Int. J. Cancer: 52,967-973 (1992) hl 1992 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer

ANALYSIS OF TWO HUMAN MONOCLONAL ANTIBODIES AGAINST MELANOMA Newman M. Y E I L D I N GCarolyn ~. GERSTNER and John M. KIRKWOOD? Division of Medical Oncology, Department of Medicine; and Pittsburgh Cancer Institute, University of Pittsbiqh, School of Medicine. Pittsburgh, PA 15213 USA. B cells derived from peripheral-bloodlymphocytes (PBL) and tumor-infiltrating lymphocytes (TlL) from a patient with a high serum antibody titer to autologous melanoma were transformed with Epstein-Barr virus (EBV) and evaluated for reactivity against autologous tumor. B cells producing antibody reactive with autologous tumor and unreactive with normal fibroblasts were detected both in TIL and in PBL. One cell line derived from PBL and another derived from TIL sustained production of tumor-reactive antibody for I 0 weeks and over I 5 months respectively. The cell line derived from PBL, 2DI I, produced an antibody reactive with a tryprin-resistant antigen expressed on the cell membrane of autologous and allogeneic melanoma cell lines. The cell line derived from TIL, IF6, produced an antibody reactive with a cell-surface glycoprotein expressed by 5 autologous melanoma cell lines derived from 5 different metastases and I6/ I 9 allogeneic melanoma cell lines. IF6 also showed reactivity with cell lines derived from a blue nevus, a congenital nevus, an astrocytoma, and I /4 renal-cell carcinomas; but it was not reactive with 5 foreskin melanocyte cell lines, 2 normal fibroblast lines, 5 leukemia/lymphomalines, 8 lung-cancer lines, 8 glioblastoma lines, or lines derived from I ovarian carcinoma, I colon carcinoma, I vulvar carcinoma, I fibrosarcoma, I murine melanoma, or 4 murine leukemia/ lymphomas. We describe here an antibody that detects a new melanoma specificity obtained by EBV transformation of tumorinfiltratingB cells.

1992 Wiley-Li.~~. Inr

A basic tenet of tumor immunology holds that the immune system can distinguish between tumor and normal tissue. Clinical and laboratory investigations suggest that both the cellular and the humoral components of the immune system are capable of tumor recognition. However, the antigens recognized by the immune response are poorly characterized. A number of murinc monoclonal antibodies (MAbs) have been dcveloped which detect human melanoma antigens, but none of these is known to represent antigens that are immunogenic in the autologous host, or the target of biologically rclevant immune responses which may alter the natural history of the malignant disease. We havc focused our investigations on the human humoral response to melanoma. Analysis of sera from melanoma patients has demonstrated tumor-reactive antibodies which rccognizc antigens with expression variably restricted to autologous tumor, to autologous tumor and allogeneic tumors of similar histogcnesis, or expressed widely among a variety of tumor and normal tissues (Shiku et al., 1976, 1977; Pfreundschuh rt al., 1978; Carey et al., 1979; Real et al.. 1984; Kan-Mitchell et al., 1989). As a means of characterizing the antigens recognized by the tumor host, we have sought to dcvclop mclanoma-reactive human monoclonal antibodies from B cells of patients with melanoma. Attempts to clone human B cells for production of MAbs against a range of antigens havc met with varying success (Cote et al., 1983; Houghton et al.. 1983; Kan-Mitchell et al., 1986,1989; Yamaguchi et (11..1987; Furukawa et al., 1989; Schadendorf et al., 1989; Irie and Morton. 1986; Irie et al., 1989). Lack of a human hybridoma fusion partner which reliably generates stable long-term antibody producing human cell lines has hampered dcvelopment of human MAbs. Fusion of human B cells to murinc fusion partners has also met with limited success in producing human MAbs. Epstein-Barr-virus (EBV) transfor-

mation of human B cells has proven to be a useful method of short-term culture and analysis of the humoral response, and EBV transformation has occasionally yielded a stable B-lymphoblastoid line producing melanoma-reactive antibody. We have reported our experience using EBV-transformation for short-term culture of human B cells (Kirkwood and Robinson, 1990). In a melanoma patient with a high titer of serum antibody reactive with autologous tumor, we demonstrated B cells producing tumor-reactive antibody both in peripheral-blood (PBL) and in tumor-infiltrating lymphocytes (TIL). One melanoma-reactive B-cell culture derived from PBL and one culture derived from TIL yielded long-term antibody-producing lines enabling more detailed analysis of reactivity. We report here an analysis of B-cell reactivity of these 2 human B-lymphoblastoid lines. MATERIAL AND METHODS

