Antigens Identified by Autologous Antibody

Transcription

1 solaton and Partal Characterzaton of Melanoma-assocated Antgens dentfed by Autologous Antbody Danel R. Vlock, Deborah Scalse, Nancy Megln, John M. Krkwood, and Byron Ballou Dvson ofmedcal Oncology, Pttsburgh Cancer nsttute, Unversty ofpttsburgh School ofmedcne, and Veterans Admnstraton Medcal Center, Pttsburgh, Pennsylvana 1524 Abstract The study of the autologous mmune response to cancer avods the dffcultes encountered n the use of xenoantsera and may dentfy antgens of physologcal relevance. However, the low tter and ncdence of autologous antbody to melanoma have hampered further evaluaton. By utlzng acd dssocaton and ultrafltraton of serum, we have been able to augment the detectable autologous mmune response to melanoma n the majorty of patents studed. n autologous system Y-Mel 84:42, serum S15 demonstrated a rse n tter from 1:32 n natve sera to 1:262,44 after dssocaton. The antgen detected by S15 was found to be broadly represented on melanoma, gloma, renal cell carcnoma, neuroblastoma, and head and neck carcnoma cell lnes. t dd not react wth bladder or colon carcnoma, fetal fbroblasts, pooled platelets, lymphocytes and red blood cells, or autologous cultured lymphocytes. Usng polyacrylamde gel electrophoress, S15 detects a 66,-mol wt antgen n spent tssue culture meda and serum ultrafltrate. n cell lysate two bands between 2, and 3, mol wt are detected by S15. The 66,-mol wt antgen s senstve to trypsn dgeston and but s resstant to pepsn and heat nactvaton. Exposure of spent meda to trypsn results n the development of a 24,-mol wt band that appears to correspond to the antgen detected n the cell lysate. The dfference between the antgens detected n the cell lysate as compared wth spent meda and serum ultrafltrate may be due to degradaton durng cell lyss. We conclude that melanoma-assocated antgens are present n the serum of patents wth melanoma and are shed or secreted by ther tumor cells. ntroducton Numerous nvestgators have utlzed a varety of antbody probes n the hopes of characterzng antgens that may dstngush melanoma and other tumors from ther normal counterparts. The antbodes employed have been derved from polyclonal xenogenec and allogenec antsera (1-7) as well as murne and human monoclonal antbodes (8-14). Although these heterologous antbodes have been found to detect tumor-assocated antgens, when suffcently studed they have also been noted to react wth normal cell consttuents (15). Our nvestgatons have focused upon the humeral mmune Address reprnt requests to Dr. Vlock, Hematology/Oncology, VA Medcal Center, Unversty Drve C, Pttsburgh, PA Receved for publcaton 4 June 1987 and n revsed form 19 October J. Cln. nvest. The Amercan Socety for Clncal nvestgaton, nc /88/6/1746/6 $2. Volume 81, June 1988, response produced by the host. The advantage of studyng autologous antbody reactvty s that t may detect antgens not apparent to heteroantsera. n addton, because the antbodes are produced by the host, they are drected aganst physologcally relevant antgens that may provde a better understandng of the role of the host-tumor nteracton. Antbody aganst autologous tumor cell surface antgens was frst reported by Carey et al. (16, 17) and Shku et al. (18, 19) usng senstve mcroserologc assays and cultured tumor cells. Antgens descrbed n those studes were dvded nto three classes: those that are restrcted to the tumor of a sngle ndvdual (class ); those that are shared among tumors of a smlar hstotype or ontogeny and rarely, f ever, found n nonneoplastc cells (class ); and those that are found n nonneoplastc cells as well as tumors (class ). These authors showed