Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen

Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.     

Syngeneic antiidiotypic antisera to murine antihuman high-molecular-weight melanoma-associated antigen monoclonal antibodies

BALB/c mice were immunized with the murine antihuman high-molecular-weight melanoma-associated antigen monoclonal antibodies (MoAbs) 149.53, 225.28, 653.25, and 763.74. Antiidiotypic antibodies could be detected in bleedings obtained 3 days following the first booster, increased in titer in bleedings obtained following the second booster, and persisted at high level in bleedings obtained 38 days following the second booster. Cross-blocking studies with a panel of anti-melanoma-associated antigen, anti-HLA Class I, and anti-HLA Class II monoclonal antibodies showed that the antisera recognize private idiotypes. The latter are located within the antigen combining site, since antiidiotypic antisera specifically inhibited the binding of the corresponding immunizing anti-human high-molecular-weight melanoma-associated antigen monoclonal antibody to cultured human melanoma cells Colo 38 in a dose-dependent fashion. The spectrotype of the anti-MoAb 149.53 antiserum comprises eight major components in the range of pH 6.2 to 7.0; those of the anti-MoAb 225.28 antiserum and of the anti-MoAb 653.25 antiserum, two major components in the ranges of pH 6.4 to 6.6 and 6.5 to 6.7, respectively; and that of the anti-MoAb 763.74 antiserum, three major components in the range of pH 6.2 to 6.4.     

Binding parameters and idiotypic profile of the whole immunoglobulin and Fab' fragments of murine monoclonal antibody to distinct determinants of the human high molecular weight-melanoma associated antigen.

The nonspecific accumulation of radioactivity in bone marrow, liver, and spleen in patients with melanoma injected with radiolabeled whole IgG of anti-high molecular weight-melanoma associated antigen (HMW-MAA) monoclonal antibody (mAb) hampers the application of immunoscintigraphy to visualize melanoma lesions. This nonspecific background can be reduced by utilizing fragments which do not contain the Fc portion of anti-HMW-MAA mAb. Since the in vivo targeting to melanoma lesions of radiolabeled F(ab')2 and Fab' fragments of anti-HMW-MAA mAb is critically dependent on their binding parameters, we have analyzed the effect of fragmentation on the binding characteristics of the anti-HMW-MAA mAbs 225.28, 763.74, and TP41.2. The three mAbs recognize distinct and spatially distant determinants with a heterogeneous distribution on the pool of HMW-MAA molecules synthesized by melanoma cells. The determinant recognized by mAb TP41.2 is detectable on a markedly smaller population of HMW-MAA molecules than those recognized by mAbs 225.28 and 763.74. 125I-labeled F(ab')2 fragments of the three mAbs displayed an immunoreactive fraction similar to that of the whole IgG, while Fab' fragments displayed a lower one. Fragmentation of mAbs 225.28 and TP41.2 to F(ab')2 produced a 2- and 1.5-fold reduction in their association constants but did not cause a significant change in that of mAb 763.74. Cleavage of F(ab')2 fragments to Fab' fragments produced 2-, 40-, and 7-fold reductions in the association constants of mAbs 225.28, 763.74, and TP41.2, respectively. These changes qualitatively fit the predictions of theory for univalent and bivalent mAb binding, since mAbs 763.74 and TP41.2 appear to show bivalent binding to melanoma cells and mAb 225.28 to show univalent binding. The affinity constant of IgG, F(ab')2 and Fab' fragments of mAbs 225.28, 763.74, and TP41.2 displays an inverse relationship with the extent of their time-dependent release from the membrane of melanoma cells. Since no endocytosis of mAb could be detected, the latter results suggest that radioactivity remains bound to melanoma cells in vivo for a longer time following injection of F(ab')2 fragments than following that of Fab' fragments of each of the anti-HMW-MAA mAb tested. Radiolabeled Fab' fragments of mAbs 763.74 and TP41.2 displayed a marked reduction in their reactivity with some of the antiidiotypic mAb tested. The loss of some idiotopes is likely to be caused by changes in the conformation of the molecules associated with the fragmentation of IgG and by damage during the iodination procedure.     

