Analysis and Identification of Serological Tumor Antigens in Patients Treated with the GM-CSF-Gene Transduced-Autologous Tumor Vaccines (GVAX®) for Renal Cell Carcinoma.
Authors: Nakazaki Yukoh , Hashiguchi Takao , Takano Fuyumi , Kurita Ryo , Clift Shirley , Ando Dale G. , Asano Shigetaka , Tani Kenzaburo 
Published: October 2019

Abstract
Vaccination of GM-CSF-transduced tumor cells (GVAX®) has been demonstrated to induce cellular and humoral immune response to tumors in animal studies as well as human clinical trials. We have recently done clinical trials of GVAX® using autologous renal cell cancer (RCC) cells. Four patients with advanced RCC, who were entered to our clinical gene therapy trial, received multiple GVAX® injections every two weeks. Two out of four patients, Cases 2 and 4, are long term survivors living more than 62 and 42 months, respectively, after the start of vaccination. To examine whether the therapeutic regimen induced antitumor antibody responses in them, we compared the serum antibody reactivity against autologous tumor cell lysates before and after the vaccinations by immunoblot analysis. Using post-therapy serum as probes, proteins of around 250 kDa and 60 kDa generated clear signals, whereas the pre-vaccination serum showed no or only weak signals at the same position, suggesting that the gene therapy induced an antibody response in them. In three of them (Cases 2–4), the induction of antibodies immunoreactive with these two proteins was significant, while the results were less clear in Case 1. The suspected common antigen was actually demonstrated in both RCC lysates and normal renal cell lysates, and in human lip-derived fibroblasts, but not in H69 lung cancer cells. The strongest signal was observed in Case 2 serum obtained at 67 days after the initial vaccination, between the 5th and 6th vaccinations. This response was maintained from day 67 until day 281, just after the 17th vaccination, although it decreased slightly. After the last vaccination, the immunoreactivity remained at a lower level, although it did not disappear completely. Based on the above results, we applied SEREX, the serological identification of antigens by recombinant expression cloning, using patient’s serum, to isolate the serological RCC tumor antigen genes. We constructed RCC cDNA-expression libraries on lambda phage vector, and screened the plaques with Case 2 serum pooled between days 25 and 80 after the first vaccination. The mRNA source was Case 2 RCC or VMRC-RCW RCC cell line. From above libraries, totally two million recombinant phages screened, twenty-eight SEREX positive clones, which passed secondary or further screening, were isolated. Sequencing analysis revealed the clones were composed of thirteen independent gene products, some of them have not been reported as SEREX defined antigen in previous studies. Expression profiling experiment showed, one of the newly defined SEREX antigen, testicular nuclear autoantigenic sperm protein (tNASP) gene expression had tendency to be dominantly expressed in tumor tissue compared with corresponding normal tissue. To precisely determine the serum antibody titre against the isolated SEREX defined antigens, we constructed the recombinant antigen/epitope tag expression vector and expressed in E. coli. The antibody titration in cancer patients or healty donor against these antigens using obtained recombinant antigen protein has been under investigation. The identification of the new anti-RCC antigen might cast a new light on the treatment of patients suffered from advanced RCC.