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(CANCER RESEARCH 52, 5948-5953, November 1, I992j Identification of Immunogenic Human Melanoma Antigens in a Polyvalent Melanoma Vaccine' Jean-Claude Bystryn,2 Milagros Henn, Joseph Li, and Susan Shroba Kaplan Comprehensive Cancer Center and the Department ofDermatology, New York University School ofMedicine, New York, New York 10016 ABSTRACT An essential element in the developmentof effectivevaccines against human malignant melanoma is the identification of antigens which are relevant for vaccine construction as evidenced by their ability to stimu late antimelanoma immune responses in humans. In this study, we identified immunogenic melanoma antigens using as probes antibodies induced in patients immunized with a vaccine which contains a broad range of potential immunogens. By immunoprecipitation/sodium dode cyl sulfate-polyacrylamide gel electrophoresis analysis of detergent ly sates of radioiodinated melanoma cells, we found that 17 (65%) of 26 patients sequentially immunized with a polyvalent melanoma antigen vaccine developed antibodies to one or more melanoma cell surface antigens with approximate molecular weights of38,000—43,000, 75,000, 110,000, 150,000, and 210,000. The immunodominant antigens which most frequently stimulated antibody responses were the Mr 110,000 antigen However, there is a need to develop more potent vaccines, because the current generation fails to augment immunity to melanoma or to prevent the progression of this cancer in many patients. An essential element in this endeavor is the identifi cation of melanoma antigens which are relevant for vaccine construction, as evidenced by their ability to stimulate antimel anoma immune responses in humans and by their expression on a broad spectrum of different melanomas. The purpose of this study was to identify such antigens, using as probes antibodies elicited by active immunization of patients with a vaccine for mulation containing a broad range of potential immunogens. followed by the Mr 210,000 and 38,000—43,000 antigens, which induced antibody responses in 62%, 27%, and 19% of patients, respec MATERIALS AND METhODS Melanoma Vaccine. A soluble, partially purified, polyvalent, mela noma antigen vaccinewas prepared from the material shed into culture medium by four lines of melanoma cells, as previously described (7). tively. These three antigens were commonly expressed on different mel anomas but rarely on nonmelanoma cells and are unrelated to class I or II human leukocyte antigens or to the previously described p97 or Mr 240,000 proteoglycan melanoma-associated antigens. Thus, these three antigens are attractive candidates for the construction of melanoma vaccines, because they are immunogenic in humans and are preferen flatly expressed on melanomas. The cells were adapted to long term growth in serum-free medium to prevent contamination of the vaccine with fetal bovine serum proteins. For vaccine production, the shed material was collected, concentrated by vacuum dialysis, pooled, treated with 0.5% NP-403 detergent to break up aggregates, and ultracentrifuged at 100,000 X g for 90 mm to remove transplantation alloantigens. The supernatant was concentrated by vacuum dialysis, filter sterilized, adjusted to a protein concentration of200 @tg/ml, bound to alum as an adjuvant, dispensed into sterile glass INTRODUCTION There is increasing interest in the development of vaccines to treat, and possibly to prevent, malignant melanoma. The pro gression of melanoma depends in part on host immune re sponses to melanoma (1), so that specific stimulation of anti melanoma immune responses with melanoma antigen vaccines may increase resistance to melanoma. The most convincing evidence that this concept is correct is that immunization to melanoma vaccines can prevent this cancer in syngeneic mice (2—4).The protection conferred is specific (2), i.e., mice immu nized with control vaccine are not protected against melanoma and melanoma vaccine-immunized mice are not protected against unrelated syngeneic tumors, indicating that the protec tion is mediated by immune mechanisms. Based on these observations a number of first-generation melanoma vaccines have been developed. These have proven to be safe to use and are capable of inducing immune responses to melanoma in some individuals (5—9).The clinical effectiveness of melanoma vaccines is still not established, although several studies suggest that they can slow the progression of this cancer vials, and stored at —4°C until used. The biochemical and antigenic properties ofthe vaccine have been published (7). The vaccine is free of fetal calf serum proteins and of human