Human high-molecular-weight melanoma-associated antigen mimicry by mouse antiidiotypic monoclonal antibody TK7-371.

Since the human high-molecular-weight melanoma-associated antigen (HMW-MAA) represents a useful target to implement active specific immunotherapy with mouse antiidiotypic monoclonal antibody (mAb), the present study is aimed at developing and characterizing mouse antiidiotypic mAbs which bear the mirror image of the determinant defined by the anti-HMW-MAA mAb TP61.5. To this end, a BALB/c mouse was immunized with the syngeneic mAb TP61.5. Screening of the 703 generated hybridomas showed that 13 of them secrete antiidiotypic mAbs which inhibit the binding of the immunizing mAb TP61.5 to melanoma cells by at least 75%. The dose of antiidiotypic mAb required to inhibit by 50% the binding of mAb TP61.5 to melanoma cells ranged between 4 and 200 ng. The 13 antiidiotypic mAbs recognize conformational idiotypes, since they did not react in Western blotting with the heavy and light chain of mAb TP61.5 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Furthermore, the 13 antiidiotypic mAbs did not react with 23 mAbs which recognize 13 determinants of the HMW-MAA distinct from that defined by mAb TP61.5. Analysis of the 13 antiidiotypic mAbs for their ability to elicit cellular and humoral anti-HMW-MAA immunity showed that only mAb TK7-371 elicited a delayed-type hypersensitivity reaction to HMW-MAA-bearing cells in syngeneic hosts and anti-HMW-MAA antibodies in BALB/c mice and in rabbits. Since rabbits express the HMW-MAA, the present results indicate that the antiidiotypic mAb TK7-371 can induce humoral immunity to self HMW-MAA. Therefore, the antiidiotypic mAb TK7-371 may be an efficacious immunogen to implement active specific immunotherapy in patients with melanoma.     

Analysis of the antigenic profile of uveal melanoma lesions with anti-cutaneous melanoma-associated antigen and anti-HLA monoclonal antibodies

The reactivity of 12 surgically removed uveal melanoma lesions with monoclonal antibodies (MoAb) to 14 membrane-bound and 2 cytoplasmic cutaneous melanoma-associated antigens (MAA), to the 2 subunits of HLA Class I antigens and to the gene products of the HLA-D region was compared with that of cutaneous melanoma lesions and correlated with their histiotype. The membrane-bound determinants defined by the anti-Mr 92,000 and 45,000 MAA MoAb TP39.1, anti-Mr 110,000 MAA MoAb M111, anti-Mr 118,000 MAA MoAb TP36.1, anti-Mr 115,000 MAA MoAb 345.134, anti-ICAM-1 MoAb CL203.4 and anti-Mr 31,000 MAA MoAb M2590, and the cytoplasmic determinants defined by the anti-MAA MoAb 465.12 and 2G-10 display a distribution in uveal melanoma lesions similar to that in cutaneous melanoma lesions. On the other hand, membrane-bound determinants defined by the anti-Mr 100,000 MAA MoAb 376.96, anti-9-O-acetyl-GD3 ganglioside MoAb ME311 and anti-GD2-GD3 ganglioside MoAb ME361 were not detected in the uveal melanoma lesions tested. Furthermore, the membrane-bound determinants defined by the anti-GD3 MoAb R24, anti-nerve growth factor receptor MoAb ME20.4, anti-Mr 97,000 MAA MoAb 140.240, anti-carcinoembryonic antigen MoAb B1.1 and anti-HMW-MAA 149.53, 225.28, and 763.74 have a markedly lower expression in uveal than in cutaneous melanoma lesions. Incubation of uveal melanoma lesions with the pool of the MoAb 149.53, 225.28, and 763.74 recognizing distinct and spatially distant determinants of the HMW-MAA increased the intensity of staining of six lesions and stained four lesions which were not stained by the individual monoclonal antibodies. The distribution of HLA Class I antigens in uveal melanoma lesions resembles that in cutaneous melanoma lesions, since they are expressed in all the lesions of the mixed and epithelioid type but were not detected in those of the spindle type, i.e., the counterparts of nevocellular nevi. HLA Class II antigens are expressed with a lower frequency in uveal than in cutaneous melanoma lesions, since they were detected only in 2 of the 12 lesions. One of them is of the mixed type and the other one of the epithelioid type. Besides HLA antigens the determinants defined by the anti-carcinoembryonic MoAb B1.1, anti-ICAM-1 MoAb CL203.4, and anti-GD3 MoAb R24 displayed a differential distribution in the different histiotypes of uveal melanoma, since they are preferentially expressed in lesions of the mixed and epithelioid type.     

