METHOD OF PREPARING SURFACE MEMBRANE ANTIGENS FROM HUMAN CARCINOMA CELLS

Abstract: A method of membrane extraction for preparing surface membrane antigens from human carcinoma cells is described. The method of membrane extraction utilizes multiple sets of saline extraction wherein the salt concentration or molarity is decreased with each set of extractions. The extraction procedure utilizes a single cell suspension of frozen or fresh carcinoma tissue. No water extraction is utilized. The method of membrane extraction avoids cell lysis and the presence of contaminants in the membrane preparation.

Description: METHOD OF PREPARING SURFACE MEMBRANE ANTIGENS FROM HUMAN CARCINOMA CELLS FIELD OF THE INVENTION The present invention is directed to a method of preparing surface membrane antigens from human carcinoma cells utilizing extraction. BACKGROUND OF THE INVENTION Prior to 1966 the preparation of surface membrane antigens from cells required homogenization or physical rup- ture of the cells. The removal and preparation of antigenic material from cells utilizing these procedures were ineffi¬ cient in that (1) the cells still contained a large amount of the antigen, and (2) large amounts of the internal cell contents would be present in the antigen preparation as a contaminant. In an article entitled "Mouse Histocompatibi ' lity Isoantigens Derived from Normal and from Tumour Cells" by Davies, Immunology, 11: 115-125 (1966), a method of surface membrane antigen removal was described wherein the cells from which the antigens were removed were washed with a hypotonic salt to a point just short of lysis. The antigen preparations were disclosed as being obtained in high yields, highly active in terms of antigenic reactivity, substantial¬ ly free of internal cellular contents, and having the cor¬ rect histocompatibility specificities. It was found that the necessary concentration of the salt used in the washing procedure, however, differed depending on the specific cell type in order to avoid cell lysis. For example, mouse lym- phoid cells lysed in 0.6% sodium chloride. Further, mouse leukemia cells could be extracted with 0.1% sodium chloride solution without lysis and mouse sarcoma cells lysed only after washing four times with water. A specific example discussed by Davies involved the collection of spleens, mesenteric lymph nodes and thy- muses in iced normal saline. The iced tissues were chopped and pressed through sieves to give a cell suspension in 0.8% sodium chloride. After standing for thirty minutes in the cold, the cells were centrifuged out at 600 g (g = gravita¬ tional force or force of gravity) for ten minutes. The cells were then resuspended in 0.7% sodium chloride. After standing an additional thirty minutes in the cold, the cells were removed by centrifugation at 600 g for ten minutes. The supernatants recovered from the centrifugations were pooled and centrifuged at 55,000 g for sixty minutes. The recovered sediment was resuspended in 30 mg/ml in-water to give an opalescent stable suspension. The suspension, how¬ ever, was described as sedimenting in a few days in the pre¬ sence of a salt. The suspension could be stored frozen or at 2°C with thymol as a preservative without loss of activ¬ ity. When the suspension was prepared from freeze dried cells, however, the suspension could not be frozen because it flocculated on thawing. In the article entitled "Delayed Cutaneous Hyper- sensitivity Reactions to Membrane Extracts of Human Tumour Cells" by Oren and Herberman, Clin. exp. Immunol., 9: 45-56 (1971), a modification of the Davies hypotonic salt tech¬ nique for the preparation of cell surface membrane antigens was described. The modified technique was described as being useful with a variety of human tumors including leuke¬ mia, Hodgkin's disease, melanoma, carcinoma, and Wilm's tumor. The concept behind their investigation and method was that human tumors possess cell surface membrane carried tumor associated antigens (TAA) and that the tumor bearing host elicits an immune response against these TAA. In Biochimica et Biophysica Acta by Herberman, 473: 93-119 (1977), it was suggested that the surface membrane should be intact or at least in large fragments for its antigens to be active. In the study by Oren and Herberman, it was found that hypotonic salt membrane preparations produce immunolo- gical delayed hypersensitivity reactions in patients with leukemia and Hodgkin's disease indicating that the membrane preparations from these cell types contained active TAA. However, it is noted that only eight patients with carcino¬ ma, melanoma, and Wilm's tumor combined were tested so that no conclusions can be drawn whether TAA was present in these membrane preparations. The specific method of hypotonic saline extraction described by Oren and Herberman is as fol¬ lows : 1. Freeze-thaw cells. 2. Centrifuge at 500 g for 10 minutes. 3. Repeated extraction* with 0.14 M NaCl. 4. Extraction* with 0.07 M NaCl (x2). 5. Extraction with 0.035 M NaCl (x2). 