The Isolation and Characterization of Tumor-Specific Antigens of Rodent and Human Tumors'

[CANCER RESEARCH 36, 3518-3525, September 1976] The Isolation and Characterization of Tumor-specific Antigens of Rodent and Human Tumors'

D. M. P. Thomson,2 P. Gold, S. 0. Freedman, and J. Shuster

Montreal General Hospital Research Institute, McGill University, Montreal, Quebec, Canada H3G 1A4

Summary similarities to normal histocompatibility antigens (4, 7, 8, 16, 24). Putative tumor-specific transplantation antigens (TSTA) Although the rejection of transplantable tumors among from both a carcinogen-induced rodent tumor (MC-i ) and 2 syngeneic animals has been used profitably in the study of human tumors were purified. The antigens were solubilized animal tumors, this type of experimental design has been from the tumor cell membranes by limited papain digestion precluded in humans by both ethical considerations and the in a manner similar to that described for the isolation of lack of a syngeneic donor-host relationship. Thus, a num normal histocompatibility antigens. The antitumor immune ber of in vitro assays of the host's cellular and humoral response of the tumor-beaming host was used to monitor the immune responses against putative TSTA have been devel purification of the putative TSTA in both the rodent and oped which are applicable to both animal and human tumor human tumor systems. In the case of the rodent tumor, a systems (1, 13, 14). Because the TSTA appear to be only major step in the purification of the TSTA involved affinity weakly immunogenic, the antitumor immune responses chromatography on Sephamose beads coupled to autolo evoked on the part of the tumor host are correspondingly gous antitumom antiserum. A comparable procedure was weak (12, 23). Nevertheless, these immune responses are utilized in the purification of the TSTA from human tumors apparently far more tumor specific than can be evoked by by using affinity chromatography on anti-human @t32-micmothe immunization of xenogeneic animals. globulin antiserum coupled to a solid phase. The data ob On the basis of the foregoing observations, the expemi tamed indicate that the TSTA of human tumors contains a ments to be described were designed with the following @32-micmogIobulinchainthat is immunochemically identical objectives: (a) to use the host's antitumor immune response with, and very similar in size to, that found in normal human in order to monitor the purification of the putative TSTA of histocompatibility antigens. A subunit of similar size was rodent and human tumor tissues; (b) to determine whether also identified in the carcinogen-induced rodent tumor. any structural and/or immunochemical similarities exist be These results suggest that the TSTA in both humans and tween human and rodent TSTA; and (c) to determine rodents may well be altered histocompatibility antigens. whether there are any immunochemical and/or structural relationships between the TSTA and normal histocompati bility antigens in either the rat or human. Introduction

Investigations based on the rejection of experimentally Materials and Methods induced tumors into previously immunized syngeneic recip ients have established the existence of TSTA1 in most ani Tumor Tissues mal tumor systems studied (12, 23). Whereas the TSTA of tumors induced by the same virus are common, those in In the experiments involving rodents, the methylcholan duced by chemical carcinogens are most usually unique to threne-induced sarcomas, MC-1 and MC-i 1, 2 TSTA-dis the individual tumor (23). In both cases, however, the TSTA tinct tumors serially passaged in syngeneic male hooded sites of major functional importance, as concerns immuno rats, were used (31). Early generations of MC-1 and MC-li logical interaction, would appearto be on the surface mem were stored in liquid nitrogen and removed at intervals for brane of the tumor cell (2). In mumine tumor systems, at passage in syngeneic hooded rats. Human malignant mela least, a good deal of evidence has accumulated that sug noma tissue and breast cancer tissue were obtained either gests that the TSTA beamsimmunochemical and structural at surgery or at autopsy and stored at â€”40Â°untilprocessed.