Human high molecular weight-melanoma associated antigen mimicry by an anti-idiotypic antibody: characterization of the immunogenicity and the immune response to the mouse monoclonal antibody IMel-1

The mouse anti-idiotype (anti-id) monoclonal antibody (mAb) IMel-1 recognizes an idiotope in the antigen combining site of the immunizing anti-human high molecular weight melanoma-associated antigen (HMW-MAA) mAb 225.28. The mAb IMel-1 is able to induce an immune response against self cross-reacting HMW-MAA in rabbits that express HMW-MAA in normal tissues. Most of the rabbit anti-anti-id antibodies recognize a spatially distant determinant(s) from that defined by anti-HMW-MAA mAb 225.28. The immunogenicity of mAb IMel-1 is enhanced by its administration with the muramyl dipeptide-derived adjuvant. Anti-HMW-MAA antibodies were not detected in sera from rabbits immunized with HMW-MAA bearing cultured human melanoma cells. The differential immunogenicity of mAb IMel-1 and cell membrane bound HMW-MAA may account for the ability of anti-id mAb to induce anti-HMW-MAA immunity in patients who have not mounted such a response to HMW-MAA present in their lesions. Rabbit anti-HMW-MAA antibodies induced by anti-id mAb IMel-1 inhibited interactions of melanoma cells with elements of extracellular matrix. This may represent an additional mechanism by which anti-HMW-MAA immunity may affect the biology of melanoma cells in patients with melanoma immunized with anti-id mAb IMel-1.     

Biosynthesis and intracellular processing of four human melanoma associated antigens

By analyzing human melanoma cells with monoclonal antibodies (MoAb) we have identified four melanoma associated antigens with distinct tissue distribution and structural properties. They include the high molecular weight melanoma associated antigen (MAA), the Mr 120,000 MAA, the Mr 100,000 MAA, and the cytoplasmic MAA defined by MoAb 465.12. Previous studies have shown that these antigens may be useful markers to characterize the biology of melanoma cells and to develop immunodiagnostic and immunotherapeutic approaches to melanoma. In the present investigation pulse-chase intrinsic labeling studies combined with the biosynthetic inhibitor tunicamycin and with enzymatic degradations with endoglycosidase H have shown that the determinants recognized by the MoAb utilized are expressed on the core protein of the molecules. Furthermore the four MAAs are highly glycosylated with N-linked carbohydrate chains and are synthesized as precursors which bear endoglycosidase H-sensitive chains. The high molecular weight (Mr 500,000/280,000) MAA displays a major precursor with a molecular weight of 240,000 which expresses the epitopes recognized by the anti-high molecular weight MAA MoAbs 149.53, 225.28S, and 763.74T. This precursor has an apparent molecular weight of 220,000 when cells are grown in the presence of tunicamycin. The Mr 89,000 and Mr 36,000 subunits of the Mr 125,000 MAA have biosynthetic precursors with molecular weights of 76,000 and 25,000. Endoglycosidase H digestion of the Mr 76,000 precursor produces a Mr 46,000 polypeptide. The Mr 100,000 MAA has a Mr 87,000 biosynthetic precursor. The cytoplasmic MAA (Mr 100,000, 75,000, 72,000, and 25,000) has a single precursor with a molecular weight of 75,000 which appears as a Mr 60,000 polypeptide after endoglycosidase H digestion. Characterization of the biosynthesis of the four MAAs will contribute to the development of approaches to modulate their expression and shedding by melanoma cells.     

