Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library

Abstract

Cancer/testis (CT) antigens-immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types-are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5' end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Figure 1

RT-PCR analysis of CT antigen expression in the established melanoma cell line SK-MEL-37. SK-MEL-37 showed expression of all CT products tested, i.e., NY-ESO-1, MAGE1, MAGE-2, MAGE-3, MAGE-4, BAGE, SSX1, SSX2, SSX4, SSX5, and SCP1. The minor band of lower molecular mass in SSX4 represents an alternate-spliced variant (18).

Figure 2

Predicted amino acid sequence of CT7, illustrating the repetitive structure encoded by the 5′ sequences of this gene. The sequence has a number of repeating elements, most of them containing a (P)QS(P)LQ(I) core sequence. The most highly conserved repeating element consisted of a 35-aa unit that was repeated 10 times in tandem (amino acid positions 125–475). The carboxyl-end sequence of 208 aa (908–1115), which is homologous to MAGE-10 gene, is underlined.

Figure 3

RT-PCR analysis of CT7 expression in normal tissues. High-level expression is seen only in testis. Trace amounts of PCR products were detected in kidney, liver, placenta, and fetal brain. Two additional bands of lower molecular mass also were seen in fetal brain; by sequencing, these products were proven to be nonspecific amplification products.

Figure 4

Southern blot analysis of CT7 gene. Genomic DNA extracted from normal tissues of two individuals were digested with EcoRI and HindIII and analyzed with a CT7 probe derived from the MAGE-unrelated 5′ sequences. Two bands of similar intensity were seen in HindIII digests, whereas EcoRI digests showed one strong band and three weaker bands, indicating that CT7 does not belong to a multigene family.