immunodeficiency virus and hepatitis virus. All lots were tested for aerobic and anaerobic bacteria, fungi, and pyrogens and were injected into mice and guinea pigs for general safety tests prior to use. Immunization. The vaccine was used to treat sequentially 26 patients with surgically resected stage II (regional) malignant melanoma. Eligi biity criteria included histologically confirmed regional metastases; adequate bone marrow, renal, and liver function; and the absence of residual metastatic disease as documented by physical examination and normal computerized tomography scans of brain, chest, abdomen, and pelvis. The patients were older than 18 years and were not receiving steroids or other potentially immunosuppressive medications. All pa tients had intact cellular immunity, as evidenced by skin test reactivity to at least one of a standard panel of recall antigens (purified protein derivative intermediate strength, mumps, candida, and str cytokinase streptodornase) or by the ability to be sensitized to dinitrochloroben zene. All patients signed written informed consent forms prior to treat ment, as approved by the New York University Medical Center Institutional Review Board. Patients were immunized with 40 @g of vaccine bound to alum as an adjuvant. This dose was divided into four equal aliquots and adminis tered intradermally, in 0.5 ml of saline, into each extremity every 3 or cause the regression of established metastasis in some pa tients (7—14). weeks for four cycles. Sera and Monoclonal Antibodies. Serum was collected from each Received 4/8/92; accepted 8/21/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by USPHS Research Grants patient immediately prior to the first and 1 week after the fourth vaccine immunization. Murine MoAb 96.5 to the p97 MAA was kindly pro vided by Dr. K. E. Hellstrom (University ofWashington, Seattle, WA), and MoAb 9.2.27 to a Mr 240,000 proteoglycan MAA was provided by P3OCA16087, AR39749, FD-R-000632, and A27663 and by grants from the Rose M. Badgeley ResiduaryTrust. 2 To whom requests 3 The abbreviations for reprints should be addressed, at Department of Derma tology, New York University School of Medicine, 560 First Aye, New York, NY, 10016. used are: NP-40, Nonidet P-40; BSA, bovine serum albu mm; FBS, fetal bovine serum MAA, melanoma-associated antigen; MoAb, monoclonal antibody, PBS, phosphate-buffered saline; SDS, sodium dodecyl sal fate; PAGE, polyacrylamide gel electrophoresis; HLA, human leukocyte antigen. 5948 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. IMMUNOGENIC MELANOMAANTIGENS Dr. R. Reisfeld (Scripps Clinic, La Jolla, CA). Murine MoAb W6/32 to incubated in the presence of competing proteins with that in a control class I HLA antigens and MoAb L243 to class II HLA antigens were sample incubated with normal saline. prepared from the corresponding hybridomas obtained Immunodepletion Experiments. These were performed by incubat from the Amer icaii Type Culture Collection (Rockville, MD). Cells. Antibody studies were conducted using as target SK-Mel-28 ing 0.12 ml (containing 1—2x l0@ cpm and approximately 180 @g protein) of detergent lysate of radioiodinated melanoma (SK-Mel-28) cells (kindly provided by Dr. J. Fogh, Sloan-Kettering Institute, Rye, NY), one of the human melanoma cell lines used to construct the melanoma vaccine. Specificity analysis was conducted using a panel of control cells consisting of allogeneic pigmented (SK-Mel-23 and SK Mel-30) and nonpigmented (SK-Mel-28, M14, and M20) human mel anoma cells; xenogeneic melanoma cells (hamster HM54 and murine B16 melanoma); normal allogeneic cells (melanocytes and peripheral blood lymphocytes (HLI and HL2)J; allogeneic unrelated tumors [neu roblastoma (IMR 32), glioma (u87MG), colon carcinoma (SK-Co-l and SW620), lung carcinoma (A549), rhabdomyosarcoma (RD), eryth roleukemia (K562), and breast carcinoma (T47D)J; and xenogeneic cells with 0.265 ml of monoclonal baby hamster kidney cells (BHK). M20 and M14 cells were provided by Dr. Donald Morton (University ofCalifornia, Los Angeles, Los Ange les, CA), HM54 by Dr. G. Lipkin (New York University School of Medicine, New York, NY), and u87MG, SW620, and T47D by Dr. Roberta Hayes (New York University School of Medicine). Human melanocytes (Clonetics, San Diego, CA) were cultured in melanocyte growth medium containing basic fibroblast growth factor (1 ng/ml), bovine pituitary extract (0.2%, v/v), insulin (bovine, 5 @gIml), hydro cortisone (0.5 @tg/ml),and phorbol myristate acetate (10 ng/ml). Nor mal human peripheral blood lymphocytes were obtained and separated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) on the same day as used. All other cells were obtained