A survey of the humoral immune response of cancer patients to a panel of human tumor antigens

Abstract

Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1(+) antibody had NY-ESO-1(+) tumors, and no patients with NY-ESO-1(-) tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20-40% and only patients with NY-ESO-1(+) tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1(+) tumors develop an antibody response to NY-ESO-1.

Figure 2

Representative results of ELISA reactivity with sera from melanoma patients NW29, NW38, and NW33, against a panel of seven recombinant tumor antigens.

Figure 1

Western blot analysis of mouse mAbs against recombinant tumor antigens. NY-ESO-1 (full length), SSX2, MAGE-3, and carbonic anhydrase (lanes a, b, c, and d) were purified and reacted with mAbs against NY-ESO-1 (mAb E978), SSX2 (mAb HM498), and MAGE-3 (mAb M3-6), respectively. Arrowheads, the main reactive protein species in each lane, migrating at the expected mol wt of these proteins (see Table 1).

Figure 3

RT-PCR analysis of NY-ESO-1 expression in tumor specimens and Western blot analysis for anti–NY-ESO-1 antibodies in patient sera. Of the five cases illustrated, three (lanes a, b, and c, NW178, NW33, and NW38, respectively) were NY-ESO-1 mRNA positive, showing the expected 355-bp RT-PCR product (top; m, molecular standard). Of these three cases, two were anti–NY-ESO-1 antibody positive, showing 22-kD NY-ESO-1 recombinant protein on Western blot (bottom, lanes a and c, 1:1000 serum dilution), whereas one (lane b) was negative. Positive control on Western blot was provided by using anti–NY-ESO-1 mouse mAb (lane Co). Two cases (lanes d and e, NW309 and NW145) were negative for both NY-ESO-1 mRNA and anti–NY-ESO-1 antibody. ELISA and Western blotting gave identical results.

Figure 3

RT-PCR analysis of NY-ESO-1 expression in tumor specimens and Western blot analysis for anti–NY-ESO-1 antibodies in patient sera. Of the five cases illustrated, three (lanes a, b, and c, NW178, NW33, and NW38, respectively) were NY-ESO-1 mRNA positive, showing the expected 355-bp RT-PCR product (top; m, molecular standard). Of these three cases, two were anti–NY-ESO-1 antibody positive, showing 22-kD NY-ESO-1 recombinant protein on Western blot (bottom, lanes a and c, 1:1000 serum dilution), whereas one (lane b) was negative. Positive control on Western blot was provided by using anti–NY-ESO-1 mouse mAb (lane Co). Two cases (lanes d and e, NW309 and NW145) were negative for both NY-ESO-1 mRNA and anti–NY-ESO-1 antibody. ELISA and Western blotting gave identical results.