6. Crude membrane pellet obtained from pooled supernatant fluids by centrifugation at 105,000 g for 1 hour. 7. Suspension of membranes in 0.14 M NaCl. * Extraction consisted of suspension in saline, incubation at 4°C for 10 minutes, and centrifugation at 500 g for 10 minutes. Breast cancer cells having cell surface TAA that elicit host cell-mediated immune response can be specifi¬ cally measured by Jji vitro assays such as leukocyte migra¬ tion inhibition ("Inhibition of Leukocyte Migration Inhibi¬ tion by Tumor-Associated Antigens in Soluble Extracts of Human Breast Carcinoma" by McCoy et al, J. Nat. Cancer Inst. , 53: 11-17 (1974)) and lymphocyte blastogenesis ("Detection of Cell-Mediated Immunity Against Tumor-Associated Antigens of Human Breast Carcinoma by Migration Inhibition and Lym- phocyte Stimulation Assays" by McCoy et al, Pergamon Press, Oxford, England, pages 77-86 (1976)). Accordingly, the iso¬ lation of TAA without contaminants from breast cancer tissue is useful so that the non-contaminated TAA can be utilized in assay testing. In "Lymphoproliterative Responses to Autologous Tumor Extracts as Prognostic Indicators in Patients with Resected Breast Cancer" by Cannon et al, Int. J. Cancer, 27: 131-138 (1981), a modified method of hypotonic salt antigen preparation for use with breast cancer tissue was described. The method described by Cannon et al utilized tumors which were trimmed of fat and minced into small pieces or cell clusters under sterile conditions. The sus¬ pension of cells and tissue fragments were then spun in a refrigerated centrifuge at 650 g for ten minutes. The supernatant was discarded and the pellet resuspended and frozen in at least 5 ml of sterile isotonic saline. The cell suspension was thawed in a 37°C water bath and centri¬ fuged at 1,500 g for ten minutes at 5°C. The supernatant and subsequent supernatants were collected, combined, and kept on ice. The pellet was resuspended in 0.9% sterile isotonic saline, mixed well and incubated on ice with fre¬ quent mixing for five minutes and centrifuged as above- described. The supernatant was collected and the procedure repeated until the supernatant was relatively clear, i.e., free of particulate material as determined by turbidity and color. The pellet was then resuspended in a solution of 0.33% isotonic saline. This procedure was repeated (as many as 6 to 8 times) until the supernatant was relatively clear. The pellet was subjected to two further dilutions of 0.25% and 0.125% isotonic saline-water with repeated washings as described above. Finally, the pellet was resuspended in distilled water, which ruptured any remaining cells and nuclei and resulted in viscous material that was not easily pelleted upon centrifugation. The viscous material was carefully excluded from the pool of supernatants. The com¬ bined supernatants from the various extractions were then centrifuged at 105,000 g for sixty minutes. The supernatant was removed and the pellet resuspended into approximately 0.5 ml isotonic saline or just enough to suspend the pellet. The total volume was recorded and an aliquot removed for protein determination and for tests for bacterial contamina¬ tion. The methods as hereinbefore described provide mem- brane antigen assays which are low in yield of TAA and which contain substantial contaminants. Accordingly, there is a need for an improved method of providing non-contaminated membrane antigen preparations which are high in TAA yield and free of contaminants. GENERAL DESCRIPTION OF THE INVENTION The method of the present invention provides for optimum non-contaminated membrane preparations rich in tumor associated antigens (TAA) obtained from the surface membrane of human carcinoma cells, in particular human breast carcin- omas. According to the present invention, single cell sus¬ pensions are extracted separately with a plurality of salt solutions having decreasing salt concentrations. This pro- cedure avoids substantial cell and nuclei rupture, and pro¬ vides a membrane antigen assay which is high in TAA and is relatively pure. More specifically, the preparation of the surface membrane antigens of human carcinoma cells of the present invention utilizes membrane extraction of frozen or fresh single cell suspensions. The use of single cell suspensions provides for a more simple extraction and, accordingly, for higher yields of TAA. The use of cell clusters or tissue fragments as disclosed in the art does not provide for as high a yield of TAA as achievable using single cell suspen¬ sions. The method of the present- invention provides for a clean membrane preparation which avoids lysis and thereby avoids the presence of contaminants in the preparation. Previous methods required use of water in the extraction method which resulted in the rupture of the cell and cell nuclei. The rupture of the cells allowed internal cellular components to contaminate the membrane preparations. The method of the present invention excludes water extraction steps and, accordingly, most cells remain intact