from the American Type Culture Collection and were cultured in RPM! 1640 supplemented with 10% FBS and antibiotics. Radlolodination. Cell surface macromolecules on melanoma and control cells were radioiodinated by the lactoperoxidase technique, as previously described (15). The cells were labeled in suspension while in logarithmic phase growth, washed thoroughly, and then lysed at 10@ cells/ml in 0.5% NP-40 detergent containing 0.025 M NaEDTA, sodium azide, and 0.174 mg/ml phenylmethylsulfonyl antibody for 18 h at gation at 12,000 X g for 20 mm. This procedure was repeated, and the cleared supernatant was tested by immunoprecipitation/SDS-PAGE analysis for the presence of residual melanoma antigens defined by vaccine-induced antibodies, as described above. RESULTS Antibodies to Melanoma prior to Vaccine Treatment. The presence of antibodies to melanoma in 26 patients with post surgical stage II malignant melanoma was investigated by im munoprecipitation and SDS-PAGE analysis. In this assay, the presence of antimelanoma antibodies is evidenced by their abil ity to precipitate radiolabeled, detergent-soluble, melanoma macromolecules, and the antigen(s) to which they are directed are identified by the location of labeled band(s) on the gel, as illustrated in Fig. 1. Prior to vaccine treatment, antibodies to melanoma were detected in 8 (31%) ofthe 26 patients (Table 1). The results obtained for a representative patient with pretreat ment antimelanoma antibodies to M@75,000, 150,000, and 210,000 antigens are illustrated in Fig. 1 (patient 124). Other patients were found to have pre-existing antibodies to one or more other antigens with approximate molecular weights of 38,000—43,000, 90,000, and 210,000. The antibodies were di rected most often to the Mr 75,000 and the Mr 90,000 antigens, 0.02% AB fluoride. Insol uble material was removed by centrifugation at 12,000 x g for 20 mm. Total acid-insoluble radioactivity was measured by precipitation with trichloroacetic antimelanoma 4°C.Bound antigens were precipitated by incubation with 0.5 ml of Protein A-Sepharose, diluted 1:2 in PBS, for 2 h at 4C and centrifu AB AB AB . I.. acid. AB Immunopreclpltatlon/SDS-PAGE Analysis. The presence of anti @ bodies to melanoma and the identity ofthe antigens to which they were directed were measured by immunoprecipitation/SDS-PAGE analysis. S * For assay, 10 @i1 of undiluted human serum were incubated with 0.1ml of radioiodinated melanoma lysate (diluted to contain approximately 200,000 cpm oftrichloroacetic acid-insoluble radioactivity) in the pres ence of 0.1 ml of 2% BSA overnight at 4C. Then 0.05 ml of goat anti-human IgG, 1gM, and IgA (KPL, Gaithersburg, MD) in 0.1 ml of 2% BSA was added to all tubes and incubated at 4C for 1 h, followed by 0.05 ml ofProtein A-Sepharose (Pharmacia LKB, Uppsala, Sweden) diluted 1:2 in PBS; incubation continued for 2 h at 4C with continual mixing. The immunoprecipitates were collected by centrifugation at 12,000 x g for 3 min, washed extensively, extracted by boiling for 5 mm with 0.05 ml ofmodified Laemmli's buffer (16) containing 5% mercap toethanol, and run on 8% homogeneous SDS-polyacrylamide gels (15). Bound labeled antigens were visualized by autoradiography using Kodak XAR-5 film and Cronex fast screens, at —70C,and the relative amount present was expressed on a scale ofO to 3+ based on the density 124 ofthe labeled bands. Molecular weight standards were from Sigma (St. Louis, MO). Competition Experiments. These experiments were performed by incubating 0.01 ml of undiluted post-vaccine treatment serum (serum 134)containing antibodies to immunogenic melanoma antigens with 1, 10, or 100 @ig of concentrated (100-fold) complete culture medium, lactoperoxidase, BSA, or FBS in 0.1 ml of PBS and with 0.1 ml of a 1:10 dilution of NP-40 lysate (15 gig) of radioiodinated melanoma (SK-Mel-28)cells for 18 h at 4C. Protein A-Sepharose was then added, and the presence of antigens bound by residual melanoma antibodies was determined by SDS-PAGE analysis as described above. Inhibition was determined by comparing the presence ofantigen bands in samples 129 133 134 136 Fig. 1. Identification ofmelanoma antigens defined by vaccine-induced human melanoma antibodies. Five patients (patients 124, 129, 133, 134, and 136) were immunized 4 times at 3-week intervals by intradermal injections of 40 @gof vaccinewith alum as an adjuvant.Sara collectedprior to vaccinetreatment (lanes A) and 1 week after the final immunization (lanes B) were tested concurrently for antibodies to radiolabeled melanoma antigens (SK-Mel-28 cells) by immuno precipitation/SDS-PAGE analysis. Induction of antibodies to individual mela noma antigens is evidenced by the presence of labeled antigen bands precipitated by post-vaccine treatment serum (lane B) which are absent or present in lesser amounts in pretreatment serum (lane A). Patient 124 had pre-existingantibodies to melanoma which were not further increased by vaccinetreatment. The other four patients developedstrong antibody responses to one or more antigens with molecular weights of approximately 38,000—43,000, 75,000, 110,000, 150,000, and 210,000. 