Human P97 Research Articles

Cyclophosphamide potentiates the antitumor activity of v-p97NY.

Previous work has demonstrated that a recombinant live vaccinia virus-based tumor vaccine, v-p97NY, induces an immune response in mice which can lead to the rejection of transfected lines of mouse melanoma cells expressing the human melanoma antigen p97 (S.-L. Hu et al., J. Virol. 62, 176, 1988; C. D. Estin et al., Proc. Natl. Acad. Sci. USA 85, 1052, 1988). We now show that the ability of v-p97NY to induce delayed-type hypersensitivity to p97 improved if the vaccinated mice were given cyclophosphamide (Cy) on the day of vaccination. Likewise, treatment of vaccinated mice with Cy increased the antitumor activity of vaccination so that tumor colony formation in the lungs was inhibited even when v-p9NY plus Cy was not given until 7 days after intravenous injection of tumor cells.

Transfected mouse melanoma lines that express various levels of human melanoma-associated antigen p97.

The gene for the human melanoma-associated antigen p97 has been introduced by cDNA transfection into cells from clone M2 of the K1735 mouse melanoma, which metastasizes to the lung when injected iv into syngeneic C3H/HeN mice. Tumor clones were established from the transfected cells and found to differ in the level of p97 expression. Their outgrowth in immunocompetent syngeneic mice was shown to inversely correlate with p97 antigen expression, and lines that express higher p97 levels elicited a stronger delayed-type hypersensitivity response when injected into the footpads of mice immune to p97. Five clones which expressed very high levels of p97 failed to grow in immunocompetent C3H/HeN mice while they formed tumors in nude (nu/nu) mice. The highest expressing clone, 2A, grew slightly faster than any of the other clones when cultured in vitro. Since several of the transfected clones were found to express a stable level of p97 and have consistent in vivo growth behavior, they provide a useful model for various forms of antigen-specific active and passive immunotherapy with the same agents as those intended for human application.

Recombinant vaccinia virus vaccine against the human melanoma antigen p97 for use in immunotherapy.

We have constructed a recombinant vaccinia virus, v-p97NY, which expresses the human melanoma-associated glycoprotein p97. Immunization with v-p97NY could induce humoral and cell-mediated immunity to p97, including delayed-type hypersensitivity, in mice and in two of two monkeys (Macaca fascicularis). The fact that an immune response was induced also in monkeys is important because normal cells from monkeys, but not from mice, express a low level of cross-reactive p97. Mice immunized with v-p97NY rejected transplants of syngeneic mouse melanoma expressing p97. A rejection response could be detected also when immunization was started 2 days after tumor transplantation, irrespective of whether the transplanted cells grew subcutaneously or as lung metastases. Evidence was obtained that melanoma cells lacking p97 may be killed as "bystanders" at the site of an immune response to melanoma cells expressing p97.

Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.

Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence.

p97 is a cell-surface glycoprotein that is present in most human melanomas but only in trace amounts in normal adult tissues. To determine the structure of this tumor-associated antigen and to identify its functional domains, we have purified and cloned p97 mRNA and determined its nucleotide sequence. The mRNA encodes a 738-residue precursor, which contains the previously determined N-terminal amino acid sequence of p97. After removal of a 19-residue signal peptide, the mature p97 molecule comprises extracellular domains of 342 and 352 residues and a C-terminal 25-residue stretch of predominantly uncharged and hydrophobic amino acids, which we believe acts as a membrane anchor. Each extracellular domain contains 14 cysteine residues, which form seven intradomain disulfide bridges, and one or two potential N-glycosylation sites. Protease digestion studies show that the three major antigenic determinants of p97 are present on the N-terminal domain. The domains are strikingly homologous to each other (46% amino acid sequence homology) and to the corresponding domains of human serum transferrin (39% homology). Conservation of disulfide bridges and of amino acids thought to compose the iron binding pockets suggests that p97 is also related to transferrin in tertiary structure and function. We propose that p97 be renamed melanotransferrin to denote its original identification in melanoma cells and its evolutionary relationship to serotransferrin and lactotransferrin, the other members of the transferrin superfamily.