Suppression of human melanoma growth in nude mice injected with anti high-molecular-weight melanoma-associated antigen monoclonal antibody 225.28S conjugated to purothionin

The cytotoxic agent purothionin purified from barley was covalently conjugated to the anti-human high-molecular-weight melanoma-associated antigen (HMW-MAA) monoclonal antibody (MoAb) 225.28S by utilizing water-soluble carbodiimide. Injection of the conjugate (0.1 mg/injection) significantly increased the life span of nude mice injected with ascitic-form human melanoma cells (Colo 38), and caused 40% inhibition on day 25 of the growth of solid-form human melanoma cells in nude mice. The effect is specific and is markedly influenced by the site of growth of tumors and by the schedule of administration of the conjugate.     

Phenotypic diversity of murine B16 melanoma detected by anti-B16 monoclonal antibodies

A panel of monoclonal antibodies (MoAbs), produced against the murine B16 melanoma, has been used to characterize its phenotypic diversity. Six MoAbs that did not bind to primary cultures of kidney, brain or liver, spleen cells, thymocytes, 3T3 fibroblasts, melanin, or transferrin receptors were selected for further evaluation. Five MoAbs, which recognized surface antigens expressed on parental B16 cells and the B16-F1, B16-F10, B16-F10 FLR, and B16-BL6 sublines, did not appear to cross-react with each other, suggesting that they identified antigenically distinct epitopes. Four MoAbs, designated as IB16-2, IB16-4, IB16-8, and IB16-10, recognized B16 surface antigens that were variably expressed over short periods of time. This variable expression was independent of the cell cycle and was characteristic of four B16 sublines. Two of these MoAbs, both of the IgG2b isotype, fixed rabbit and guinea pig complement and were cytolytic in the presence of rabbit complement. One MoAb, designated IB16-6, recognized a surface antigen consistently expressed on greater than 90% of cells of both the parental tumor and the sublines. This MoAb bound to several murine and one human melanoma cell line, but not to other histopathological types of tumors or normal tissues. The cellular antigen that this antibody recognized was not detected in the cytoplasm, did not modulate in the presence of IB16-6, and was sensitive to trypsin, pronase, alcohols, acetone, and detergents, thereby suggesting that it was a protein. Our data are among the first that directly show the extent of phenotypic diversity of the B16 melanoma and sublines that have been derived from it.     

Imaging with 131I-labeled monoclonal antibodies to a high-molecular-weight melanoma-associated antigen in patients with melanoma: efficacy of whole immunoglobulin and its F(ab')2 fragments

In vitro experiments selected optimal conditions to radiolabel with 131I the whole immunoglobulin and F(ab')2 fragments of the monoclonal antibody (MoAb) 225.28S to a high-molecular-weight melanoma-associated antigen (HMW-MAA). Injection of the radiolabeled whole immunoglobulin and F(ab')2 fragments of the MoAb 225.28S into eight patients with melanoma resulted in the accumulation of radioactivity in 10 of 18 metastases. This localization is specific because of the close relationship between detection of HMW-MAA in lesions by immunohistochemical techniques and outcome of immunoscintigraphy and because of the different distribution in tumors and adjacent tissues of radiolabeled F(ab')2 fragments of MoAb 225.28S compared with 99mTc-pertechnetate and with radiolabeled F(ab')2 fragments of MoAb 4C4 to hepatitis B surface antigen. F(ab')2 fragments are superior to whole immunoglobulins to perform immunoscintigraphy, since they markedly reduce the background in bone marrow, liver, and spleen. The sensitivity of the procedure allows the detection of lesions with a diameter of at least 1.5 cm and is influenced by the level of the HMW-MAA in lesions and by their anatomical site.     

Development of MoAb HMSA-2 for melanosomes of human melanoma and its application to immunohistopathologic diagnosis of neoplastic melanocytes