5949 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. IMMUNOGENIC MELANOMA ANTIGENS antibodies to which were present 110,000 antigen, which induced an antibody response in 16 (62%) patients. Antibodies to this antigen were detectable prior to vaccine treatment in only 2 (8%) patients. The next most immunogenic antigens were the Mr 38,00043,000 and Mr 210,000 antigens, antibodies to which were induced or aug mented by vaccine treatment in 7 (27%) and 5 (19%) patients, respectively. Specificity of Immunogenic Melanoma Antigens. The speci ficity of the immunogenic antigens defined by vaccine-induced antibodies was evaluated by measuring the expression of each on a panel of control cells consisting of allogeneic pigmented and nonpigmented human melanomas, xenogeneic melanomas, unrelated allogeneic tumors, and normal allogeneic cells. The cells were radioiodinated by lactoperoxidase, solubilized in NP 40, and assayed for expression of melanoma antigens by immunoprecipitation/SDS-PAGE analysis using a panel of post-vaccine treatment sera with vaccine-induced antibodies to the immunogenic antigens described above and, as controls, pre-vaccine treatment sera obtained from the same patients. As in 7 (27%) and in 6 (23%) patients, respectively (see Table 1). Antibodies to these two antigens were usually present concurrently in the same patient. Antibody Response to Melanoma Vaccine Immunization. The ability of vaccine treatment to stimulate antimelanoma antibodies was measured by comparing the pattern of mela noma antigens bound by sera collected from the same patient prior to vaccine treatment and 1 week following four vaccine immunizations. A vaccine-induced antibody response was evi denced by the presence of an antigen bound to a greater extent by post- versus pre-vaccine treatment sera from the same pa tient, as illustrated in Fig. 1 (lanes A and B, respectively). The overall results are summarized in Table 1. Some patients had pre-existing antibodies to melanoma which were not further augmented by vaccine treatment, as illustrated in Fig. 1 (patient 124). By contrast, other patients developed strong antibody responses to melanoma antigens following vaccine treatment, as illustrated in Fig. 1 (patients 134 and 136). Overall, vaccine treatment induced a new antibody response, or augmented the level of pre-existing antimelanoma antibodies, in 17 (65%) of the 26 patients. The antibody response was directed to various combinations of one or more antigens, as described an additionalcontrol, the panelincludedpre- and post-vaccine treatment sera collected from a patient (patient 124) who had pre-existing antibodies to melanoma which were not further below. Identification of Immunogenic Melanoma Antigens. The antigens defined by vaccine-induced antibodies had approxi mate molecular weights of 38,000—43,000, 75,000, 110,000, 150,000, and 210,000. The immunodominant antigen which most frequently induced an antibody response increased by vaccine treatment. Representative results are illus trated in Fig. 2. The Mr 38,00043,000, 75,000, 110,000, and 210,000 antigens defined by vaccine-induced antibodies were strongly expressed on melanoma cells (see Figs. 1 and 2a), but not on melanocytes (Fig. 2b) or on colon carcinoma cells (Fig. was the M@ 2c). However,the melanocytesand colon carcinomacells did Table 1 Incidence ofantibodies to melanoma prior to and following immunization with a polyvalent melanoma antigen vaccine strongly express antigens defined by pretreatment serum 124, which served as a positive control for the labeling procedure and for the antigen assay. The tissue distribution of the antigens defined by vaccine-induced antibodies in a representative serum (patient 134) is summarized in Table 2. The M@38,00043,000, 75,000, 110,000, and 210,000 antigens were commonly ex melanomaaBefore (%) of patients with antibodies to byantigenimmunization MelanomaNo. After immunizationMr Increased immunization (27%)Mr 210,0002 (8%)Mr 150,0002 (62%)Mr 110,0002 (8%) 9 (35%) 7 (8%) (8%) 4 (16%) 18(69%) 2 16 0Mr 90,0006 (8%)Mr 75,0007 (19%)Any 38,000-43,0001 antigen (23%) 6 (27%) 9 (4%) 6 (31%) 23 a Measuredby immuno8precipitation/SDS-PAGE (23%) (35%) 2 (23%) 5 (88%) 17 (65%) analysis (n = 26). pressed by human melanomas, being expressed to various de grees by the five lines studied. These four antigens were selec tively associated with melanoma, because they were expressed on two or fewer of 11 nonmelanoma control cell lines studied. C AB a AB AB AB AR b AB AB AB AB AB AB AB AB AB AB @@-2OO V@ @q! -200 @-2OO w ..