Purification of the Putative TSTA of Rodent and Human I Presented at the Symposium â€œCancer and Chemistry' â€˜as part of the Fourth Conference on Embryonic and Fetal Antigens in Cancer, November 2 Tumors to 5, 1975, Charleston, S. C. This work was supported in part by the Medical Research Council of Canada and the Cancer Society of Montreal. Preparation of Tumor Cell Membranes. The tumor tissue 2 Presenter. 3 The abbreviations used are: TSTA, tumor-specific transplant antigens; was thawed and finely minced with stainless steel scissors. PBS, phosphate-buffered saline containing 0.14 M NaCI-O.OiMsodium phos The mince was suspended in 4 volumes of 0.25 M sucrose phate buffer, pH 7.3; $,m, $,-microglobulin; LAI, leukocyte adherence inhibi tion; NAI, nonadherence index; SDS, sodium dodecyl sulfate; TSA, tumor containing 0.05 M Tris, 2 mt@iCaCl2,and 2 mM MgCI2, pH specific antigens. 7.5, at 4Â°.The tumor tissue was homogenized with an Ultra

3518 CANCER RESEARCH VOL. 36

turrax homogenizer as previously described (30, 31). The jected to chromatography on a DEAE-Sephadex A-SOcol process was monitored by phase-contrast microscopy, after umn equilibrated with the buffer described above. lon-ex staining with 0.2% trypan blue, to achieve the release of change chromatography on DEAE-Sephadex removes con more than 90% of the nuclei, but with minimal disruption of taminants of highly negative charge, and there appears to these structures. The homogenate was then centrifuged for be a separation of protein from nucleic acid components 10 mm at 980 x g in 250-mI plastic bottles in a refrigerated (22). The unbound fraction, which contained the TSTA ac IEC PR 6000 centrifuge. The supemnatant fluid, which con tivity, was then dialyzed against a 0.1 M Tmis-0.3 M glycine stituted two-thirds of the volume in each bottle, was de 0.2 M NaCi buffer, pH 8.0, containing 0.001 M disodium canted, and the pellet was resuspended in an equal volume EDTA, and was then concentrated by ultrafiltration in an of the same buffer. Both the homogenization and centrif u Amicon chamber as described above. The concentrate was gation steps were repeated as described above, and the centrifuged at 120,000 x g for 1 hr, and the supernatant was decanted supemnatants were pooled. An aliquot of 3 M lith then subjected to molecular sieve chromatography on a ium chloride in distilled water was gradually added to the calibrated Sephadex G-iSO column equilibrated with the total supemnatant to a final concentration of 0.1 M. The same Tris-glycine-NaCI buffer described above. The frac membranes in the supemnatant were sedimented by centrif tions obtained were concentrated by ultrafiltration and as ugation for 1 hr at 75,000 x g in a fixed-angle rotor in a MSE sayed for tumor antigenic activity. Superspeed-65 centrifuge (19). The supemnatant fluid was Affinity Chromatography of the Tumor Antigenic Prepa discarded, and the crude membrane preparation was sus rations. The â€˜y-giobulinfraction from the sera of syngeneic pended in 0.5 M Tris buffer, pH 7.5, containing 0.1 M lithium hooded rats, hyperimmunized to the MC-i sarcoma as pre chloride, to a volume equal to about one-half of the original viously described (30, 31), was precipitated by 50% satu pooled supemnatant. The pellets were then homogenized to rated ammonium sulfate. This material was covalently cou a smooth suspension with the Ultraturrax homogenizer. The pied to cyanogen bromide-activated Sephamose4B in 0.2 M resuspended crude membranes were again sedimented at citrate buffer, pH 6.5 (6, 31). 