Cultured cell lines Analysis of antibody reactivity was conducted using cell lines initiated as in vitro cultures at the Pittsburgh Cancer Institute, including: foreskin melanocytes (5 lines), a congenital nevus, a blue nevus, one primary melanoma, 24 metastatic melanomas, an astrocytoma, 4 renal-cell carcinomas, a colon carcinoma, a breast carcinoma, an ovarian carcinoma, a fibrosarcoma, a vulvar carcinoma, 3 small-cell lung carcinomas, 8 non-smallcell lung carcinomas, 5 leukemia/lymphomas, 2 head-and-neck carcinomas, and 2 normal fibroblasts. Additionally, a murine melanoma cell line, BL-6, and 4 murine lcukemia/lymphomas maintained in culture at the Pittsburgh Cancer Institute were used for specificity analysis. Eight glioblastoma lines were kindly provided by Dr. R. Selker, Department of Neurology, West Penn Hospital. Human monoclonal antibodies Two melanoma-reactive human monoclonal antibodies were developed from EBV-transformed B cells (Miller, 1971; Kirkwood and Robinson, 1990) of a melanoma patient with a high serologic titer of antibody reactive with autologous tumor. 2 D l l is an IgMh produced by a lymphoblastoid line derived from PBL cultured at limit dilution, and IF6 is an IgMh produced by a TIL-derived lymphoblastoid line. Prior other B-cell lines from the initial screen studied in more limited fashion all yielded IgMh and K antibodies that bound tumor cells preferentially. Iinniiitiohistochemistry Confluent-cultured cell lines were harvested either with a cell scraper or with 0.02% wiv EDTA in PBS, washed in PBS, 'Present address: Hematology-Oncology Section, 7th Floor, Silverstein Pavilion. Philadelphia. PA 19104, USA.

2To whom correspondence and reprint requests should be addressed, at Division of Medical Oncology, Department of Medicine. University of Pittsburgh, 3459 Fifth Avenue, 7 Main North, Pittsburgh, PA 15213 USA. Received: July 28. 1992




August Bx=Primary


January Bx=Primary


Apr i 1 Bx=LLne 136.1 (Lymph Node)


January BX=DN X 2

1 1 July February Bx=Line Bx=Line 136.32 136.15 (Abdominal Met) (Buttock Met) 1 September Bx=Line 136.26 .

une x=DN X 2


February Bx=Line 136.34 (Axil. Met)


July Bx=Tonsillar Met

FIGURE 1 - Clinical history of patient 136. = = =, time on IL-2IALAK therapy; 1 above the time line represents blood draws; *, PBLs, January 1989, source of EBV-transformed lymphocytes.

and suspended at loh cellsiml in PBS. Cytospins were prepared in a Shandon Cytospin 2 centrifuge (Sewickley, PA), fixed in acetone, and blocked with 10% FCS in PBS. Cytospins were incubated with primary antibody for 1 hr at room temperature. Antibody reactivity was assessed using peroxidaselabelled goat anti-human IgM and AEC (3-amino-9-ethylcarbazole). Antibo& absorption The titer of reactivity of B-cell culture supernates was determined using serial 2-fold dilutions of the supernate for immunohistochemical analysis. The titer of supernate 2 logz dilutions below the highest titer showing reactivity against autologous tumor was used for absorption studies (Vlock and Kirkwood, 1985). For absorption, titered supernate was incubated with the absorbing cells at 1:l volivol for 30 min at 37°C. The cells used for absorption testing were sedimented at 500g for 10 min, and the supernate recovered and analyzed immunohistochemically for reactivity with autologous tumor.