To characterize the biologic and molecular nature of melanosomal proteins, we recently developed two mouse monoclonal antibodies (MoAbs), human melanosome-associated antigen (HMSA)-1 and HMSA-2, for melanosomes isolated from human malignant melanoma. This study describes the methods of development for MoAb HMSA-2 and the specificity and distribution of antigen(s) reactive with it in normal and pathological tissues. Characteristically, MoAb HMSA-2 was found to detect neoplastic melanocytes on formalin-fixed and paraffin-processed specimens. The reaction patterns for benign and malignant melanocytic tumors were different, i.e., homogeneous reactivity in the dermal cells of common melanocytic nevi but heterogeneous reactivity in the same type of cells in malignant melanoma. MoAb HMSA-2 reacted positively with the epidermal melanocytes in the lesions of dysplastic melanocytic nevi, but not with those of common melanocytic nevi. Reaction products with MoAb HMSA-2 were localized in the cytoplasm. MoAb HMSA-2 did not react with normal melanocytes even on frozen sections. Benign and malignant epithelial and soft tissue tumors of the skin were usually negatively stained with MoAb HMSA-2. Findings indicate that MoAb HMSA-2 is a unique MoAb which identifies neoplastic melanocytes and which can be useful in differentiating each type of melanocytic tumor.     

Inhibition of tumor cell growth in vitro by murine monoclonal antibodies that recognize a proliferation-associated cell surface antigen system in rats and humans

The mouse monoclonal antibody (MoAb) B3 raised against a rat bladder cancer cell line and the MoAbs HBJ127 and HBJ98 raised against a human bladder cancer cell line recognize homologous antigens predominantly present on proliferating cells of the corresponding species. Examination of MoAb-defined antigen and epitopes revealed that both HBJ127 and HBJ98 MoAbs defined a human cell surface glycoprotein complex having an apparent molecular weight of 125,000-130,000 which was composed of a heavy subunit of a glycoprotein nature (Mr 90,000-95,000) and a disulfide-linked light subunit of protein nature (Mr 30,000-35,000), but the HBJ127 and HBJ98 MoAbs recognized a protein epitope and a sugar epitope on the heavy subunit, respectively. Likewise, the B3 MoAb recognized a protein epitope on the heavy subunit of a rat cellular glycoprotein complex of similar composition to the HBJ127/HBJ98-defined human antigen. Addition of the B3 MoAb to rat and the HBJ127 or HBJ98 MoAb to human tumor cells inhibited the nucleic acid synthesis or the proliferation of the tumor cells in vitro in a dose-dependent manner. The target tumor cells exposed to MoAb could regrow when they were freed from the antibody, indicating that the effect of these MoAbs on the tumor cells is cytostatic and reversible. These MoAbs did not cause down-regulation of the cell surface antigen and did not arrest the cell cycle in a certain phase. These observations indicate that the Mr 125,000 glycoprotein cell surface component detected in both rat and human systems may play a requisite role for cell proliferation and that our MoAbs could inhibit the function by binding to the functionally proximal region of the component.     

Syngeneic monoclonal antibodies to B16 melanoma viral antigens

Four stable IgM monoclonal antibody-producing hybridomas were generated by fusing mouse myeloma cells with spleen lymphocytes from C57BL/6 mice hyperimmunized against the syngeneic B16 melanoma. All four monoclonal antibodies (R31/15, R37/4, R37/6, and R37/7), in common with polyclonal antiserum from immunized mice, recognized antigens on the same complex of related cell surface molecules specified by endogenous AKR-type murine leukemia virus, designated the B16-gp/70/80/85 antigen complex. Reactivity with this antigen complex was demonstrated by radioimmunoprecipitation. Specificity for viral Mr 70,000 glycoprotein-related antigens was indicated by absorption of antibody activity by endogenous AKR virus and by inhibition of antibody binding to B16 melanoma cells by monospecific antiserum to murine leukemia virus Mr 70,000 glycoprotein. Neither polyclonal nor monoclonal antibodies recognized antigens on fish, guinea pig, swine, or human melanoma cell lines. Polyclonal antiserum reacted with several other mouse melanomas and with certain mouse lymphoma lines induced by, or harboring, endogenous murine leukemia viruses, but the monoclonal antibodies were unreactive except for recognition of antigens on Harding-Passey mouse melanoma cells by antibody R37/4 and on RL male 1 mouse lymphoma cells by antibody R37/7. Only monoclonal R37/7 was cytotoxic for cultured B16 melanoma cells in an antibody- and complement-dependent assay with guinea pig complement, although all antibodies were cytotoxic with rabbit complement. In reflecting the predominant humoral immune response to the B16 melanoma detected in syngeneic mice during tumor growth, these monoclonal antibodies will permit experimental amplification of that response to help determine how that immunity influences tumor growth and metastatic dissemination.     