antbody aganst autologous antgens n a thrd of melanoma patents studed-a fndng confrmed n our nvestgatons (2). However, the low ncdence and weak tter of autologous antbody to melanoma have hampered contnued nvestgaton and has rased questons regardng the relevance of humeral mmunty n melanoma. We postulated that our falure to detect autologous antbody to melanoma wth greater frequency may be due to the presence of crculatng antgen formng mmune complexes. To pursue ths we modfed a technque reported by Sjorgen et al. (21) to dssocate antbody from antgen n the sera of patents wth melanoma. We have prevously reported that ths technque s successful n enhancng autologous antbody reactvty n 9 of 1 cases studed (2, 22). Seral studes of autologous antbody reactvty, n natve and dssocated sera, have demonstrated correlatons between clncal course and dsease progresson (22). n addton, we have found that the presence host-derved antbody reactvty aganst an allogenec melanoma cell lne s a predctor of eventual dsease recurrence (23). At present, the analyss of antgens detected by autologous antbody depends prmarly upon determnaton of specfcty n tests aganst a varety of malgnant and nonmalgnant cell lnes. A more defntve approach s to characterze the antgen by mmunochemcal means. To date ths has been dffcult because of the low tter of antbody detectable n natve sera and nsuffcent quantty of antgen. Only two successful mmunochemcal characterzatons of class melanoma antgens have been reported. Carey et al. (17) partally characterzed a 25,-4,-mol wt class antgen (AU) by gel fltraton chromatography. More recently, Real et al. (24) reported a class glycoproten of 9, mol wt solated by mmunoprecptaton wth autologous antbody and radolabeled melanoma cells. Our ablty to enhance autologous antbody tter by dssocaton of mmune complexes has allowed mmunochemcal analyss to proceed. We report the successful use of polyacrylamde gel electrophoress (PAGE) wth polyclonal autologous 1746 Vlock et al.

2 antbody n the detecton of a class melanoma antgen solated from cell lysate, serum, and spent meda. Methods Cell lnes Melanoma cells. Sterle tumor specmens were obtaned, establshed, and mantaned n a manner reported prevously (2, 22). Melanoma lnes Me 1447, Me 9, and Me 43 were kndly provded by Dr. Theresa Whtesde of the Unversty of Pttsburgh School of Medcne, Pttsburgh, PA. Cultures establshed at three or more passages were used n serologc assays. Nonmelanoma cells. Acquston and mantenance of nonmelanoma cell lnes have been prevously reported (2). For specfcty testng the followng addtonal cell lnes were used: gloma cell lnes LN-18, GLL-19, and CL-15, neuroblastoma cell lnes JMC-32 and SK-N-MC, and fetal fbroblast cell lnes P-FF 86:1 and P-FF 86:1 1 suppled by Dr. Theresa Whtesde; renal cell carcnoma cell lnes 516W, 63D, 53 1W, and GR3 suppled by Dr. Byron Ballou; bladder carcnoma cell lne HTB4 kndly provded by Dr. Crag McCune (Unversty of Rochester, School of Medcne, Rochester, NY); and squamous cell carcnoma cell lnes UMSCC 17A, 23, 35, and 36 provded by Dr. Thomas Carey (Unversty of Mchgan, School of Medcne, Ann Arbor, M). Proten A hemadsorpton The proten A hemadsorpton assay was performed after the method descrbed by Pfreundschuh et al. (25). Brefly, ndcator cells for the proten A mxed hemadsorpton tests were prepared by conjugatng staphylococcal proten A (Pharmaca Fne Chemcals, Pscataway, NJ) to the surface of selected human blood group O-Rh' red blood cells wth. 1% CrCl3 at ph 5.. ndcator cells were washed twce n PBS plus 1% agamma FCS (Gbco, Grand sland, NY) and resuspended for use n ths medum. Target monolayers were seeded n mcro-teresak assay plates and