75,000 x g for 1 hr. The supemnatant was discarded and the Antiserum to human f32m was raised in horses, using membrane pellet was suspended in 0.01 M Tris-HCI, pH 7.5, purified f32m kindly provided by Dr. M. D. Poulik of the containing 1 unit of penicillin and 1 @gofstreptomycin per William Beaumont Hospital, Royal Oak, Mich. Serial bleed ml. The volume was adjusted to approximately 200 ml and ings were obtained from these animals, and their serum stirred overnight at 4Â°.The membranes were sedimented at antibody activity against @32mwasmeasured by radioimmu 120,000 x g for 1 hm,and this fraction was then separated by noassay as previously described (28). At the earliest evi centrifugation through a dextman-polyethylene glycol dence that these animals were producing anti-/32m anti aqueous 2-phase system adapted from that described by body, presumably of relatively low affinity, the y-globulin Brunette and Till (3). On completion of this centrifugation fraction of the horse serum was obtained by batch chroma step, the membranes were found at the interface of the 2- togmaphy on Whatman DE-52 DEAE-ceiiulose equilibrated phase system. The membrane fraction was collected, di with 0.01 M phosphate buffer at pH 7.6. This material was luted with approximately 4 times its volume of cold distilled then coupled to Sephamose4B using cyanogen bromide as water, and sedimented by centrifugation at 75,000 x g for 1 described above (6, 31). hr. The membrane pellet was resuspended in cold distilled The TSTA-containing fractions obtained from the Sepha water and the centmifugation step was repeated. The pellets dex G-1 50 columns described above were subjected to af were then resuspended in cold PBS, pH 7.3, and stored at finity chromatography on an appropriate antibody-bound â€”40Â°. column. After the column effluent had been thoroughly Papain Solubilization of Membrane Protein. The purified washed with 0.01 M Tris-0.14 M NaCI buffer, pH 7.4, the membranes were suspended in PBS, pH 7.3, at a protein antibody-bound fraction was then eluted with 2.5 M MgCI2, concentration of approximately 10 to 12 mg/mI, as deter pH 6.8, and 3 M KCNS for the rodent tumor antigens and the mined by the procedure of Lowry et al. (20). The suspension human tumor antigens, respectively. The MC-1 TSTA was was incubated at 37Â°ina water bath, and twice-crystallized trace-labeled with 1251before application to the affinity col papain (Worthington Biochemical Corp., Freehold, N. J.) umn. The material that did not adhere to the affinity column preactivated in cysteine was then added to a final concen was designated as the unbound fraction, while the material tration of0.5 unit per mg of membrane protein. The incuba that was bound to the column and then eluted with the tion was continued for 1 hr at 37Â°,during which period the chaotmopic agent was designated as the bound fraction. suspension was stirred continuously. Proteolysis was am The dialyzed eluate was centrifuged for 1 hr at 75,000 x g rested by the addition of iodoacetamide to a final concen and concentrated by ultrafiltration in an Amicon chamber tration of 0.05 M. The suspension was rapidly cooled in an fitted with PM-b membrane. The concentrated eluate was ice bath and was then centrifuged at 120,000 x g for 3 hr at again centrifuged at 100,000 x g for 1 hr. This final centrifu 4Â°.The supernatant recovered after centrifugation was di gation step removed any Sepharose particles that may have alyzed overnight at 4Â°against a buffer containing 0.02 M escaped from the column and/or any denatured and aggre Tris-0.38 M NaCI, pH 8.0, and was then concentrated by gated proteins that had been eluted, both of which may ultrafiltration in an Amicon chamber fitted with a PM-10 nonspecifically interfere with the LAI assay for tumor anti membrane, to a final volume of approximately 10 ml. genicity to be described below. Table 1 summarizes the Chromatographic Procedures in the Purification of steps in the isolation of the putative rodent and human TSTA. The soluble tumor membrane components were sub tumor antigens.