Cross-reactivity of mimotopes with a monoclonal antibody against the high molecular weight melanoma-associated antigen (HMW-MAA) does not predict cross-reactive immunogenicity

The high molecular weight melanoma-associated antigen (HMW-MAA) is highly expressed in advanced primary and metastatic melanoma. An epitope of the core protein of HMW-MAA is recognized by the murine monoclonal antibody (mAb) 225.28S. In this study, we aimed to characterize peptides that antigenically mimicked this epitope and to determine their efficacy as components of an HMW-MAA-based anti-melanoma vaccine. Therefore, we screened a constrained 10 mer phage display peptide library against mAb 225.28S. Selected phage-displayed peptides were then tested for their specificity for the antibody's antigen-binding site. DNA sequences coding for specific peptide ligands were determined. Binding of mAb 225.28S to HMW-MAA was inhibited in a dose-dependent manner by phage-displayed peptides from 51 to 83% and by synthetic peptides from 38 to 87%. Subsequently, the immunogenicity of the five mimotopes with the highest inhibition capacity was examined in rabbits. Immunizations with synthetic mimotopes conjugated to tetanus toxoid resulted in peptide-specific antibodies, but none of the highly antigenic mimotopes induced HMW-MAA cross-reactive antibodies. This report describes an example of disparity between antigenicity and cross-reactive immunogenicity, complicating the selection of potential vaccine candidates.     

Monoclonal antibody to human carcinoma-associated protein complex: quantitation in normal and tumor tissue

We have identified and quantitated a tumor protein complex, TSP-180, on murine carcinomas with two monoclonal antibodies (MoAbs) (Cancer Res., 46: 707-712, 1986). One of the two MoAbs, 135-13C, recognizes a TSP-180-like protein complex on several human carcinomas in culture. MoAb 135-13C has been used to purify the human TSP-180 complex from A431 cells and the purified material used to immunize F344 rats to produce another MoAb, 439-9B, to the human TSP-180 complex. This MoAb does not precipitate the murine TSP-180 or bind to murine cells. Both MoAb 135-13C and 439-9B precipitated the same proteins from A431 cells but did not compete with each other for binding sites, indicating that they recognize different epitopes on the same protein. The two MoAbs have been used in a two-site assay to quantitate TSP-180 proteins on human cells and tissues. Carcinoma cell lines A431, SW948, and A549 all give high values (46 to 443 ng/mg of protein) while murine tumors, a human melanoma, and human fibroblasts are negative (less than 10 ng/mg of protein). Most tissues from autopsy of 2 normal individuals are negative for human TSP-180 at the levels tested (less than 10 ng/mg of protein). Some organs have intermediate range expression: spleen, 5 to 111 ng/ml of protein; colon, 24 to 111; and small intestine, 39 to 99. One primary colon and one larynx tumor were positive (144 to 372 ng/mg of protein) while 5 breast carcinomas, a stomach tumor, a metastatic melanoma, and a kidney tumor were negative. These data indicate that human TSP-180 may be preferentially expressed in certain malignant carcinomas of diverse origin. The potential for TSP-180 as a tumor marker requires further study.     

Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy

Two 111indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.     

Fine specificity of high molecular weight-melanoma-associated antigen-specific cytotoxic T lymphocytes elicited by anti-idiotypic monoclonal antibodies in patients with melanoma

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)(+) melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11-30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11-30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11-30 VH chain, VH (3-11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76-84). Lymphocytes from HLA-A2(+)-immunized patients proliferated to VH (3-11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76-84). Significant increase in IFN-gamma production but not in interleukin 10 production in response to VH (3-11) and to variant HMW-MAA peptide (76-84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2(+) patients with the two peptides induced CTL, which lysed HMW-MAA(+)/HLA-A2(+) A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.     