-97. .._-97 @. @-68 ..-43 w_* @ SKCO1 124 129 133 134 136 124 129 133 134 136 124 129 133 134 136 Fig. 2. Expression of melanoma antigens on different cells. SDS-PAGE analysis of lactoperoxidase-radioiodinated, NP-40-soluble antigens on SK-Mel-23 mela noma cells (a), normal melanocytes (b), and colon carcinoma (SK-CO-l) cells (c) precipitated by pre- (lanes A) and post-vaccine treatment (lanes B) sera from five patients is shown. The antigens defined by vaccine-induced antibodies in patients 129, 134, and 136 are expressed on melanoma cells but not on the control cells. The antigens defined by antibodies present prior to vaccine treatment in patient 124 are expressed by control as well as melanoma cells. 5950 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. IMMUNOGENIC MELANOMA ANTIGENS Table 2 Tissuedistributionofmelanoma antigens definedby antibodiesinducedby vaccineMelanoma immunizationwithmelanoma antigenaCell lines 38,000@43,000Melanoma, Mr 210,000 humanSK-MeI-23 ++SK-Mel-30 ++ ++++SK-Mel-28 Mr 110,000 ++ + ++ ++ + ++ ++ + +++ 0 0 + 0 00Normal 0 0 00Normallymphocytes (HL1) 00Colon lymphocytes (HL2) 0 0 0 0 0 0 ++ 0 0 0 0 0 + + 0 0 0 0 0 0 ++M14 ++M20 +++++Control Mr 75,000Mr cellsXenogeneic melanomaMurine +++Hamster melanoma (B16) 00AllogeneicMelanocytes melanoma (HM54) 00Colon carcinoma (5K-Co-I) 00Erythroleukemia carcinoma (5W620) ++++++Rhabdomyocarcoma (K562) ++Lung (RD) 00Breast carcinoma (A549) 00Glioma carcinoma (T47D) 00XenogeneicBaby (u87MG) 00a hamster kidney (BHK) Defined by vaccine-induced 134.Table antibodies from patient 3 Tissuedistributionofmelanoma antigens definedby antibodiespresentprior treatmentMelanoma to vaccine antigenaCell lines 38,000@43,000Melanoma, Mr 210,000 Mr 150,000 Mr 110,000 HumanSK-Mel-23 ++++SK-Mel-30 + + + ++++SK-MeI-28 0 0 ++0M20 ++0Control +++0Ml4 0 Mr 75,000Mr 0 + 0 + 0 + + 0 0 0 0 + + 0 0 0 cellsXenogeneic melanomaMurine +++0Hamster melanoma (Bl6) ++0AllogeneicMelanocytes melanoma (HM54) ++0Normal 0 0 +++0Normal lymphocytes (HL1) ++ ++ 0 +++0Colon lymphocytes(HL2) +++0Colon carcinoma (SK-Co-l) +++0Erythroleukemia carcinoma (SW620) ++++Rhabdomyocarcoma (K562) +++0Lung (RD) ++++Breast carcinoma (A549) ++ +++ ++ 0 + +++ 0 ++ 0 0 + ++ 0 0 0 0 0 0 0 + 0 0 0 0 0 0 0 +++0Glioma carcinoma (T47D) +++0XenogeneicBaby (u87MG) +0a hamster kidney (BHK) Defined bynatural antibodies frompatient 124. nonmelanoma cell lines studied. The Mr 110,000 antigen was The Mr 110,000 antigen was the antigen most selectively asso expressed, but only weakly, by one melanoma control cell line. ciated with melanoma, because it was the most strongly cx The tissue distribution of the Mr 90,000 antigen defined by pressed on human melanoma cells and the most weakly cx pre-existing melanoma antibodies could not be studied since pressed on control cells. The expression of the Mr 150,000 antibodies to it were not present in the serum used for this antigen on control cells could not be measured reliably because analysis. the antibody response to it was weak. The tissue distributions of the Mr 38,00043,000, 75,000, The tissue distribution of antigens defined by melanoma an and 210,000 antigens defined by pre-existing and by vaccine tibodies present prior to vaccine treatment, using as a probe induced antibodies differed. As an example, the Mr 75,000 and serum 124, is summarized in Table 3. The Mr 75,000, 150,000, and 210,000 antigens defined by this serum were expressed on 210,000 antigens defined by pre-existing antibodies were most tissues tested, including normal cells. The expression of strongly expressed on lymphocytes and on colon and lung car cinoma cells, whereas the antigens of similar molecular weight the Mr 38,00043,000 antigen was more restricted. It was de tected on two of the five melanoma lines and on two of the 11 defmed by vaccine-induced antibodies were not detectable on 5951 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. IMMUNOGENIC MELANOMA ANTIGENS these cells. Conversely, K562 cells, which expressed the Mr 210,000 antigen defined by vaccine-induced antibodies, did not express the Mr 210,000 antigen defined by pre-existing anti bodies. Similarly, M20 cells, which expressed the Mr 38,000 43,000 antigen defined by vaccine-induced antibodies, did not express the antigen of similar molecular weight defined by pre existing antibodies. Relation of Immunogenic Antigens to Culture Medium Corn