Successful Isolation of Novel Cancer-Testis Antigens Suitable for Immunotherapy of Hematologic Malignancies Using a Yeast Two-Hybrid System in a Testicular cDNA Library.
Authors: Meklat Farouk , Li Wei , Wang Zhiqing , Zhang Yana , Zhang Jian , Lim Seah H. 
Published: October 2019

Abstract
Most intracellular proteins are expressed with their interacting ligands. If a protein shows restricted normal tissue expression, its interacting ligand will likely also follow a restricted normal tissue expression pattern. We hypothesized that protein molecules interacting with CT antigens may also be testicular restricted and potential CT antigens. Identification of these proteins provides the opportunity for their application in polyvalent tumor vaccines to overcome the problems associated with antigen heterogeneity within a tumor specimen. We have applied two known CT antigens, Sperm protein 17 (Sp17) and SEMG1, as baits in yeast two-hybrid systems of a testicular cDNA library to identify the protein interacting with these two antigens and determine whether the interacting protein are also CT antigens. To do so, we first isolated and amplified cDNA encoding Sp17 and SEMG1. Following successful amplification and sequence confirmation, the cDNAs were sub-cloned into pGBKT7 and transformed into yeast strain AH109 and selected on SD/-Trp plates. Mating was performed between AH109-pGBKT7-Sp17 or pGBKT7-SEMG 1 and pre-transformed human testis cDNA library in yeast strain Y187. Following mating, the culture was first selected on SD/-His/-Leu/-Trp plates and then on SD/-Ade/-His/-Leu/-Trp/X-a -Gal plates. A total of 17 positive clones were isolated using Sp17 and 24 positive clones using SEMG 1 as the bait. Following confirmation of interaction, the colonies were expanded and the the plasmids subjected to sequence identification by nucleotide analysis. All 17 clones isolated using Sp17 encoded Ropporin 1 and all 24 clones isolated using SEMG 1 encoded Protamine 1. Using RT-PCR on total RNA derived from a panel of normal tissues, we demonstrated the very restriction normal tissue expression of Protamine 1 and Ropporin 1, being present only in normal testis, indicating that they are also testicular-specific genes. Analysis of a panel of fresh tumor cells, we showed the aberrant expression of both Protamine 1 and Ropporin 1 in a proportion of hematologic malignancies, including acute myeloid leukemia, multiple myeloma and chronic lymphocytic leukemia, supporting the notion that Ropporin 1 and Protamine 1 are both novel CT antigens in hematologic malignancies. Furthermore, these antigens were also able to elicit high titer B-cell responses in vivo in these patients, suggesting their immunogenicity in the autologous host, even in cancer-bearing patients. Interestingly, the expression of one partner CT antigen within an individual tumor specimen does not necessary predict for the co-expression of the interacting CT antigen. In conclusion, we have described a novel approach to the identification of CT antigens that could be used for immune targeting. This approach could be applied using other known CT antigens to identify other tumor antigens. The lack of a good correlation between the expressions of the partner protein with the interacting protein suggests two important points. First, the aberrant expression of these interacting pair of molecules is not a result of coordinated intracellular regulatory mechanisms but likely due to random processes. Second, if the function of one protein is dependent on the presence of its ligand, then these individual molecules expressed within the tumor cells are unlikely to be of any functional significance in the tumor cells from most patients.