during the entire extraction procedure and a minimum of internal cellu¬ lar components leak into the membrane preparations. Additionally, the membrane preparations of the present invention remain highly active as TAA allowing for their use in lymphocyte blastogenesis assays as described in "Lymphocyte Blastogenesis Assay as a New Prognostic Test in Predicting Breast Cancer Recurrence and Survival" by McCoy et al, Am. Soc. Clin. Oncology, (1988). The membrane preparation will remain active even after three months in storage at -70°C. The method of the present invention utilizes a decreasing salt concentration extraction procedure to pre- pare surface membrane antigens from human carcinoma cells. The procedure utilizes frozen or fresh single cell suspen¬ sions. The single cell suspensions are prepared according to conventionally known methods. A particular single cell suspension is subjected to four sets of two saline extractions on ice. Following each extraction, the suspension is centrifuged providing a super¬ natant and a precipitated insoluble portion or pellet. The supernatant is separated off and the pellet is subjected to the next extraction step. All of the collected supernatants are pooled. Each set of extractions utilizes a saline solu¬ tion having decreased salt concentration or molarity from the previous extraction set. A minimum of cell lysis occurs in each extraction. Following the four sets of extraction, the pooled supernatants, which are the extracted fragments of cell sur¬ face membranes containing carcinoma TAA, are centrifuged. The resultant pellet is resuspended in a minimum volume of saline solution and the protein concentration determined. Any conventional physiological saline suitable for temporarily maintaining living cells, such as an isotonic aqueous solution of sodium chloride, is suitable for use as the saline solution in the extractions described above. The molarity of the saline solutions utilized, as stated above, are present in decreasing amounts with each set of extractions. The molarity of the initial saline so¬ lution used in the first extraction set can be present in a range of from 0.160 to 0.150, and is preferably 0.156 M. The molarity is decreased by one-half for each subsequent set of saline extraction and resuspension. DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENT A non-contaminated membrane preparation rich in breast cancer TAA was prepared utilizing the method des¬ cribed below. A single cell suspension of either -70°C frozen or fresh breast carcinoma tissue was prepared utilizing any suitable conventionally known method. A 0.156 M saline extraction of at least about 1 x 10^ cells from the breast tumor tissue, now in the form of a single cell suspension, was performed for ten minutes on ice. Following extraction, centrifugation of the cells at 200 g was carried out. The supernatant was separated. A 0.156 M saline extraction and centrifugation in the same manner as described above was then performed on the remain¬ ing precipitated insoluble material. The supernatants were pooled after each centrifugation. Minimum cell lysis oc¬ curred. A 0.078 M saline extraction of the cells from the first set of extractions was then performed for ten minutes on ice. Thereafter, centrifugation of the cells at 200 g was carried out. The supernatant was separated. A second 0.078 M saline extraction and centrifugation as described above was then performed. The supernatants collected in the second set of extractions were then pooled with the super¬ natants from the first set of saline extractions. Minimum cell lysis occurred. A third and fourth set of saline extractions, fol¬ lowed by centrifugation in the same manner as described above, is then carried out. Specifically, the third set of extractions utilizes 0.039 M saline solution and the fourth set of extractions utilizes 0.0195 M saline solution. The 0.039 M and a 0.0195 M saline extractions were performed twice each for ten minutes on ice. The supernatants were collected and pooled with the supernatants from the first and second set of extractions following centrifugation of the cells at 200 g. Minimum cell lysis occurred. The pooled supernatants, i.e., the extracted frag¬ ments of cell surface membranes containing breast cancer TAA, were then centrifuged at 105,000 g for sixty minutes. The resultant pellet was then resuspended in a minimum volume (approximately 0.3-0.5 ml) of 0.9% saline and the protein concentration determined. The TAA was highly active and is useful in lympho¬ cyte blastogenesis assays to predict breast cancer recur¬ rence and survival as reported in "Lymphocyte Blastogenesis Assay as a New Prognostic Test in Predicting Breast Cancer Recurrence and Survival" by McCoy et al, supra. As will be apparent to one skilled in the art, various modifications can be made within the scope of the aforesaid description. Such modifications being within the ability of one skilled in the art form a part of the present invention and are embraced by the appended claims.

