A Yeast Two-Hybrid System Using Sp17 Identified Ropporin as a Novel Cancer-Testis Antigen in Hematologic Malignancies.

Cellular proteins are usually expressed with their interacting counterpart proteins. If the interacting proteins for Cancer-Testis (CT) antigens could be identified, these proteins could also be CT antigens that may be suitable targets for tumor vaccine. We have applied Sperm protein 17 (Sp17), a CT antigen identified in our laboratory, as the bait in a yeast two-hybrid system to screen a testicular cDNA library constructed in yeast strain Y187 to identify the protein interacting with Sp17. A total of 17 positive colonies were isolated, 11 encoded for the AKAP-binding protein, Ropporin. Ropporin is a spermatogenic cell-specific protein that serves as an anchoring protein for the A-kinase anchoring protein, AKAP3. Ropporin has never been reported to be expressed in tumor cells. Using a pair of sequence-specific primers in PCR, we determined the expression of Ropporin in hematologic malignancies. Ropporin mRNA was aberrantly expressed in 6/16 (37.5%) multiple myeloma (MM), 6/14 (43%) chronic lymphocytic leukemia (CLL) and 2/11 (18%) acute myeloid leukemia (AML). Ropporin is another novel CT antigen because the mRNA could not be detected in normal tissues except in testis, fetal brain and fetal liver.

The coding sequence of human Ropporin was then cloned into the pQE30 vector to generate a recombinant human Ropporin fusion protein with an N-terminal 6-His tag from E coli. Successful generation of the fusion protein was confirmed on Western blot analysis using an antibody directed at the 6-His tag and also rabbit anti-mouse Ropporin antisera that cross react with the human Ropporin.

We then used the recombinant Ropporin protein to determine the immunogenicity of Ropporin in patients with hematologic malignancies by detecting for high titer (1:1000) anti-Ropproin antibodies using sera from patients in an ELISA system. We established the basal level of positivity in 31 healthy individuals. A very low level of signal was obtained (mean OD450nm ± SD = 0.0736 ± 0.0147). Using mean ± 2SD as the cut off signal intensity, high titer anti-Ropporin antibodies were detected in 8/30 (26.7%) MM, 7/24 (29%) AML and 18/31 (58%) CLL. Signals obtained from the two sets of control wells consisting of either another E coli-derived recombinant protein or bovine serum albumin were consistently < 0.01. The specificity of the signals was confirmed in Western blot analysis. Not surprisingly, due to the high sensitivity of the ELISA system, not every ELISA-positive specimen produced a positive Western blot signal. However, all positive Western blot signals were ELISA positive specimens. All ELISA negative specimens did not produce positivity in Western blot analysis.

Finally, we carried out a correlative study on paired specimens. Fourteen paired specimens with both tumor RNA and patient sera were available. The detection of positive Ropporin antibodies predicted for the expression of Ropporin gene in the tumor cells, suggesting the translation of the Ropporin mRNA into protein in vivo and that the B-cell immunity in these patients are likely due to the tumor Ropporin.

Conclusions:

1. The yeast two-hybrid system may be used to identify protein interacting with CT antigens and these proteins may themselves also be CT antigens;

2. Ropporin is a novel CT antigen in hematologic malignancies;

3. Ropporin is immunogenic in these patients.

Further work is warranted to determine the suitability of Ropporin as a candidate antigen for tumor vaccine.