A purification of 10,000-fold over crude 3 M KCl extracts and 2,500-fold over crude butanol extracts was obtained. These results suggest that TSTA from murine tumor cells is preparatively purified by following extraction with pIEF and then pITP.

Keywords: Tumor Cell, Tumor Growth, Cancer Research, Butanol, Dose Range

References

• 1.Baldwin RW, Harris JR, Price MR. Fractionation of plasma membrane-associated tumor-specific antigen from an aminoazo dye-induced rat hepatoma. Int J Cancer. 1973;11:385. doi: 10.1002/ijc.2910110216. [DOI] [PubMed] [Google Scholar]
• 2.Basombrio MA. Search for common antigenicities among twenty-five sarcomas induced by methylcholanthrene. Cancer Res. 1970;30:2458. [PubMed] [Google Scholar]
• 3.Bier M, Cuddeback RN, Kopwillem A. Preparative plasma protein fractionation by isotachophoresis in sephadex columns. J Chromatogr. 1977;132:437. doi: 10.1016/s0021-9673(00)82908-8. [DOI] [PubMed] [Google Scholar]
• 4.Billing R, Terasaki PI. Human leukemia antigen. I. Production and characterization of antisera. J Natl Cancer Inst. 1974;53:1635. [PubMed] [Google Scholar]
• 5.Blackstock R, Schimpff RD, Smith RT. Heterogeneity of KCl-solubilized antigens of chemically induced sarcomas. Proc Soc Exp Biol Med. 1978;157:354. doi: 10.3181/00379727-157-40052. [DOI] [PubMed] [Google Scholar]
• 6.Brandchaft PB, Aoki T, Silverman TN. Immunogenic and immunosuppressive components in soluble preparations from Moloney murine leukemia virus-induced tumor cells. Int J Cancer. 1976;17:678. doi: 10.1002/ijc.2910170518. [DOI] [PubMed] [Google Scholar]
• 7.Carbone G, Boicchi M, Parmiani G. Lack of anti-TSTA antibodies in mice immunized with a methylcholanthrene-induced fibrosarcoma. Cancer Immunol Immunother. 1981;11:31. [Google Scholar]
• 8.Coggin JH., Jr Induction of transplantation resistance with soluble Simian virus 40-induced hamster tumor-specific transplantation antigen. Cancer Res. 1979;39:2952. [PubMed] [Google Scholar]
• 9.Davey GC, Currie GA, Alexander P. A serologically detected tumor-specific membrane antigen of murine lymphomas which is not the target for syngeneic graft rejection. Br J Cancer. 1979;40:168. doi: 10.1038/bjc.1979.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Foley EJ. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Res. 1953;13:835. [PubMed] [Google Scholar]
• 11.Gehan EA. A generalized Wilcoxon test for comparing arbitrarily singly-censored samples. Biometrika. 1965;52:203. [PubMed] [Google Scholar]
• 12.Hjalmarsson SG. Preparative isotachophoresis — The effect of using ampholine of different pH ranges as spacer ions in the fractionation of serum proteins. Science Tools. 1975;22:35. [Google Scholar]
• 13.Klein G, Sjogren HO, Klein E, Hellstrom KE. Demonstration of resistance against methylcholanthrene-induced sarcomas in the primary autochthonous host. Cancer Res. 1960;20:1561. [PubMed] [Google Scholar]
• 14.Law LW, Appella E, Dubois GC. Immunogenic properties of a solubilized partially purified tumor rejection antigen (TSTA) from a chemically induced sarcoma. In: Ruddon R. W., editor. Biological markers of neoplasia: Basic and Applied Aspects. Amsterdam: Elsevier/North Holland; 1978. p. 35. [Google Scholar]
• 15.LeGrue SJ, Kahan BD, Pellis NR. Extraction of a murine tumor-specific transplantation antigen with 1-butanol. I. Partial purification by isoelectric focusing. J Natl Cancer Inst. 1980;65:191. [PubMed] [Google Scholar]
• 16.Lennox E, Cohn J, Lowe T. Syngeneic monoclonal antibodies to a methylcholanthrene-induced mouse sarcoma. Transplant Proc. 1980;12:95. [PubMed] [Google Scholar]
• 17.Martin WJ, Gipson TG, Conliffe MA, Cotton WG, Dove LF, Rice JM. Histocompatibility differences between C3HfeB/HeN and C3H/HeN mice: tumor induced in C3HfeB/HeN mice expresses C3H/HeN-associated alloantigens. J Immunogenet. 1978;5:255. doi: 10.1111/j.1744-313x.1978.tb00653.x. [DOI] [PubMed] [Google Scholar]