ponents and HLA Antigens. Because the M@ 38,00045,000, 110,000, and 210,000 antigens defined by vaccine-induced an tibodies were preferentially expressed on human melanoma cells, their specificity was studied further. They were not related to FBS proteins, because the vaccine was prepared from cells which had been maintained in serum-free medium for several years prior to use for vaccine production, nor were they related to components of the medium used to grow cells for vaccine production, because the binding of these three antigens by spe cific antisera was not blocked by a large excess of concentrated (100-fold) complete culture medium (including growth factors and dyes) used to grow the cells for vaccine production or by a 100-fold excess of unlabeled FBS proteins or bovine serum albumin. Immunodepletion of detergent lysates of radioiodinated mel anoma cells with monoclonal antibody W6/32 to class I HLA antigens or with MoAb L243 to class II HLA antigens com pletely removed the respective antigens from the lysate (see Fig. 3, lanes5 and 9) but did not removethe M@38,00043,000, 75,000, 110,000, or 210,000 antigens defined by vaccine-in duced antibodies (Fig. 3, lanes 4 and 7). In additional experi ments, the Mr 38,00043,000, 75,000, 110,000, and 210,000 antigens defined by vaccine-induced antibodies were found to be expressed strongly (2+ to 3+) by cells which expressed poorly or not at all (0 to 1+) the previously described p97 MAA de fined by MoAb 96.5 or the Mr 240,000 high-molecular weight proteoglycan MAA defined by MoAb 9.2.2. Conversely, cells PBS anti-HLA 1 which expressed the Mr 97,000 and the Mr 240,000 MoAb defined antigens did not express the M@ 110,000 and Mr 210,000 antigens defined by vaccine-induced antibodies. These data indicate that the immunogenic Mr 38,00043,0®, 110,000, and 210,000 melanoma antigens defined by vaccine induced antibodies are not related to FBS proteins, to compo nents of the culture medium used to grow the cells for vaccine production, to class I or II HLA antigens, or to previously described melanoma-associated antigens with closely related molecular weights. DISCUSSION The identification of human melanoma-associated antigens which are immunogenic in humans is a critical element in the design of effective vaccines against this cancer. While mela noma is known to express a broad spectrum ofdistinct antigens, most of these have been defined by murine monoclonal anti bodies. Such antibodies identify human antigens which are im munogenic in mice but reveal little about the immunogenic potential of the antigens in humans. One approach to identify immunogenic antigens which are capable of stimulating antimelanoma immune responses in hu mans is to utilize as probes antibodies which are induced by active immunization ofpatients with melanoma vaccines which contain a broad range of potential immunogens. Using this approach, and an assay which permits the identification of in dividual antigens defined by polyvalent antisera, we have pre viously been able to identify several antigens in murine B16 melanoma which are immunogenic in syngeneic mice (15). In the current study, we have applied this strategy to identify human melanoma antigens defined by antibodies induced in patients immunized with a polyvalent melanoma antigen vac cine. The melanoma vaccine was made from surface material shed into culture medium by a pool of four melanoma cell lines and contains a broad range of potential immunogens (7). Prior studies have shown that this vaccine is safe to use (5), immu anti-HLA 2 nogenic in humans (5, 7, 13, 14), and capable of stimulating a ‘-200 cellular immune response toapatient's owntumor(17).There is a statistically @- A I delayed-type 4 5 6 B 7 (P < 0.02) and clinically meaningful ability of the responses vaccine and to induce a delay strong in tumor months, respectively) (14,18).ByCoxmultivariate analysis, @--@ __ hypersensitivity the patients with a strong delayed-type hypersensitivity response to vaccine immunization was 4.7 years longer than that of nonre sponders (>72 months versus 15 months, respectively) and overall survival was 3.7 years longer (>89 months versus 44 ..,-43 — significant between @@-68sected stage II melanoma, themedian disease-free survival of V @LL correlation progression (14). In a study of 94 patients with surgically re .