reacted wth seral dlutons of autologous sera. After ncubaton of target cells wth sera at 37 C, wells were washed three tmes wth PBS contanng 2% agammaglobulnemc FCS at 37 C and.1 ml of an.15% suspenson of ndcator cells was added to each well. Plates were washed two to four tmes wth PBS-2% agamma FCS after 45 mn, and postve cells were read as follows: () cells were those wth a > 5% erythrocyte rosette. The end pont of the assay was the last well wth 1% of target cells (). Acd dssocaton and ultrafltraton (AD& U)' The method, ntally descrbed by Sjogren et al. (21), was used to dssocate mmune complexes. Brefly, a 6-ml ultrafltraton chamber and Amcon HP-3 membrane (Amcon Corp., Danvers, MA) were used. 2-3 ml of prefltered (.45,um, Mllpore Corp., Bedford, MA) serum was added to 5 ml of glycne-salne buffer,.1 M, ph 3.1, n the ultrafltraton chamber. Ultrafltraton was performed at 4 C under 1 ps N2 untl the orgnal volume of the test soluton was reached. Ths process was repeated twce and then the serum was washed three tmes wth 5 ml of PBS, and concentrated to the orgnal volume of serum. The total tme nvolved vared from 24 to 48 h dependng on the serum used. Absorpton analyss Absorptons were performed usng a wde range of fresh, cryopreserved, and cultured heterologous, allogenec, and autologous cells (see Table ). These were washed three tmes and mxed at a 1:1 vol/vol (1 X 18 cells/ml mnmum) wth the serum to be tested (.1 ml, at a serum dluton two doublngs above the endpont of the ttraton for that serum). Absorptons were carred out for 3 mn at 4 C and then 3 mn at 37 C. Specmens were then centrfuged at 2, rpm for 2 mn n a Sorvall RC3B refrgerated centrfuge (DuPont-Sorvall, New- 1. Abbrevatons used n ths paper: AD & U, acd dssocaton and ultrafltraton; NC, ntocellulose; TBS, Trs-buffered salne. town, CT). Supernatant serum was retested, wth an alquot of control unabsorbed serum ncubated n parallel, usng proten A to assess the degree of specfcty antbody absorpton. solaton ofmelanoma antgens Solublzaton of melanoma cells. Preparaton of melanoma antgens followed the method reported by Lloyd et al. (26). Brefly, cells from a confluent 15-cm2 flask (17 cells) were detached wth EDTA and washed twce usng PBS. 1.5 ml of lysate buffer (.15 M NaCl,.1 M Trs,.1 M Mg C12, 1 mm phenylmethysulfonyl fluorde, 2 U/ml aprotnn,.5% NP-4, ph 7.2) was added to each.1 ml of pellet. After ncubatng 15 mn at 2'C, the cells were spun at 1, g for 3 mn. The soluton was clarfed through a.22-jum flter, alquotted, and stored at -7'C. solaton ofthe ultrafltratefrom serum. The ultrafltrate, obtaned by acd dssocaton and ultrafltraton, was concentrated by lyophlzaton. Prelmnary work (22) and the absorpton analyss llustrated n Fg. 1 has shown that ths method allows for the preservaton of antgencty. solaton ofantgen shedfrom autologous tumor cells. Spent autologous tumor cell meda was obtaned from a confluent 15-cm2 flask. Meda contanng FCS were removed and the flask was washed twce wth serum-free meda, HB12 (New England Nuclear Research Products, Boston, MA). 5 ml of serum-free meda was added and ncubated for 3 d at 37C. The vablty of cells placed n serum-free meda was > 95% at ncubatons of up to 5 d. The meda were then removed and concentrated X2 n an Amcon spnal flud concentrator (Amcon Corp.). PAGE ofcell surface antgens extractedfrom SDS solublzed whole cells and elutedfrom acd-dssocated serum by ultrafltraton. One-dmensonal SDS PAGE was performed after the method of Laemmel (27). 