1% bovine hemoglobin in PBS at room temperature for 1 hr, followed by incubation with primary antibody (1F6) overnight at 4°C. Reactivity of the primary antibody was analyzed with alkaline-phosphatase-conjugatedgoat anti-human IgM (Tago, Burlingame, CA) and developed with a BCIPiNBT (5-bromo4-chloro-3-indoyl phosphate p-toluidine/p-nitro blue tetrazolium chloride) Color Development Kit (Bio-Rad, Richmond, CA).

Siib-cellularfrcrctionatioti Cultured cells grown to confluence were harvested, washed in PBS, re-suspended in lysis buffer (0.32 M sucrose, 0.02 M Tris, 0.5 M NaCl containing 0.2 mM EDTA, 0.2 mgiml aprotinin, and 1 mM PMSF) at 4"C, and lysed in a PotterElvehjem homogenizer (Kontes, Vineland, NJ). The lysate was layered on a sucrose gradient (1.2 M and 0.8 M sucrose in Tris-buffered saline) and centrifuged at 50,OOOg for 90 min to separate cytoplasm, plasma membrane and nuclear fractions. Plasma membrane fractions were harvested from the 2-step gradient interfaces, washed in PBS, and re-suspended in 0.6% Biochemical characterization of antigens NP-40 in PBS. NP-40 insoluble membrane components were Cell-surface antigen(s) were characterized according to pelleted and solubilized in Laemmli buffer containing 2% their susceptibility to denaturation prior to the performance of SDS. Sub-cellular fractions were aliquotted and stored at absorption studies. Cells were exposed to: ( a ) 56°C for 1 hr, ( b ) -70°C until needed. Protein concentrations were determined 40 mM N-ethylmaleimide (Sigma, St Louis, MO) for 30 min at by the Lowry procedure. Reactivity of 1F6 against the subRT, ( c )40 mM dithiothreitol (Boehringer Mannheim Biochem- cellular fractions was assessed by immunoblot. icals, Indianapolis, IN) for 30 min at RT, ( d ) 10 mU/ml endoglycosidase H (Boehringer Mannheim) for 1 hr at 3 7 T , RESULTS ( e ) trypsin 0.25% (GIBCO, Grand Island, NY) for 45 min at 37°C followed by inhibition with a 4-fold excess of soybean Clinical history of patient PCI-MEL 136 Patient PCI-MEL 136 initially presented in August 1986 trypsin inhibitor (Fluka, Buchs. Switzerland), or ( f )neuraminidase 0.05 Uiml (Boehringer Mannheim) for 45 min at 37°C. with a non-familial primary melanoma of Breslow depth 3 mm, Treated cells were tested for absorption capacity using titered Clark level 111, on his posterior trunk; the melanoma was supernate, which was then assayed immunohistochemically resected. A second Breslow-depth-0.85 mm Clark-level-I11 against autologous tumor. Additionally, cytospins were made primary melanoma of the right ear was resected in January of the variously treated cells to test antibody reactivity directly 1987. A right suprascapular soft-tissue metastasis was resected in April 1987. The melanoma line PCI-MEL 136.1 was derived by immunohistochemistry. by in vitro monolayer culture of the tissue. After developing Immunoblots liver. nasopharyngeal and S.C. abdominal metastases in DecemFor immunoblot studies, cultured cells were grown to ber 1988, the patient underwent therapy with systemic adminconfluence, washed with PBS, and harvested with a cell istration of interleukin-2 and adherence-purified lymphokinescraper. Pelleted cells were lysed in 0.6% Nonidet P-40 activated-killer (ALAK) cells. He experienced a major clinical (Sigma) in PBS, and nuclei removed by pelleting at 3,000g for anti-tumor response to therapy, with all lesions regressing 10 minutes. Lysates were aliquotted and stored at -70°C until completely, except for a I-cm S.C. abdominal metastasis which needed. Protein concentration was determined using the showed partial regression and was resected. The melanoma Lowry procedure (protein assay kit, Sigma). For each immuno- line PCI-MEL 136.15 was derived from this resected metablot, cell lysates were prepared under reducing conditions in static tumor specimen. Metastasis to the nasal pharynx reLaemmli buffer (0.0625 M Tris, 2% SDS, 10% glycerol, 5% curred in April 1989 and was also resected. The resected tumor 2-ME, 0.002% bromophenol blue, and 0.001% phenol red) was cultured in vitro yielding melanoma line PCI-MEL 136.26. and heated at 95°C for 3 min; 50 pg samples were electropho- In July of 1991 the patient had a recurrence in the right rescd on a 6% SDS-polyacrylamide gel with a stacking gel of buttock; it was resected. The resected tumor was cultured in 4% SDS-polyacrylamide and transferred by electroblot to vitro yielding melanoma cell line PCI-MEL 136.32 (Fig. 1). The nitrocellulose paper. Electroblotted lysates were blocked with most recent recurrence to date was a left axillary mass,