@ 123 .7 8 9 C Fig. 3. Relation between antigens defined by vaccine-induced antibodies and HLA antigens.NP-4Olysatesofradioiodinated melanomacells(SK-Mel-28)were immunodepleted with PBS (A), with MoAb W6/32 to class I HLA antigens (B), or with MoAb L243 to class II HLA antigens (C). Remaining antigens were precipitated with vaccine-induced antibodies defined by serum 134 (lanes 1, 4, and 7), by MoAb W6/32 (lanes 2, 5, and 8), or by MoAb L243 (lanes 3, 6, and 9) and were analyzed by SDS-PAGE and autoradiography. The immunodepletion pro cedure selectively removed the class I and II HLA antigens defined by W6/32 or L243, without altering the level ofantigens defined by vaccine-induced antibodies. these relations are independent of disease severity or of overall immunological competence, suggesting that this is a vaccine effect. These observations suggest that the vaccine may be din ically effective in slowing the progression of melanoma in some patients. In this study, we found that antibodies to melanoma were present in 31% of 26 sequentially tested patients with surgically resected stage II (regional disease) malignant melanoma prior to vaccine treatment. The antibodies were directed to one or more antigens with approximate molecular weights of 38,000— 43,000, 75,000, 90,000, 110,000, 150,000, and 210,000. These antigens were expressed as often on nonmelanoma tumor cells as on melanoma cells and were also expressed on some normal cells, suggesting that they are normal tissue antigens expressed 5952 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. IMMUNOGENIC MELANOMA on melanoma cells. Because only a limited number of normal individuals were studied, it is not possible to determine whether these antibodies represent “natural― antibodies to melanoma or reflect patients' spontaneous immune responses to their mela noma. The vaccine contains melanoma antigens which are immu nogenic in humans, since vaccine treatment induced new anti body responses or augmented the level of pre-existing antibod ies to antigens expressed by melanoma cells in 65% of the 26 patients. The immunogenic antigens had approximate molecu lar weights of 38,000—43,000, 75,000, 110,000, 150,000, and 210,000. Because these were antigens defined by polyclonal sera, there is a possibility that the antigens of similar molecular weight immunoprecipitated by different sera may not be iden tical. All antigens appeared to be expressed on the external surface of melanoma cells, because they were radioiodinated by the lactoperoxidase technique. The immunogenic potency of these antigens, as evaluated by the frequency with which they induced antibody responses in patients, was variable. The most potent immunogen was the Mr 110,000 antigen, which induced antibody responses in 62% of patients. Antibody responses in individual patients were directed to various combinations of one or more antigens, indicating that the immunogenicity of the antigens depends in part on host factors, as well as on the properties of the antigens. An interesting but unexplained oh ANTIGENS and 210,000 melanoma antigens defined by vaccine-induced antibodies appear to differ from previously described melanoma antigens of similar molecular weight which are defined by mu rine monoclonal antibodies. In summary, the antigens which appear to be the most at tractive candidates for the construction of melanoma vaccines are the Mr 210,000, 1 10,000, and 38,000—45,000 antigens, be cause they possess a combination of properties which are par ticularly desirable for this purpose (1), i.e., the ability to elicit immune responses in humans, expression on the surface of melanoma cells where they can be seen by host effector immune mechanisms on viable cells, selective expression on human mel anoma, and common expression on different melanoma cells so that a generic vaccine can be used to treat different patients. The Mr 110,000 antigen is a particularly attractive candidate for vaccine construction, because it is the immunodominant antigen. REFERENCES 1. Bystryn, i-C. Tumor vaccines. Cancer Metastasis Rev., 9: 81—91,1990. 2. Bystryn, i-C. Antibody response and tumor growth in syngeneic mice immu nized to partially purified B16 melanoma-associatedantigens. Immunology, 120:96—101, 1978. 3. Avent, i., Vervaert, C., and Seigler, H. F. Non-specific and specific active immunotherapy in a Bl6 murine melanoma system. i. Surg. Oncol., 12: 87—96,1979. 