• 18.Meltzer MS, Leonard EJ, Hardy AS, Rapp HJ. Protective tumor immunity induced by potassium chloride extracts of guinea pig hepatomas. J Natl Cancer Inst. 1975;54:1349. doi: 10.1093/jnci/54.6.1349. [DOI] [PubMed] [Google Scholar]
• 19.Minami A, Mizushima Y, Takeichi N, Hosokawa M, Kobayashi H. Dissociation of antitumor immune responses in rats immunized with solubilized tumor-associated antigens from a methylcholanthrene-induced fibrosarcoma. Int J Cancer. 1979;23:358. doi: 10.1002/ijc.2910230314. [DOI] [PubMed] [Google Scholar]
• 20.Natori T, Law LW, Appella E. Immunochemical evidence of a tumor-specific surface antigen obtained by detergent solubilization of the membranes of a chemically induced sarcoma. Meth A. Cancer Res. 1978;38:359. [PubMed] [Google Scholar]
• 21.Pasternak L, Pasternak G, Karsten V. Immunogenicity of soluble extracts from a UV light-induced mouse sarcoma. Cancer Immunol Immunother. 1978;3:273. [Google Scholar]
• 22.Pellis NR, Kahan BD. Specific tumor immunity induced with soluble materials: restricted range of antigen dose and of challenge tumor load for immunoprotection. J Immunol. 1975;115:1717. [PubMed] [Google Scholar]
• 23.Pellis NR, Kahan BD. Methods to demonstrate the immunogenicity of soluble tumor-specific transplantation antigens. I. The immunoprophylaxis assay. Methods in Cancer Research. 1976;13:291. [Google Scholar]
• 24.Pellis NR, Tom BH, Kahan BD. Tumor-specific and allospecific immunogenicity of soluble extracts from chemically induced murine sarcomas. J Immunol. 1974;113:708. [PubMed] [Google Scholar]
• 25.Pellis NR, Yamagishi H, Macek CM, Kahan BD. Specificity and biological activity of tumor-specific transplantation antigens. Int J Cancer. 1980;26:443. doi: 10.1002/ijc.2910260409. [DOI] [PubMed] [Google Scholar]
• 26.Pellis NR, Yamagishi H, Shulan DJ, Kahan BD. Use of preparative isoelectric focusing in a Sephadex gel slab to separate immunizing and growth facilitating moieties in crude 3 M KCl extracts of a murine fibrosarcoma. Cancer Immunol Immunother. 1981;11:53. [Google Scholar]
• 27.Prehn RT, Main JM. Immunity to methylcholanthrene-induced sarcomas. J Natl Cancer Inst. 1957;18:769. [PubMed] [Google Scholar]
• 28.Price MR, Preston VE, Robins RA, Zoller M, Baldwin RW. Induction of immunity to chemically induced rat tumors by cellular or soluble antigens. Cancer Immunol Immunother. 1978;3:247. [Google Scholar]
• 29.Ransom JH, Schengrund CL, Bartlett GL. Solubilization and partial characterization of tumor-rejection antigen from an ultraviolet-induced murine tumor. Int J Cancer. 1981;27:545. doi: 10.1002/ijc.2910270419. [DOI] [PubMed] [Google Scholar]
• 30.Rao VS, Bonavida B. Specific enhancement of tumor growth and depression of cell-mediated immunity following sensitization to soluble tumor antigens. Cancer Res. 1976;36:1384. [PubMed] [Google Scholar]
• 31.Reisfeld RA, Kahan BD. Biologic and chemical characterization of human histocompatibility antigens. Fed Proc. 1970;29:2034. [PubMed] [Google Scholar]
• 32.Rogers GT, Searle F, Bagshawe KD. Carcinoembryonic antigen: isolation of a sub-fraction with high specific activity. Br J Cancer. 1976;33:357. doi: 10.1038/bjc.1976.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Seon BK, Negoro S, Minowada J, Yoshizaki K. Human T cell leukemia antigens on the cell membranes: purification, molecular characterization and preparation of specific antisera. J Immunol. 1981;127:2580. [PubMed] [Google Scholar]
• 34.Tanaka T, Yamagishi H, Pellis NR, Kahan BD. Production of a tumor-specific xenoantiserum from partially purified immunoprotective tumor antigen. Cancer Immunol Immunother. 1982;13:48. doi: 10.1007/BF00200200. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35.Zar JH. Biostatistical analysis. Englewood Cliffs: Prentice-Hall; 1974. p. 151. [Google Scholar]