Reactivity of 2D1 1 0.30





o v1 0














Weeks of Culture FIGURE2 - Microculture 2 D l l yielded a tumor-reactive IgMh for 10 weeks continuous culture. Loss of reactivity of the culture

supernate paralleled decreasing antibody production. TABLE I


FIGURE 3 - Reactivity of 2Dll examined by immunohistochemistry. Cytospins of cultured cell lines were incubated with IF6 and reactivity assessed using peroxidase-labelled goat anti-human IgM and AEC. Reactivity is demonstrated against 2 autologous melanoma lines, PCI-MEL 136.26 ( a ) and PCI-MEL 136.15 (b), an allogeneic melanoma line, PCI-MEL 306.2 (c), and a normal fibroblast line, CCD187SK (d).


Target cell lines ~

lines tested' ~~

Autologous melanoma Allogeneic melanoma Blue nevus Normal fibroblasts

313 313 111


'Values represent the number of cell lines against which 2D11 reactsinumber of cell lines tested. resected in February 1992, yielding melanoma cell line PCIMEL 136.34. Serologic testing revealed a high titer of reactivity against autologous tumor (1:1024), and a more detailed study of B-cell antibody reactivity in blood and tumor specimens was undertaken using EBV transformation of B cells.

Production and analvsis of"2011 , EBV-transformed PBL from patient PCI-MEL 136 were cultured at limit dilution (25 B cells/well) yielding growth in 13% of wells (231180 wells). One microculture, 2Dl1, produced an antibody reactive with autologous tumor but unreactive with normal fibroblasts as determined by ELISA. The culture produced IgMh. 2 D l l retained tumor reactivity for eight weeks without sub-culture (Fig. 2). Decreased reactivity of the culture supernate paralleled decreasing antibody production by the culture. Production of an inhibitor, such as a blocking antibody, does not appear to be involved, as it was possible to reconstitute baseline levels of reactivity to lowertitcred late supernate specimens by concentration of these supernates. Thus, production of a blocking antibody does not appear to be the cause of loss of reactivity. Tumor reactivity was not recovered in sub-cultures of the 2 D l l B cells, in attempts to select for enhanced antibody yield. By imrnunohistoehemical analysis, 2 D l l was reactive with 3 lines of autologous tumor (PCI-MEL 136.1. PCI-MEL 136.15 and PCI-MEL 136.26), 3 lines of allogeneic melanoma, a blue nevus line, and one of 3 normal fibroblast lines (Table I). Against the melanoma lines, immunohistochemistry showed strong staining around the cell circumference, suggesting membrane staining, and weak staining of the cytoplasm (Fig.

3). Staining of the blue nevus line suggested weak antigen expression on the cell membrane of approximately 20% of cells. In contrast, the normal fibroblast line with which 2 D l l reacted showed trace cytoplasmic staining of a few cells, but no membrane staining. Exposure of autologous tumor cells to trypsin prior to testing for antibody reactivity decreased intensity of staining only slightly, suggesting that the antigenic determinant recognized by antibody 2D11 may not be proteinaceous.