4. Livingston, P. 0., Calves, M. 1., and Natoli, E. J. Approaches to augmenting the immunogenicity of the ganglioside GM2 in mice: purified GM2 is supe servation is that vaccine treatment rarely induced an antibody response to the Mr 75,000 and Mr 90,000 antigens, to which pre-existing antibodies were most common. Because ofthe lim ited number of patients studied, the clinical efficacy of pre existing or vaccine-induced antibodies has not been established. The Mr 38,00043,000, 75,000, 110,000, and 210,000 anti gens defined by vaccine-induced antibodies appear to be selec tively expressed on melanoma. All four of these antigens were expressed on all human melanoma cells studied but were cx pressed on only one or two of 11 nonmelanoma control cell lines. This is in contrast to antigens defined by pre-existing antimelanoma antibodies, which were widely distributed on nonmelanoma cells. There were differences in tissue distribu tion between the Mr 38,00043,000, 75,000, 110,000, and 210,000 antigens defined by vaccine-induced antibodies and antigens of similar molecular weight defined by pre-existing melanoma antibodies, suggesting that these antigens are differ ent even though they migrate similarly on SDS-PAGE. None of the antigens defined by vaccine-induced antibodies is related to culture medium components which may be present in vaccines prepared from cultured cells, since the binding of these anti bodies was not inhibited by excess concentrated complete me dium or FBS proteins. The Mr 38,00043,000 antigen co-mi grates closely with HLA class I and II antigens but differs from these on the basis of immunodepletion experiments. The Mr 110,000 antigen co-migrates near, but not with, the Mr 97,000 9. Wallack, M. K., Bash, i. A., and Leftheriotis, E. Positive relationship of clinical and serologic responses to vaccine melanoma oncolysate. Arch. Surg., 122:1460—1463, 1987. 10. Hersey, P., Edwards, A., Coates, A., et a!. Evidence that treatment with vaccinia melanoma cell lysates (VMCL) may improve survival of patients with stage II melanoma. Treatment of stage II melanoma with viral lysates. Cancer Immunol. Immunother., 25: 257—265,1987. 11. Mitchell, M. S., Kan-Mitchell, J., Kempf, R. A., et al. Active specific immu notherapy for melanoma: phase I trial of allogeneic lysates and a novel adjuvant. Cancer Res., 48: 5883—5893,1988. 12. Berd, D., and Mastrangelo, M. J. Active immunotherapy of human mela noma exploiting the immunopotentiating effects ofcyclophosphamide. Can cer Invest., 6: 377—379,1988. 13. Bystryn, J-C., Dugan, M., Oratz, R., et a!. Vaccine immunotherapy of human malignant melanoma: relationship between method of immunization, immu nogenicity, and tumor progression. In: C. F. Fox (ed.), Human Tumor An tigens and Specific Tumor Therapy, pp. 307—315.New York: Alan R. Liss, Inc., 1989. 14. Bystryn, i-C., Oratz, R., Roses, D., Harris, M., Henn, M., and Lew, R. Relation between immune response to melanoma vaccine immunization and clinical outcome in stage II malignant melanoma. Cancer (Phila.), 69: 1157— 1164, 1992. transferrmn-like 15. iohnston, D., Schachne,J. P., and Bystryn,i-C. Identification of immuno melanoma antigen defined by MoAb 96.5, and nor to whole cells. i. Immunol., 138: 1524—1529, 1987. 5. Bystryn,i-C., Oratz, R., and Harris, M. N. Immunogenicityof a polyvalent melanoma antigen vaccine in man. Cancer (Phila)., 61: 1065—1070,1988. 6. Morton, D. L. Active immunotherapy against cancer. present status. Semin. Oncol., 13:180—185, 1986. 7. Bystryn, i-C., iacobsen, S., and Harris, M. Preparation and characterization of a polyvalent human melanoma antigen vaccine. J. Biol. Response Modif., 5: 221—224,1986. 8. Livingston, P. 0., Natoli, E. i., Calves, M. i., et aL Vaccines containing purified GM2 ganglioside elicit GM2 antibodies in melanoma patients. Proc. Nail. Acad. Sci. USA, 84: 2911—2915, 1987. genic B16 melanoma-associated antigens. i. Biol. Response Modif., 6: 108— the Mr 210,000 antigen co-migrates near the Mr 240,000 pro 120, 1987. teoglycan antigen defined by MoAb 9.2.27. However, the M@ 16. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680—682,1970. 110,000 and 210,000 antigens are highly expressed by cells 17. Oratz, R., Cockerall,C., Speyer,i., Harris, M. N., Roses,D. F., and Bystryn, which express little or none of the Mr 97,000 and 240,000 i-C. Induction of tumor-infiltrating lymphocytes in malignant melanoma antigens, and conversely the expression of the Mr 110,000 and metastasis by immunization to melanoma antigen vaccine. i. Biol. Response Modif., 8: 355—358,1989. 210,000 antigens is very low on cells which strongly express the Mr 97,000 and the Mr 210,000 antigens, indicating antigens are different. Thus, the Mr 38,00043,000, that these 110,000, 18. Bystryn, J-C., Oratz, R., Roses, D. F., Harris, M. N., Henn, M., and Lew, R. Improved survival of melanoma patients with delayed type hypersensitivity response to melanoma vaccine immunization. Clin. Res., 39: 503A, 1991. 5953 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research. Identification of Immunogenic Human Melanoma Antigens in a Polyvalent Melanoma Vaccine Jean-Claude Bystryn, Milagros Henn, Joseph Li, et al. Cancer Res 1992;52:5948-5953. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/21/5948 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research.