SEPTEMBER1976 3@19

Assays for TSTA Activity. Indirect Membrane Immuno and analyzed by SDS-poiyacrylamide gel electrophoresis in fluorescence. Ind imect membrane immunofluomescence was 7.5 and 10% gels (32), and in 12.5% 8.0 M urea gels (29). The performed on washed viable single tumor cell suspensions gel was then cut into individual i-mm sections and counted prepared from finely minced solid tumor digested in 0.04% in a Nuclear-Chicago y-scintillation spectrometer. These trypsin and 0.05% collagenase in the presence of a small procedures provided information concerning both the de amount of DNase (30). The presence of antigenic activity in gmeeof purity of the preparations in question and the molec the papain-soluble tumor digest of MC-1 membranes was ular weights of the molecules, or their subunits, involved. detected and quantitated by the capacity to inhibit MC-i tumor-immune antiserum from binding to cell surface anti gens on viable MC-1 tumor cells. The fluorescence index Results was computed and expressed as previously described (30). Tube LA! Assay. Isolated membranes, cell sap, and pa Purification of the Papain-solubilized Tumor Antigen of pain-solubilized membrane fractions obtained upon gel the MC-1 Rat Sarcoma. Table 2 reveals that the TSTA activ chromatography were assayed for human tumor antigen ity of the MC-1 tumor is present on the tumor cell mem activity by determining their capacity to inhibit the adher branes rather than in the cell sap. Moreover, this surface ence of leukocytes in test tubes. The assay was performed, associated tumor antigen was solubilized by limited papain and the NAI was calculated and expressed as previously digestion and was partially purified by ion-exchange and described (10, 21). The NAI represents the difference in molecular sieve chromatography. From the data obtained reactivity to the specific and nonspecific tumor extracts. from molecular sieve chromatography on Sephadex G-i50, The higher the NAI, the greater is the differential in reactivity the maximum TSTA activity appeared to reside in the molec between the tumor and control tissue extracts, whereas a uiamweight range of 40,000 to 60,000 daltons (Table 2; Chart low NAI indicates minimal or no difference in reactivity. A 1). After affinity chromatography on the anti-MC-i affinity NAI of 30 or more is indicative of a positive response, i.e. , a significant inhibition of leukocyte adherence to glass based Table 2 on a large series of breast cancer, melanoma, and control Isolation of MC-1 TSA monitored by inhibition of membrane subjects (10, 21). Leukocytes from patients with limited immuno fluorescence cancer (Stage I or II) that reacted in the tube LAI assay to the indexâ€• appropriate PBS extracts of either breast cancer or mela Extract used for inhibition of syngeneic with MC-1 sarcoma noma were used to determine whether the isolated tumor anti-MC-1cellsPBS9.6Normal immune serumâ€•Fluorescence materials contained tumor-specific activity. Control sub jects had either benign surgical diseases or unrelated can extract9.0Purifiedtissue cers. membranesMC-i2.0MC-118.4Papain-soluble Electrophoretlc Analysis of Tumor Antigens

The papain-solubilized membrane preparations, obtained MC-1'Sephadex as described above, and those isolated at each step of G-150Fraction purification were subjected to assays for tumor antigen. In 18.0Fraction addition, those fractions containing tumor antigenic activity 26.6Fraction were madiolabeled with 1251by the chloramine-T method (9) 34.0Fraction 47.1Papain-solubie

Table 1 MC-ii'Sephadex G-150Fraction Isolation of papain-solubilized tumor-specific antigens 18.5Fraction 1. Solid tumor, Ultraturrax homogenization 28.9Fraction 2. Differential centrifugation 38.3Fraction a. 980 x g for 10 mm 48.8Affinity b. 75,000 x g for 1 hr 3. Membrane purification by aqueous 2-phase polymer system chromatographyMC-1 4. Papain digestion of Membranes, 0.5 unit papain activity per mg TSAEluate, membrane protein at 37Â°for 1 hr bound4.6Effluentâ€•, 5. Icebath cooling unbound8.9MC-11 6. Centrifugation, 120,000 x g for 3 hr TSAEluate, 7. Supernatantdialysis, 0.38 M NaCi-0.02MTris, pH 8 bound9.1Effluent, 8. DEAE-Sephadex-A-50 ion-exchange chromatography, unre unbound9.0 tamed fraction aAnti-MC-1,immuneserumwasusedata finaldilutionof1:10 9. SephadexG-150chromatography with tissue extracts. 10. Affinitychromatography b Number of cells stained with test serum divided by number of a. Syngeneictumor-immune antiserum cells stained with normal serum. All samples are read and scored as b. Xenogeneichorse anti-human f32m unknowns. 11. Tumor antigenic activity monitored by in vitro assaysof antitu C Protein contents of fractions were approximately 8 mg/mI. momimmunity d MC-1 TSA-bound fraction contained a protein content of ap a. Inhibition of indirect membrane immunofluorescence proximately500pg/mI. Theeffluent contained a protein content of b. Tube LAI assay 8 mg/mi.