Production and isotype analysis of l F 6 EBV-transformed TIL cultured at 250 B cellsiwell yielded 8 /780 microcultures producing antibody reactive with autologous tumor but unreactive with fibroblasts. One of these microcultures, 1F6, produced only one species of antibody, IgMh, and expressed only cell-surface IgMh, suggesting clonal origin. The other cell lines were polyclonal. In 4 of the microcultures, including 1F6, an IgMX was determined by ELISA to be the tumor-reactive antibody. A fifth line produced both an IgMh and an IgMK which were tumor-reactive. Three lines did not retain antibody reactivity of the desired specificity long enough to permit more thorough determination of the antibody reactivity. The oligoclonal microcultures lost tumor specificity over 5 to 7 weeks of culture, and it could not be restored by sub-culturing. 1F6 was expanded in culture and continues to produce tumor-reactive antibody after 15 months of continuous culture without sub-culture. Analysis of specificity of IF6 Immunohistochemical reactivity of 1F6 was examined against a panel of cultured cell lines. Against cell lines of similar histogenesis, IF6 was reactive with 5/51 autologous melanomas, 16/19 allogeneic melanomas, a blue nevus, a congenital nevus, and an astrocytoma, but unreactive against 5 normal foreskin melanocytes as well as 8 glioblastomas (Table 11). Against human cell lines derived from tumors of diverse other embryogenic derivations, 1F6 was reactive with 114 renal-cell carcinoma cell lines, but unreactive against a panel of other tumors or normal cell lines. Similarly, IF6 showed no reactivity against a panel of m u r k tumors, including a murine melanoma.




Cell line


Human cell lines of related embryogenic origin Autologous melanoma Allogeneic melanoma Blue nevus Congenital nevus Astrocytoma Foreskin melanocytes Dysplastic nevi Glioblastoma Human cell lines of disparate embryogenic origin Leukeniia/lymphoma3 Renal-cell carcinoma Fibrosarcoma Colon carcinoma Ovarian carcinoma Vulvar carcinoma Small-cell lung carcinoma Non-small-cell lung Ca Squamous head-and-neck Ca Non-neoplastic human cell lines Normal fibroblasts Xenogeneic cell lines Leukemia/lymphoma4 Melanoma’





515 16119


lil 111 111 015 014 018



114 01 1 01 1 ni 1 01 1

111 01 1 01 1

013 018



01 1

014 01 1

‘Slides were prepared from single-cell suspensions, fixed in acetone, and incubated with antibody from B-cell supernatants. Antibody reactivity was assessed by immunoperoxide using AEC (3-amino-9-ethylcarbazole).-’Absorption of the supernatant against an equal volume of cells was conducted prior to use in immunocytology-TJ937, K562, MOLT-1, RAJI, and CEM.--‘Murine cell lines EL-I, P815. CTLL-2. and Stobo.-5Murine melanoma, BL-6. Cell-sicrfucererrctivity of 1F6 According to imniunohistochemistry, 1F6 appears to bind a cell-mcmbrane antigen on the mclanoma cell lines, giving a circumferential pattern of staining by immunohistology and a lesser degree of cytoplasmic staining (Fig. 4). To demonstrate cell-surface antigen expression, titcred antibody was preabsorbed against viable cells (Fig. 5). Table I1 shows that antibody reactivity was ablated by absorption with cultured lines of autologous tumor, an astrocytoma, or the renal-cell carcinoma, with which it showed direct reactivity. However, antibody reactivity was not ablated by absorption using lincs derived from a colon carcinoma, an ovarian carcinoma, or normal fibroblasts, against which the antibody was unreactive by direct immunohistologic analysis.