1-cm gradent gels of 5-2% acrylamde were made. Samples were dssolved n.25 M Trs-HCl (ph 6.8), 2% SDS, 1% glycerol, 5% 2-mercaptoethanol wth.1% bromphenol blue dye marker. Fnal sample volumes contaned 5-1 Mg of proten n.2-.3 ml, and electrophoress was carred out wth a current of 2 ma untl the bromphenol blue marker reached the margn of the gel. Electrophoretc transfer ofprotens from slab PAGE to ntrocellulose (NC). The methods of Towbn et al. (28) were used for transfer of protens to a Hoefer transblot apparatus (Hoefer Scentfc nstruments, San Francsco, CA). Transfer was performed at 4 V for 2-3 h (ncreased to 9 V durng the last hour). The NC (.2 um, Schecher & Schuell, nc., Keene, NH) to whch electrophoretc transfer was accomplshed was staned for proten wth nda nk (29) and mmunologcally. mmune detecton oftransferred protens. After transfer to NC, the sheet was ar dred for 3 mn at 2 C and then quenched n Trs-buf z t , NATVE SS5 C2 POST DSSOCATON S15 C3E POST ELUATE C4 POST SPENT MEDA s- POST NATVE 55 Fgure 1. Absorpton of autologous S 5 reactvty. Ths llustrates the reactvty of serum S5 aganst autologous cultured melanoma Y-Mel 84:42. Natve sera S5 (lane 1) gave a tter of 1:32. AD & U sera S15 (lane 2) resulted n a tter of 1:262,44. The addton of an equal volume of autologous eluate obtaned from ultrafltraton ablated enhanced reactvty (lane 3). A smlar reducton n enhanced reactvty was seen wth the addton equal volumes of autologous spent tumor culture meda (lane 4) and natve sera S15 (lane 5). solaton ofmelanoma Antgens 1747

3 fered salne (TBS) ph 7.5, plus 1% BSA for 45 mn on a rocker. Antbody dluted n TBS n.5% Tween-2 was overlad on the NC paper and ncubated on a rocker at 4VC for 16 h. The paper was washed fve tmes wth TBS.5% Tween-2 for 5 mn. The second peroxdase-conjugated rabbt ant-human g antbody (Cappel Laboratores, Cochranvlle, PA), dluted wth TBS plus.5% Tween 2 was overlad on the NC paper and ncubated at room temperature whle rockng for 1 h. After washng, the thrd antbody, peroxdase-conjugated goat ant-rabbt gg (Cappel Laboratores) dluted n TBS plus.5% Tween 2 was overlad on the NC paper and ncubated whle rockng at 2 for 1 h. The NC paper was developed wth 4-chloro- naphthol n methanol 3% H22 for 15 mn. The reacton was stopped by removng the NC and placng t n a fresh contaner of dh2 and washng twce for 1 mn. Results Analyss of system Y-Mel 84:42. Before AD & U, natve serum S 5 showed reactvty to a tter of 1:32 aganst autolo- gous melanoma cell lne Y-Mel 84:42. After AD&U, enhanced reactvty to 1:262,44 was noted, the hghest tter yet reported n any autologous system (Fg. 1). As can be seen, enhanced autologous antbody reactvty could be ablated by the addton of autologous ultrafltrate, natve serum, and spent tumor culture medum. We have prevously reported that sera from 1 normal ndvduals showed no reactvty aganst melanoma cells before or after AD&U (2). Specfcty analyss of serum S15. Reactvty of serum S15 was tested by drect and absorpton analyss aganst a varety of normal and malgnant cell lnes. The results are summarzed n Table. 51 was found to detect an antgen common to melanoma, globlastoma, neuroblastoma, renal cell, head and neck, and breast carcnoma cell lnes. t dd not react wth bladder and colon carcnoma, fetal fbroblasts, pooled lymphocytes, red blood cells and platelets, FCS, and cultured autologous lymphocytes. Table. Serologc Defnton ofsystem Y-MEL 84:42 by Drect* and Absorpton Analyss* Cell lne Drect Absorpton Melanoma Y-MEL 84:42* Y-MEL 81:71 ME 1447 ME 9 ME 43 P-MEL 86:15 P-MEL 86:26 P-MEL 86:25 Gloma LN 18 GLL 19 CL 15 Renal cell carcnoma 516W 63D GR3 531W Breast carcnoma, MCF 7 Bladder carcnoma, HTB4 Neuroblastoma JMC 32 SK-N-MC Head and neck carcnoma UMSCC 17A UMSCC 23 UMSCC 35 UMSCC 36 Colon carcnoma, HCT 8 Fetal fbroblasts P-FF 86:1 P-FF 86:11 Autologous lymphocytes Pooled