3520 CANCERRESEARCHVOL. 36

any time. Moreover, themewas no evidence of cmoss-meactiv 1.0 ity of the tumor Ã¤ntigenic activity between the breast and melanoma systems. 5' 1 II SDS-Polyacrylamide Gel Electrophoresis of Isolated E ++ + ++ + Melanoma and Breast Tumor Antigens. The papain-soiu @ 0.5 ble breast cancer and melanoma antigens in the 70,000- to i50,000-moleculam-weight fraction of a Sephadex G-150 column were labeled with 1251bythe chloramine-T method

0.1 and analyzed by SDS-polyacmylamide gel electrophomesis in the presence of reducing agents. Both tumor antigens had 20 41@1@1@1@2OO

FRACTION NUMIER

Chart 1. Sephadex G-150 chromatography. Papain-soluble membrane an 1.0 tigen from DEAE-Sephadex chromatography was applied to the column of Sephadex G-150 (5 x 87 cm) equilibrated with 0.3 M glycine-0.2 M NaCl-0.00i M disodium EDTA and 0.1 M Tris-HCI, pH 8.0; and 10-mI fractions were collected and assayed for MC-i TSA activity by inhibition of membrane immunofluorescence. Maximum MC-i TSA activity was in the area shown by E U thebar. C 0 S @ 0.5 S column, it was found that only the bound fraction contained + tumor antigenic activity (Table 2; Chart 2). Hence, a good deal of the papain-solubilized membrane preparation that had copumified with the TSTA to the point of molecular sieve 0.1 chromatography failed to bind to the specific antitumom to 20 30 40 50 60 antibody, appeared in the column effluent, and lacked tu momantigenic activity. FRACTION NUMBER Papain-solubie membrane material from the unrelated Chart 2. Affinity chromatography profile of the MC-1 TSA. The 1st large and non-cross-reacting MC-1i tumor applied to the MC-1 peak is the unbound or effluent fraction. The column was then washed with approximately 500 ml (10 times bed volume) of column buffer 0.14 M NaCI affinity column showed no specific binding, since the mate 0.01 M Tris, pH 7.4. The bound material was eluted with 100 ml (2 times bed mialin the eluate did not inhibit in the membrane immunofiu volume) 2.5 M MgCI,-0.01 MTris-HCI, pH 6.8. A small peak was observed. The MC-i TSA was always trace-labeled with 1251before application, and the omescence assay (Table 2). radioactivity was measured in each 10-mI fraction. The profile of 1251radioac SDS-GeI Electrophoresis of Purified MC-1 TSA. The 125l tivity was identical to the A,,,, tracing. labeled MC-i TSA eluted from the anti-MC-i affinity column

@ 0 0 0 was applied to a Sephadex G-200 column and eluted with a 0 single major peak with molecular weights of approximately 8 Sg S 45,000 to 60,000. However, an analysis of the 125l-Iabeled + ++ + MC-i TSA by SDS-polyacmyiamide gel electrophomesis me vealed that, in the presence of a reducing agent, 1251-Iabeied 0 MC-i ISA appeared to be composed of 3 poiypeptide chains with apparent molecular weights of 12,000, 18,000, U and 25,000 daltons (Chart 3). The major peak had an appar ent molecular weight of 12,000 daltons, and this peak had a mobility identical to that of â€˜25i-labeledhumanf32m. isolation of Tumor Antigenic Material from Human Breast Cancer and Malignant Melanoma. The develop ment of the LAI assay in test tubes for the detection of + antitumom immunity in patients with breast cancer (10) and 6 malignant melanoma (21) has provided an immunological technique for monitoring the purification of the tumor anti 94 gens from melanoma and breast cancer preparations. The a-I results shown in Tables 3 and 4 reveal that the tumor anti U, genic activity of these tumors, as monitored by LAI, was found in the purified tumor cell membrane preparation and not in the cell sap. The material containing this activity 15 @O 456075 eluted maximally in the early fractions from the Sephadex Distonc. Migrat.d(mm) G-i50 column, in the apparent molecular weight mangeof Chart 3. SDS gel electrophoresis of â€œI-labeledMC-i TSA eluted from anti-MC-1 affinity column. Top, profile of the â€˜251-labeledMC-1TSA run 70,000 to iSO,000 daitons (Fraction 2) (Tables 3 and 4). unreduced on 12.5% 8 PA urea gels; bottom, â€˜25l-labeledMC-1 TSA run However, when the LA1-active fractions were chromato reduced on 12.5% 8 M urea gels. Mobility of calibration proteins is indicated. graphed on anti-human /32m affinity columns, only the The fastest moving peak of the â€˜251-labeledMC-1TSA had a mobility identical with that of human $@mandwas a broad peak. The control MC-11 TSA eluted bound fraction retained this activity (Tables 5 and 6). No from the MC-i affinity column had too few 1251counts to analyze. /3MG, 1@- LAI-active material was found in the unbound fractions at microglobulin; BPB, bromphenol blue.