lntrnirrioliistologic reactivity of IF6 Immunohistologic study of tumor sections from biopsies derived at 6 time points from subject 136 shows positive staining of tumor cells with lF6 on all of these frcsh snapfrozen autologous tumor biopsies (Samples 136.1, .9, .15, .26, .32, .34-Table 11). Parallel studies of the nevic component of 4 samples of autologous dysplastic nevi were negative. Biochemical analysis of the antigen recognized by 1F6 antibody To characterize the antigen recognized by the antibody in 1F6, autologous tumor cells were enzymatically or chemically treated prior to use in absorption studies (Table 111). Treatment of autologous tumor with trypsin, dithiothreitol, endoglycosidase H, or by heating at 56°C for 1 hr ablated the ability of

autologous tumor cells to absorb reactivity from 1F6, suggesting denaturation of the antigen recognized by 1F6. Treatment of autologous tumor with N-ethylmaleimide prior to preabsorption failed to diminish their absorption activity. Neuraminidase enhanced the absorption activity of autologous tumor. Neuraminidase-treated autologous tumor demonstrated enhanced capacity to absorb antibody reactivity, with treated cells capable of removing 3 log. dilutions of binding activity, as comparcd with 2 with untreated tumor cells. In summary, these studies suggest that 1F6 reactswith a glycoprotein that is partially shielded by sialic acid on the cell surface.

Metabolic itthibitioii of IF6 antigen qnthesis Tumor cells were cultured in the prescnce of tunicamycin at 100 pgiml concentration for 4 to 18 hr. Reactivityof the tumor cell with 1F6 in Western blot assays was then studied, revealing obliteration of the antigen in the culture exposed to tunicamycin for 18 hr. This further supports the hypothesis that the antigen recognized by 1F6 is a glycoprotcin. Irnmuitoblot of IF6 Reactivity of 1F6 was assessed by immunoblot against a panel of cell lines (Fig. 6). 1F6 binds to an antigen with an apparent molecular weight of 194 kDa detected in preparations of all the melanoma lines tested and on the blue nevus. In contrast, no reactivity was detected on immunoblot against an equivalent amount of antigen derived from the astrocytoma or the renal-cell carcinoma with which 1F6 reacted immunohistochemically. A 194-kDa band of reactivity was seen with the astrocytoma and the rcnal-cell carcinoma lines when the gel was loaded with 200 kg protein per lane, suggesting relatively lcss antigen expression than in the melanoma lines (data not shown). IF6 showed differential reactivity against the 3 different autologous tumor lines. The greatest reactivity was seen against PCI-MEL 136.26, the tumor from which 1F6 was derived, while the least reactivity was seen against the earliest derived autologous tumor line PCI-MEL 136.1. Expression of major histocompatibility antigens, as detected by MAb, to framework determinants of MHC-class-I antigens revealed no significant differences among the several autologous tumor lines studicd here. Sub-cellulurfructioriation Immunoblots with 1F6 were performed against sub-cellular fractions of autologous tumor (Fig. 7). Reactivity was greatest against the cytoplasmic fraction of lysate and the Nonidet P-40 insoluble membrane fractions. DISCUSSION

The major obstacle to the characterization of the autologous human humoral immune response to melanoma is the lack of tcchnology required to sustain the growth and function of committed human B cells, thus limiting the ability to generate quantities of human MAbs sufficient for biochemical, diagnostic and therapeutic evaluation. Transformation and immortalization of human B cells with EBV yields short-term antibodyproducing lines, allowing partial charactcrization of the humoral immune response, and fusion of human B cells with murine fusion partners has met with limited success in yielding long-tcrm antibody-producing lines. The advent of new B-cellsupporting cytokines and chemical growth and differentiation factors and the development of new fusion and molecular techniques offer promising new approaches for sustaining growth of stable long-term antibody-producing cell lines. Here we have demonstrated the presence, both in PBL and in TIL, of B cells committed to the production of tumorreactive antibody in a patient with serum antibody reactive with autologous tumor. Most B-cell cultures that produced



FltirlRE 4 - Reactivity of 1F6 examined by immunohistochemistry. Cytospins of cultured cell lines were incubated with 1F6 and reactivity assessed using peroxidase-labelled goat anti-human IgM and AEC. Reactivity is demonstrated against 3 autologous melanoma lines, PCI-MEL 136.26 (a), PCI-MEL 136.15 (b), and PCI-MEL 136.1 (c); two allogeneic melanoma lines, PCI-MEL 105.1 (d), and PCI-MEL 310.2 ( e ) ; a congenital nevus line, PCI-MEL 228.1 If); an astrocytoma line A1253 (g); a renal-cell carcinoma cell line (11); a foreskin melanocyte line, PCI-MEL 167.0 (i); and a normal fibroblast line, CCD187SK 0).