platelets Pooled lymphocytes Pooled RBCs Fetal calf serum * See Methods. t Autologous tumor. STAGE TREATMENT z p 64 POST HuFN*(LE) CCNU TME N MONTHS Fgure 2. Seral autologous antbody tters Y-Mel 84:42. Ths s from a patent who ntally presented wth resected stage melanoma and progressed onto metastatc dsease. Sera obtaned at several ntervals were subjected to AD & U and tested aganst autologous melanoma by proten A hemadsorpton. n dssocated sera (POST), antbody tter paralleled progresson and recurrence of melanoma. The frst two drops n dssocated antbody tter may have been due to resecton of tumor. The last drop n dssocated antbody tter may be secondary to treatment wth human leukocyte nterferon [HuFNa(LE)]. Of note s a pretermnal rse n both natve (PRE) and dssocated antbody tters. CCNU, lomustne Vlock et al.

4 Seral autologous studes ofsystem Y-Mel 84:42. Sera obtaned at 1-4-mo ntervals were tested before and after AD&U aganst autologous melanoma cells by proten A hemadsorpton. The results were then correlated wth clncal status and are llustrated n Fg. 2. n sera subjected to AD&U (POST), antbody tter demonstrated a progressve rse that paralleled recurrence and progressve dsease. The etology of the frst two drops s not clear but may be the result of treatment wth surgery and radotherapy, respectvely. The thrd drop n antbody tter may be due to treatment wth nterferon as has been reported prevously (22). Of addtonal note s the pretermnal rse n both natve (PRE) and AD&U (POST) antbody tters. Serum obtaned at month 48 was used for subsequent mmunochemcal analyss. mmunochemcal analyss. Cell lysate, spent meda, and serum ultrafltrate underwent PAGE and subsequent transfer to NC. Melanoma antgens were detected by mmunoblottng wth autologous dssocated serum S15. The results are llustrated n Fg. 3. As can be seen, autologous antbody detected a 66,-mol wt antgen n serum ultrafltrate and spent meda (Fg. 3, strps A3 and B3). n the cell lysate, 2-3 bands between 2, and 3, MW reacted wth autologous serum (strps C2 and C3). Although other antgens were detected by autologous serum, they reacted wth normal human sera as well. Normal human serum dd not detect the 66,-mol wt antgen n serum ultrafltrate or spent meda (Fg. 3, strps Al and Y MEL 84:42 BJ). Nor dd normal human serum react wth the three 2,-3,-mol wt antgens found n the cell lysate (Fg. 3, strp Cl). n one experment, antbody S15 was exposed to 17 cells/ml of autologous melanoma Y-Mel 84:42 before mmunoblottng. After absorpton, S15 faled to detect the 66,-mol wt band whle stll reactng wth the hgher molecular weght antgen (Fg. 3, strp D). Spent meda was exposed to enzymatc dgeston wth pepsn (2,889 U/ml, Mllpore Corp.) and trypsn (1:25, Gbco) before mmunoblottng (Fg. 4). The 66,-mol wt antgen was lost by exposure to trypsn for 3 mn (strp C). Exposure of spent meda to pepsn for up to 5 h faled to ablate the antgen (strp B). Of note, exposure to trypsn resulted n the development of an addtonal band between 2, and 3, mol wt whch appeared to correspond to the bands detected n the cell lysate. The antgen was not nactvated by heatng to 1 C for 5 mn. Dscusson Ths study reports the successful detecton and partal purfcaton of a class melanoma antgen detected by autologous antbody. As shown n the specfcty analyss performed, the antgen s represented on a wde number of neoplastc cells but not normal cells (see Table ). n addton, the antgen appears to be present on the cell surface and s readly shed n vtro and '1 tf 97, 66, 45, 36, 29, 24, 2, 14, A B C Fgure 3. mmunoblots of melanoma antgens. Melanoma antgens solated from ultrafltrate (A), spent meda (B), and cell lysate (C) were subjected