SEPTEMBER1976 3521

Table 3 Antigenic activity determinedantigenNAIâ€•by tube LAI assay of papain-solubilized malignant melanoma fractionsPurifiedPBSof malignant melanomaNAr' SephadexG-150chromatographicr

ex- mom 34MelanomaLeukocytesfromtractâ€• branes Cell sap1 2 306MelanomaPatient 188 12585 115 282Control Patient 260 65 1875 60 34Controlsubject12 3 1515 12 subject1 16 10 28 No. of nonadherentcells in the presenceof specific tumor extract aNAI= â€”Noof nonadherentcellsin the presenceofnonspecifictumorextract < No. of nonadherentcells in the presenceof nonspecific tumor extract

A NAI of 30 or greater indicates significant inhibition of adherence. b A PBS extract of breast cancer was the nonspecific extract.

r Chromatographic fractions were concentrated to approximately 6 mg/mI and tested at a 1 :4 dilution in Medium 199.

Table 4 antigenLeukocytesAntigenic activity determined by tube LAI assay of papain-solubilized breast cancer ofchromatographiccfractionsPurified breast cancerNAIâ€• SephadexG-i50

PBS ex- mom 34BreastcancerPatientlfromNAIâ€• tractâ€• branes Cell sap1 2 58 8 110 31 BreastcancerPatient2 70 41 10 78 115 25 9 Control subject 4 0 2 0 13 8 10 Controlsubject92 1 3 i248 15 22 126 14 aA NAIof 30or greaterispositive. b A PBS extract of the melanoma antigen was used as the nonspecific control extract. C Chromatogmaphic fractions were concentrated to approximately 5 to 8 mg/mI protein and tested in tube LAI assay at a 1:4 dilution in Medium 199.

Table 5 Table 6 Antigenic activity of TSAof malignantmelanomaisolatedby affinity Antigenic activityaffinitychromatography of TSA of breast cancer isolated by chromatographyf3@mNA1â€• with anti-human f3@mNAI'with anti-human to NAla affinity chroma to NAIâ€•affinitychroma PBSex- tography PBSox- tography tract of tract of malignant breast can Boundâ€•MelanomaLeukocytesfrom melanoma Unboundâ€• Boundâ€•BreastcancerPatient3Leukocytes from cer Unboundâ€• Patient 3 89 11 69 63Control 54 17 MelanomaPatient 4 84 17 112 Subject â€”9 â€”22 â€”28 Control subject 5 â€”i6 â€”6 a NAI was calculated with PBS extract of breast or with isolates of