Induction of antigen-specific immune response with use of anti-idiotypic monoclonal antibodies to anti-carcinoembryonic antigen antibodies

Anti-idiotypic monoclonal antibodies (MoAbs) were prepared in a syngeneic system against anti-carcinoembryonic antigen (CEA) MoAb 5B3 (IgG1), which reacted with a carbohydrate moiety on CEA, and MoAb MA208 (IgG1), which reacted with a peptide on CEA. Anti-idiotypic MoAb T3-503 and T4-202 recognized the private idiotype of MoAb 5B3; anti-idiotypic MoAb M7-049 and M7-625 did so for MoAb MA208. Idiotype mapping showed that MoAb 5B3 has at least two distinct idiotopes at its combining site and MoAb MA208 also has two. Four different anti-idiotypic MoAbs (Ab2) could induce anti-anti-idiotypic antibodies (Ab3) specific to their respective immunizing anti-idiotypic MoAbs. Anti-anti-idiotypic MoAb M7-625 antiserum (at least a part of the antibodies) have the same reactivity as MoAb MA208, since the serum competed with the binding of MoAb MA208 against CEA and contained the antibody population reactive with purified CEA in immunoblotting assay. These results suggest that anti-idiotypic MoAb M7-625 bears the internal image of the antigen (CEA) and induces the anti-anti-idiotypic antibodies specific to CEA. Therefore, an anti-idiotypic antibody bearing the internal image of a tumor associated antigen might be used as a possible tool for vaccination or immunotherapy against malignant tumors as an antigen specific immunomodulator.     

Monoclonal antibodies against human oral squamous cell carcinoma reacting with keratin proteins

Two mouse monoclonal antibodies (MoAbs), B10 and 1H5, were generated by fusing mouse myeloma NS-1 cells with spleen cells from a BALB/c mouse immunized with Ueda-1 cells derived from human squamous cell carcinoma (SCC) of the floor of the mouth. Immunohistochemical analysis revealed that these MoAbs recognize the filamentous components of cytoplasm which were protein in nature. While the pattern of antigen distribution in various cell lines was not cell-type specific, reactivity of these antibodies with tissue sections was informative. MoAb 1H5 was preferentially reactive with well-differentiated squamous cell carcinoma, however, reaction with adenocarcinoma was observed infrequently. This antibody also preferentially reacted with the spinous layer of normal stratified squamous epithelium. MoAb B10, however, was reactive with nonepithelial tissues as well as with epithelial ones, and its level of binding bore no relationship to the grade of histologic malignancy. SDS-PAGE and Western blotting analysis, using cytokeratin extracts of Ueda-1 cells and human epidermis, demonstrated that MoAb B10 reacted with a wide range of keratin proteins of 65-67K, 58K, 56.5K, 56K, 52K, 50K, 48K, 45K, 40K, 38K, 36K, and 34K molecular weight (MW), while MoAb 1H5 reacted with keratin proteins of 65-67K, 58K, 56.5K, 56K, 52K, 48K, and 34K MW. These results suggest that MoAb 1H5 may recognize keratin subfamilies related to squamous differentiation, whereas MoAb B10 recognizes a wide range of keratin proteins, and may even react with other kinds of intermediate filament proteins (IFPs).     

Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody

We have isolated a monoclonal antibody, designated HMB-50, that is highly specific for melanomas and melanocyte derived lesions. The antibody recognizes melanomas, neonatal melanocytes, and junctional nevi but does not react with adult melanocytes, dermal nevi, or a variety of non-melanocyte derived neoplasms. In tissue culture, the antibody reacts with five of seven human melanoma lines and neonatal foreskin melanocytes but fails to recognize fibroblasts and a number of different carcinomas. HMB-50 identifies a Mr 95,000 glycoprotein that is released into the growth medium by melanoma cells and neonatal melanocytes in vitro. This molecule is unrelated to antigens recognized by a variety of antimelanoma monoclonal antibodies isolated in other laboratories. The Mr 95,000 glycoprotein has been purified by antibody affinity chromatography and a polyclonal rabbit antiserum raised that exhibits identical specificity to the monoclonal antibody. The Mr 95,000 glycoprotein is rapidly released by melanoma cells (within 60 min) and one line produces relatively large quantities of the molecule (1 microgram/10(6) cells/24 h). The molecule in normal melanocytes differs slightly in electrophoretic mobility compared to its counterpart in melanomas and this difference appears to result from posttranslational modification.