to PAGE, transferred to NC, and exposed to normal human sera (strps Al, BJ, Cl), natve autologous sera S 5 (strps A2, B2, C2), and dssocated autologous sera S 15 (strps A3, B3, C3). Dssocated autologous antbody detected a 66,-mol wt antgen n the ultrafltrate and spent meda (A3 and B3). n the cell lysate autologous sera S 5 detected two to three bands between 2, and 3,-mol wt (C2, C3). Normal human sera dd not D react wth the 66,-mol wt antgen found by autologous antbody n the ultrafltrate and spent meda (Al, BJ). Nor dd normal human sera react wth the 2,-3,-mol wt antgens detected by autologous antbody on the cell lysate (Cl). (D) Absorpton of autologous antbody reactvty. Before mmunoblottng aganst spent meda, antbody S 15 was absorbed wth 7 cells/ml of autologous melanoma Y-Mel 84:42. After absorpton, S15 faled to detect the 66,-mol wt band whle stll reactng wth the hgher molecular weght antgen (strp D). solaton ofmelanoma Antgens 1749

5 Y-MEL 84:42 't :- A L^. ^., ze! a, 1 T,.7n D : BC 66, 24, Fgure 4. Enzymatc dgeston of melanoma antgens. The excessve wdth of the 66,-mol wt band, due to overloadng of the gel, was ntentonal n order to detect any breakdown products. Antgen obtaned from spent meda was exposed to trypsn and pepsn pror to PAGE and mmunoblottng wth dssocated autologous sera S 5. Wth no enzymatc exposure (A) the prevously mentoned 66,- mol wt band s noted. Exposure to pepsn for 5 h (B) faled to destroy antgencty, however a 3-mn exposure to trypsn (C) resulted n loss of the antgen. Of addtonal note s that followng exposure to trypsn the development of a 24,-mol wt band whch appears to correspond to one of the lower molecular weght bands noted n the cell lysate (Fg. 3, strp C3). The antgen was not nactvated by heatng to 1C for 5 mn (not shown). n vvo (see Fg. 1). t s unlkely that ths represents a cytoplasmc antgen as has been reported by others (3). By usng the proten A hemadsorpton assay, we are able to vsually confrm the presence of the antgen on the surface of melanoma cells. The dffculty encountered by others n detectng and solatng autologous antgens has been attrbuted to a number of factors: loss of antgen expresson n vtro, poor mmunogencty of the antgens, antgenc heterogenety, nsuffcent antbody levels, and the falure to mmunoprecptate the antgens (17, 24, 31). Ths report and our prevous studes (2, 22, 23) would suggest an alternatve explanaton. We have found that the majorty of patents wth melanoma produce an aganst ther own antbody tumor, however, t s masked by the presence of crculatng antgen formng mmune complexes. Ths would account for the low frequency and tter of autologous n antbody natve serum and the problems encountered n antgen solaton. Our ablty to sgnfcantly enhance antbody tter acd by dssocaton and ultrafltraton has facltated the solaton of autologous antgen. The autologous antbody tter of 175 Vlock et al. 1:262,44 found n dssocated serum S15 s over 1-fold hgher than any prevously reported. The antgen solated n these studes may be smlar to the three class melanoma antgens reported prevously (2, 22). Comparsons between all four autologous systems reveals a smlar range of reactvty. Pfreundschuh et al. (25) and Shku et al. (19) noted cross-reactvty between the AJ astrocytoma antgen and the AH melanoma antgen although drect comparsons were never made. t s possble that our four autologous systems and those reported by others (19, 22, 25) may be detectng a smlar antgen. Wth the successful partal mmunochemcal purfcaton of one autologous antgen, t s now possble to proceed wth drect comparsons. The 66,-mol wt antgen that we have descrbed has not prevously been reported n autologous studes of