a NAI was calculated with PBS extract of ovarian tumor as a soluble breastcancer as specific antigen and PBSextract of ovar nonspecific control antigen. Humanf.12misolatedidenticallyfrom a ian tumor as nonspecific control antigen. Human f32misolated hepatoma was also used as a nonspecific control, and the results identically from a hepatoma was also used as a nonspecific control, were unchanged. and the results remained similar. A NAI of 30 or greater is positive. b Unbound and bound fractions were concentrated to approxi b Unbound and bound proteins were concentrated to approxi mately 2 mg/mI and tested in the tube LAI assay at a protein mately 4 mg/mI and tested in the tube LAI assay at a protein concentration of 75 to iSO @g. concentration of 75 to 150 j.@g. similar profiles on SOS electrophomesis (Charts 4 and 5). by SDS gel electrophoresis, showed the same 3 peaks of There were 2 major peaks; the peak with the fastest mobility 12,000, 25,000, and 40,000 daltons (Chart 6). moved identically with that for 1251-iabeled human f32m. The Approximately 70% of the material applied with TSTA minor intermediate peak had an electrophoretic mobility of activity was bound to the anti-human f32m column. After approximately 25,000 daitons. The slowest component had application of the chaotropic agent, 70 to 90% of the bound an approximate molecular weight of 40,000 (Charts 4 and fraction was recovered as determined by measurements of 5). human f32m or total protein. The unbound fraction con The material that bound to the anti-human /32m affinity tamed no, or minimal amounts, of human f32m as deter column and that had demonstrated LAI activity was also mined by radioimmunoassay for human f32m. In contrast, labeled with 1251The madiolabeled material, when analyzed the bound fraction was considerably enriched in /32m.

3522 CANCER RESEARCH VOL. 36

0 0 Since the papain-solubilized TSA of the MC-1 tumor 80 retains its antigenicity, as determined by its ability to neu tralize MC-i tumor-immune serum in the membrane immu :i@@ nofluomescence assay, the technique of affinity chromatog maphywas chosen to achieve selective isolation and pumifi 0 7.5% cation of the MC-i tumor-specific antigen. The Sephamose I 3 anti-MC-i antibody column, unlike conventional tech a- U niques, could selectively isolate the tumor-specific moiety from a variety of other membrane components. The isolated 1. TSA of the MC-i rat sarcoma was approximately 50,000 to

g,@ Â§. 1 2 4 BPB @ ( ( 600 A

0. U

v-@â€”11020 â€˜, â€˜@----â€”-@ 6014..Ai@/@\@ 30 4050 100

@ jf%@ 13 30 45 60 75

@ 10 f + 12.5% 0 8MUrea 2 4 BPS 2000 B a. U @ 1500 0. U l000 20 30 40 50 60 @ lbDistance Migrated (mm) @!@ 500 Chart 4. SOS gel electrophoresis of the â€œ'51-labeledpapain-solubilized breast cancer antigen from Sephadex G-i50 chromatography (Fraction 2). Fraction 2 from the column with a molecular weight range of 70,000 to 150,000 had maximum LAI activity. The 1251breastcancer antigen was run reduced on 7.5, 10, and 12.5% 8 M urea gels. The peak with the fastest mobility moved in a manner identical with the human f32mpeak. Mobility of calibration proteins is indicated. BPB, bromphenol blue. 3 4 BPB 2000 Discussion C @ 1500 The results of the present investigation indicate that the a- tumor antigens of both a carcinogen-induced rodent tumor U and those human tumors that were studied are present in ,,:;- 1000 I,' the tumor cell membrane and can be soiubilized by limited papain digestion in a manner similar to that used for the 500 purification of histocompatibility antigens from various spe cies. Moreover, it was possible to use the host's antitumor immune response as a method of monitoring the pumifica tion of the tumor antigenic preparation. The data obtained 15 30 45 60 75 90 105 suggest that, in the case of the human tumor studied, at DISTANCE MIGRATED (mm) least, the surface tumor antigens involved contain a /32m Chart 5. SDS gel electrophoresis of the â€˜251-labeledpapain-solubilized chain that is immunochemically identical with and is very melanoma antigen from Fraction 2 of Sephadex G-i50 chromatography. A, similar in size to that found in normal human histocompati 7,5% gel; B, 10% gel; C, 12.5% 8 M urea gel. The â€˜Â°â€˜I-Iabeledmelanoma antigen was run reduced. Mobility of calibration proteins is indicated: 1, bility antigens. A subunit of similar size was also identified albumin (68,000); 2, aldolase (40,000); 3, chymotrypsinogen (25,000); 4, in the carcinogen-induced rodent cancer. cytochrome c (12,400). BPB, bromphenol blue.