Absorption of 1F6 by Viable Autologous Tumor


Treatment of tumor cells used for absorption

0 - N a t i v e supernate .-Absorbed supernate Q)


Q c

None Trypsin N-ethylmaleimide Dithiothreitol Heat (56°C x 1 hr) Endoglycosidase H Neuraminidase

Absorption of antibody






+ +'

Cultured autologous tumor cells were pre-treated prior to use in absorption assays. Positive absorption (+) demonstrates that the antigen was not altered by pre-treatment, and the cells retain the ability to absorb the antibody. Negative absorption (-) demonstrates loss of absorption capacity by antigen.-'Enhancement of antibody absorption was seen after pre-treatment of cells with neuraminidase. Neat







Titer (1/dilution) FIGURE 5 - Cell-surface reactivity of 1F6was assessed by absorption assays using viable autologous tumor. After absorption, the titered supernates were examined for immunohistochemical reactivity against autologous tumor. The percentage of cells stained was determined and compared with native (unabsorbed) supernate.

tumor-reactive antibody sustained tumor-restricted reactivity for only a few weeks. However, some EBV-transformed B-cell lines obtained here sustained antibody production long enough to allow analysis of antibody specificity and partial characterization of the bound antigen. Microculture 2 D l l was derived from PBL and sustained production of tumor-reactive antibody long enough to allow partial analysis of specificity. The lymphoblastoid line expressed on its cell surface and produced only IgMX, suggesting clonal outgrowth. The antigen recognized by antibody from 2D11 appears to be a more broadly expressed antigen present in cultures derived from autologous and allogeneic melanoma, a blue nevus, and normal fibroblasts. Immunohistochemical reactivity against autologous and allogeneic melanomas, as well as the blue nevus, was strong, and the antibody appeared to bind a cell-membrane antigen immunohistochemically. However, reactivity against fibroblasts was weak, and the

antibody recognized a cytoplasmic antigen. The antigen was trypsin-resistant, suggesting that, unlike 1F6, 2 D l l may not recognize the peptide backbone of this antigen. Unfortunately the loss of antibody production in this culture precluded further study. The 1F6 B cell lymphoblastoid line was derived from TIL of patient Pitt Melanoma Unit, PCI-MEL 136. This line continues to be a stable source of tumor-reactive antibody after 15 months of culture. 1F6 appears to be of clonal origin, expressing only IgMX on the cell surface and producing only IgMX antibody. The antigen recognized by 1F6 appears to be an antigen of limited tumor-associated distribution. It is expressed in cells of neural-crest origin, including melanomas, an astrocytoma, a blue nevus cell line, and a congenital nevus cell line. The antibody was unreactive with 5 foreskin melanocyte cell lines, but showed reactivity against nevocytic melanocyte lines derived from a congenital nevus and a blue nevus. In tests against a panel of other cell lines derived from tissues not of neural-crest origin, the only cell line recognized by 1F6 was a renal-cell carcinoma cell line. However, against the renal-cell carcinoma line, the antibody reacted only weakly to a small percentage of cells. IF6 showed reactivity against immunoblots of the astrocytoma line and the renal-cell carcinoma line only when high quantities of protein were loaded on the gels, suggesting that the antigen is expressed in fewer cells or is only weakly expressed.