human melanoma. t s doubtful that ths antgen s smlar to those detected by monoclonal antbodes (none of whch have been < 9, mol wt) or the two prevously reported autologous class antgens. Although the antgens of smlar molecular weght have been detected utlzng allogenec and xenogenec antbodes (6, 32), the dstrbuton ofthese antgens on a wde varety of normal and xenogenec tssues dffers from what was noted n ths study. n addton, because the antgen can be solated by PAGE, t s unlkely that ths antgen s smlar to the ganglosdes GD2 or GM2 whch cannot be solated by PAGE and are detected through the use ofchloroform/methanol extracton and thn-layer chromatography (31, 33-35). n addton to the 66,-mol wt antgen detected after PAGE, other bands were noted as well (Fg. 3). However, because these antgens were detected by normal human as well as autologous sera ther sgnfcance s less clear cut. Whle antgens detected by allogenec antbodes may be sgnfcant, prevous studes (6, 32) have shown them to react wth normal cell consttuents. The fact that the 66,-mol wt antgen was not detected by normal human sera makes t all the more relevant. Because the hgher molecular weght bands are detected by normal human sera, t s unlkely that the 66,-mol wt antgen, whch was only dentfed by autologous sera, represents a breakdown product of those bands. n addton, reactvty of the hgher molecular weght band wth S15 was not removed by absorpton wth autologous melanoma Y-Mel 84:42. One dscrepancy n our fndngs relates to the dfferences noted between the antgens detected n the ultrafltrate and spent meda as opposed to those found n the cell lysate. Autologous antbody detected two to three antgens between 2, and 3, mol wt n the cell lysate. The relatonshp of these antgens to the 66,-mol wt antgen found n spent meda and ultrafltrate s not entrely clear. One possblty s that cytoplasmc antgens are beng detected as has been reported by others (3). Alternatvely, t may be that exposure to lyss buffer and ntracellular enzymes partally dgested the antgen. ndrect evdence for ths can be seen n our results n whch exposure ofspent meda to trypsn resulted n the development of a 24,-mol wt band that appears to correspond to one of those seen n the cell lysate. Further characterzaton of ths autologous antgen s currently underway. solaton of larger quanttes of ths antgen may allow the development of a more specfc monoclonal antbody for the dagnoss and therapy of melanoma. Beyond that the ultmate goal of such studes wll be to better understand the sgnfcance and bologc role of these antgens as they relate to the mmune response and natural hstory of melanoma.

6 Acknowledgments The authors wsh to thank Dr. Barry Solomon, vce presdent for research, W. R. Grace, Susan Heller, and Saul M. Wess, R. B. P. for techncal advce and assstance. Ths study was supported n part by grant CA from the Natonal Cancer nsttute. Dr. Vlock s a recpent of a Veterans Admnstraton Career Development Award. References 1. Chee, D.., A. W. Bodde, and J. A. Roth Producton of melanoma-assocated antgens by a defned malgnant cell stran grown n chemcally defned medum. Cancer Res. 36: Roth, J. A., H. K. Slocum, M. A. Pellegrno, E. C. Holmes, and R. A. Resfeld Purfcaton of soluble human melanoma-assocated antgens. Cancer Res. 36: Bystryn, J.-C., and J. R. Smalley dentfcaton and solublzaton of odnated cell surface melanoma assocated antgens. nt. J. Cancer. 2: McCabe, R. P., S. Ferrone, and M. A. Pellegrno Purfcaton and mmunologc evaluaton of human melanoma-assocated antgens. J. Nat!. Cancer nst. 6: Galloway, D. R., R. P. McCabe, M. A. Pellegrno, S. Ferrone, and R. A. 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