SEPTEMBER 1976 3523

4 after papain digestion (5, 25, 26). Furthermore, @32m,the 11 000-dalton polypeptide chain found in all histocompati 3 bility antigens, appears to be an integral part of the breast

0 and melanoma tumor antigens. It appears that rodent and human TSTA have structural similarities and, at least in man, the 11 000-dalton subunit is apparently f32m.Thus, the TSTA in rodents and humans is very similar to the AG-B and HLA species-specific histocom patibility antigens. The tumor-specific polypeptide chain 15 30 45 60 may, in fact, represent a modified histocompatibility anti gen. By analogy to the histocompatibility antigens, the larger subunits therefore appear to carry the distinctive tumor antigenic determinant. The biological significance of these observations is compatible with the hypothesis (27) that histocompatibility molecules serve as adapters that combine with non-self-components that are recognized by cytotoxic T-lymphocytes. Current work in our laboratory is Distance Migrated(mm) designed to establish a similarity, at the level of primary Chart 6. SDS gel electrophoresis of breast cancer antigen eluted from structure, between the histocompatibility antigens and tu anti-human f32maffinity column. The eluted breast antigen was labeled with momantigens. 1251 and run reduced on 10% gel. Top, profile of the â€˜25l-labeled breast antigen; bottom, profile of â€˜[email protected] of calibration proteins is indicated. Acknowledgments 60,000 daitons as determined by Sephadex G-200 chroma We thank Dr. J. Marti, Dr. N. Grosser, and Dr. Maximo Flores for testing togmaphy. On SDS-geis, when reduced, this material con the fractions. We are grateful to J. Weatherhead, P. Friedlander, and B. sisted of 3 subunits with molecular weights of approxi Chaland for able technical assistance. mately 11,000, 18,000, and 25,000 daltons. The 11,000-mo leculamweight subunit had a mobility identical with that of human f32m. References The isolated MC-i TSA of the chemically induced tumor 1. Baldwin, R. W. In Vitro Assays of Cell-Mediated Immunity to Human Solid thus appears to be composed of subunits with molecular Tumors: Problems of Quantitation, Specificity, and Interpretation. J. weights similar to those described for the rat Ag-B histo NatI. Cancer Inst., 55: 745-748, 1975. 2. Baldwin, R. W., and Embleton, M. J. Detection and Isolation of Tumour compatibility antigens (17, 18). Specific Antigen Associated with a Spontaneously Arising Rat Mammary Hailiday and Miller (11) have described a simple in vitro Carcinoma. Intern. J. Cancer, 6: 373â€”382,1970. technique of detecting host antitumom immunity called LA1. 3. Brunette, D. M., and Till, J. E. Rapid Method for Isolation of L Cell Surface Membranes Using an Aqueous 2-Phase Polymer System. J. Holan et a!. (15) described a modified version of this assay Membrane Biol., 5: 215-224, 1971. in matsthat was performed in test tubes. In our laboratory 4. Comoglio, P. M., Bestini, M., and Forni, G. Evidence for a Membrane this technique was adapted and modified for the detection Carrier Molecule Common to Embryonal and Tumour-Specific Antigenic Determinants Expressed by a Mouse Transplantable Tumour. Immunol of sensitization to human breast cancer and malignant mel ogy, 29: 353-364, 1975. anoma tumor antigens (10, 21). With this assay, leukocytes 5, Cresswell, P., Springer, T., Strominger, J. L., Turner, M. J. , Grey, H. M., and Kubo, R. T. Immunological Identity of the Small Subunit of HL-A sensitized to the tumor antigen respond by not adhering to Antigens and @32-Microglobulinand Its Turnover on the Cell Membrane. glass. It appeared feasible, therefore, to use the same me Proc.NatI.Acad.Sci.U.S.,71:2123-2127,1974. sponding cells to monitor the purification of the sensitizing 6. Cuatrecasas, P. Protein Purification by Affinity Chromatography. Deriva tizations of Agarose and Polyacrylamide Beads. J. Biol. 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D. M. P. Thomson, P. Gold, S. O. Freedman, et al.

Cancer Res 1976;36:3518-3525.

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