Human melanoma-associated antigens: Analysis of antigenic heterogeneity by molecular, serologic and flow-cytometric approaches

The relationship of antigenic heterogeneity to the epitope recognized by an antibody was examined with monoclonal antibodies to human melanoma-associated antigens. Expression of the human melanoma-associated antigens, 250-Kd glycoprotein/proteoglycan and p97, was examined quantitatively by flow cytometry on fresh cell suspensions of human melanoma. Percent positive cells and mean fluorescence intensity were consistently higher with antibody 9.2.27 to the 250-Kd glycoprotein/proteoglycan than with antibody to p97. In addition, assessment of percent positive cells in multiple skin lesions biopsied from individual patients indicated that in 26 of 30 lesions, greater than 90% of the cells stained positively with 9.2.27. This relative lack of antigenic heterogeneity with antibody 9.2.27 contrasted with previous reports which showed considerable antigenic heterogeneity with other antibodies to the 250-Kd glycoprotein/proteoglycan. The explanation for this distinction was sought by quantitative flow cytometric and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. Comparison by flow cytometry and immunoperoxidase of three antibodies, which recognized distinct epitopes of the 250-Kd glycoprotein/proteoglycan, indicated that 9.2.27 reacted more intensely with cultured cells and tissue sections than other antibodies to the same antigen. Examination by SDS-PAGE indicated that 9.2.27 could immunoprecipitate a larger proportion of 250-Kd glycorpotein molecules than other antibodies. In addition, immunodepletion experiments in gels indicated that the 9.2.27 determinant was present on a higher proportion of 250-Kd glycoprotein molecules than PG-2 antibody to a separate determinant. It is likely that 9.2.27 antibody displays less antigenic heterogeneity because its epitope is represented on a higher proportion of the antigen molecules. Thus, not only the nature of the antigen but also the epitope recognized by an antibody influences the degree of antigenic heterogeneity.

Assignment of the gene for human melanoma-associated antigen p97 to chromosome 3.

p97 is a 97,000 molecular weight cell-surface glycoprotein, which is present in human melanomas but in only trace amounts in normal adult tissues. Amino acid sequence and iron binding studies have shown that p97 is structurally and functionally related to transferrin. Reports that the genes for the transferrin receptor (TR) and possibly transferrin are located on chromosome 3 prompted us to investigate the chromosomal localization of the p97 gene. Our strategy was to characterize interspecies somatic cell hybrids derived from human fibroblasts or lymphocytes for expression of p97 and presence of human chromosomes. Although fibroblasts and lymphocytes express only small amounts of p97, we were able to type the hybrids for p97 by using monoclonal antibodies in highly sensitive and specific immunoassays. Of 14 hybrids, 6 contained chromosome 3 and expressed p97, and 8 were negative for both. We conclude that the p97 gene, like the transferrin and TR genes, is located on chromosome 3.

Human melanoma cells can be killed in vitro by an immunotoxin specific for melanoma-associated antigen p97.

Conjugates (immunotoxins) comprising ricin A-chain and monoclonal antibody 96.5, which is specific for human melanoma-associated antigen p97, inhibited protein synthesis and colony formation of cultured human melanoma cells that expressed more than 80,000 molecules of p97 per cell. Cells expressing fewer than 5,000 molecules of p97 were not killed. The presence of 10 mM ammonium chloride significantly increased the efficiency of the immunotoxin, tumor cells expressing high levels of p97 being killed at immunotoxin concentrations as low as 10(-10) M.

Detection of a human melanoma-associated antigen, p97, in histological sections of primary human melanomas.

The unlabelled antibody technique of Sternberger was used to study the localization in histological sections of human melanoma-associated antigen p97, which is defined by a monoclonal antibody. The antigen was detected in 8 of 10 primary skin melanomas, in 6 of 7 metastatic melanomas and in 2 of 2 compound nevi. It was localized at the cell surface, the cytoplasm and the nucleus always being negative. The antigen was not seen in cells from 3 basal cell carcinomas, 1 squamous cell carcinoma, 1 leiomyosarcoma, or in samples of normal skin (including keratinocytes, connective tissue consisting of collagenous and elastic fibers, fibroblasts, sebaceous glands, blood vessels, smooth muscles, or inflammatory cells such as granulocytes, lymphocytes and macrophages), kidney or lung. There was, however, staining of some cells in the secretory segment of eccrine sweat glands from 2 patients, possibly corresponding to myoepithelial cells. Antigen expression was somewhat variable between cells from different melanomas as well as between individual cells from the same melanoma. The possible diagnostic value of this procedure for identification and classification of melanomas is discussed.

Human melanoma-associated antigen p97 is structurally and functionally related to transferrin.

p97 is a 97,000-molecular weight (MW) cell-surface glyco-protein, which is present in most human melanomas but in only trace amounts in normal tissues1–4. We describe here the purification of p97 by affinity chromatography with monoclonal antibody, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and determination of the N-terminal amino acid sequence using a new, highly sensitive protein sequencer5. The sequence was found to be homologous to the N-terminal sequences of transferrin and lactotransferrin. This structural homology was confirmed by the observation that antiserum to denatured p97 cross-reacted with denatured transferrin and lactotransferrin. We have also demonstrated that p97 is functionally related to transferrin and lactotransferrin in that it binds iron. This is one of the first reports of the amino acid sequence of a human tumour-associated cell-surface antigen and one of the few cases in which insight has been obtained into its function.

Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies.

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.