FIGURE 6 - Reactivity of 1F6 was assessed by immunoblot. Cell lysates were prepared under reducing conditions in Laemmli buffer (0.0625 M Tris. 2% SDS, 10% glycerol, 5% 2-ME. 0.002% bromophenol blue, and 0.001% phenol red). 100 kg samples were run on a 6% SDS-polyacrylamide gel and transferred to nitrocellulose paper for immunoblot with IF6 with reactivity as shown: autologous tumor lines PCI-MEL 136.26(lane l), PCI-MEL 136.15 (lane 2), and PCI-MEL 136.1 (lane 3); allogeneic melanomas PCI-MEL 105.1 (lane 4), PCI-MEL 306.2 (lane S), PCI-MEL 208.2 (lane 6), PCI-MEL 310.2 (lane 7); a blue nevus line (line 8); a fibroblast line (lane 9); an astrocytoma (lane 10); and a lung-cancer line (lane 11)

FIGURE7 - Sub-cellular fractions of autologous tumor were prepared under reducing conditions in Laemmli buffer; 50 kg protein samples were run on a 6% SDS-polyacrylamide gel and transferred to nitrocellulose paper for immunoblotting with 1F6 with reactivity as shown: nuclear fraction (lane I); membrane from the 1.2 M sucrose interface, NP-40 insoluble (lane 2) and soluble (lane 3); membrane from the 0.8 M sucrose interface, NP-40 insoluble (lane 4), and soluble (lane 5); and the cytoplasm fraction (lane 6 ) .

IF6 rccognizcs an antigen of apparent molecular weight 194 kDa expressed on the cell membrane and in the soluble cytoplasmic fraction of autologous melanoma cells. Absorption of antibody reactivity by viable cells suggests that the antigen is expressed on thc cell surface. Biochemically, the antigen appears to be a glycoprotein, as evidenced by its sensitivity to heat, trypsin, dithiothreitol, and endoglycosidase H and its absence after 8 hr of tunicamycin exposure. Enhanced antibody reactivity observed against ncuraminidasctreated cclls suggests that the antigen is partially obscured from recognition in the intact tumor cell by the presence of sialic-acid rcsidues. Murine MAbs reactive with high-molecularweight melanoma-associated antigens (gp240, p210, KD200.

and ML-05) did not block binding of 1F6 immunohistochemically, suggesting that the glycoprotein recognized by 1F6 differs from these well-described glycoprotcin species (data not shown). The absence of other stable long-term antibody-producing EBV-transformed B-cell lines may be due to outgrowth/ overgrowth of irrelevant B-cell clones in oligoclonal B-cell cultures, decrease and/or loss of Ab production from the relevant B cells, and/or change of specificity of the Ah produced. Of the 8 TIL-derived B-cell cultures producing tumor-reactive antibody studicd herc, 7 were oligoclonal. Loss of tumor reactivity of supernate produced by these cultures suggests outgrowth of contaminating B-cell clones. Subculture of these cultures at limit dilution failed to retrieve or stabilize production of tumor-reactive antibodies. Sub-cultures yielded either unreactive supernates, or supernates with antibody that was reactive both with tumor and with normal fibroblasts. In conclusion, we have demonstrated that EBV transformation of B cells provides a means of short-term B-cell culture allowing analysis of the cellular basis of the humoral immune response to melanoma. In a melanoma patient with a high titer of serum antibody reactive with autologous tumor, we have demonstrated B cells derived both from TIL and from PBL committed to production of tumor-reactive antibody. One B-cell line derived from PBL and another derived from TIL continued to yield tumor-reactive antibody long enough to allow partial analysis of antibody specificity and partial biochemical characterization of thc antigen recognized. The lymphoblastoid line 1F6 continues to produce tumor-reactive antibody after 15 months of continuous culture. Studies are being undertaken to characterize the antibody and antigen at the molecular level. ACKNOWLEDGEMENTS

We thank Ms. R. Mascari. Ms. M.L. Wastyn, Ms. C.A. Aston and Ms. N. Patuc for their assistance in preparing this manuscript.




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Analysis of two human monoclonal antibodies against melanoma.

B cells derived from peripheral-blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) from a patient with a high serum antibody titer to au...
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