Claims: THE CLAIMS : 1. A method of preparing surface membrane antigens from human carcinoma cells comprising (a) providing a single cell suspension from carcinoma tissue; (b) separately ex¬ tracting said single cell suspension with a plurality of salt solutions having decreasing concentration, and (c) col¬ lecting the extractant; said method being free of water ex¬ tractions to thereby avoid substantial cell and cell nuclei rupture to provide a membrane antigen assay which is active and relatively pure. 2. A method of preparing surface membrane antigens from human carcinoma cells comprising: (a) providing a single cell suspension from carcinoma tissue; (b) extracting cells from said single cell suspension on ice utilizing a saline solution having a molarity within the range of from 0.160 to 0.150, followed by centrifugation of the suspension and collection of the supernatant; (c) repeating step (b) with the extracted cells of step (b); (d) pooling said supernatants collected in steps (b) and (c) ; (e) extracting said extracted cells of step (c) on ice with a saline solution having a molarity one-half the value of the molarity of said saline solution utilized in step (b), followed by centrifugation of the suspension and collection of the supernatant; (f) repeating step (e) with the extracted cells of step (e); (g) pooling the supernatants collected in steps (d), (e) , and (f) ; (h) extracting said extracted cells of step (f) on ice with a saline solution having a molarity one-half the value of the molarity of said saline solution of step (e) followed by centrifugation of the sus¬ pension and collection of the supernatant; (i) repeating step (h) with the extracted cells of step (h); (j) pooling the supernatants collected in steps (g), (h), and (i); (k) extracting said extracted cells of step (i) on ice with a saline solution having a molarity one-half the value of the molarity of said saline solution of step (h), followed by centrifugation of the suspension and collection of the supernatant; (1) repeating step (k) with the extracted cells of step (k); (m) pooling the supernatants collected in steps (j), (k), and (1); (n) centrifuging the pooled supernatants of step (m) to produce a pellet and supernatant; and (o) removing the supernatant of step (n) and suspending said pellet of step (n) in a minimum volume of saline solution having a molarity one-half the value of said saline solution utilized in step (k). 3. The method according to claim 2 wherein said single cell suspension of step (a) is prepared utilizing -70°C frozen carcinoma tissue. 4. The method according to claim 2 wherein said single cell suspension of step (a) is prepared utilizing fresh carcinoma tissue. 5. The method according to claim 2 wherein said carcinoma tissue is breast carcinoma tissue. 6. The method according to claim 2 wherein said saline solution is an isotonic aqueous solution of sodium chloride. 7. The method according to claim 2 wherein the number of said cells extracted from said single cell suspen¬ sion is at least 1 x lO***-. 8. The surface membrane antigens prepared accord¬ ing to claim 1. 9. The surface membrane antigens prepared accord¬ ing to claim 2.

Inventors: MCCOY JAMES LLOYD, MCCOY, JAMES, LLOYD
Applicants: IMMUQUEST LAB INC, IMMUQUEST LABORATORIES, INC.
Application year: 1989
Main classification: A61K37
IPC codes: A61K39, C07K1, C07K14, C12P21, G01N33