Expression and Purification of cGMP grade NY-ESO-1 for Clinical Trials Adam J. Lowe Graduate Field of Microbiology, Cornell University, Ithaca, NY 14853 Cameron L. Bardliving Graduate Field of Biomedical Engineering, Cornell University, Ithaca, NY 14853 Chung-Jr Huang, Leonardo M. Teixeira, and Leonardo M. Damasceno, and Kyle A. Anderson Graduate Field of Microbiology, Cornell University, Ithaca, NY 14853 Gerd Ritter and Lloyd J. Old LICR – NY Branch, Memorial Sloan Kettering, New York, NY 10021 Carl A. Batt Department of Food Science, Cornell University, Ithaca, NY 14853 DOI 10.1002/btpr.552 Published online February 28, 2011 in Wiley Online Library (wileyonlinelibrary.com). NY-ESO-1 is a cancer testis antigen expressed in numerous cancers. Initial tests have shown its efficacy as a cancer vaccine, stimulating the body’s own immune response against the invading tumor. To produce enough material for phase I clinical trials, a process using current good manufacturing practices to produce clinical grade material was developed and executed. His-tagged NY-ESO-1 was expressed in C41DE3 Escherichia coli under control of the T-7 promoter. NY-ESO-1 was produced in a 20 L fed-batch fermentation utilizing a pH- stat control scheme. The protein was then purified from inclusion bodies using a three-col- umn process that achieved a yield of over 3.4 g and endotoxin below the detection limit of 0.005 EU/lg protein. V V C 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 435–441, 2011 Keywords: NY-ESO-1, purification, CT-antigen, vaccine, cancer Introduction The NY-ESO-1 protein, also know as CTAG1 and LAGE- 2, is a cancer testis (CT) antigen that holds significant prom- ise as an immunotherapeutic vaccine. It was first identified by serological analysis of recombinant cDNA expression libraries (SEREX) from tumor mRNA and host serum. Its expression has been detected by RT-PCR and immunohisto- chemistry in multiple cancers, including melanoma, 1 neuro- blastoma, 2 and soft tissue sarcoma. 3 The gene is located in q28 region of the X chromosome and is a member of the CT-X antigen family. 4 NY-ESO-1 is one of the most immu- nogenic CT-antigens, eliciting immunity spontaneously and after vaccine administration. 5 Clinical responses were also detected in chemorefractory cancers. 5 CT antigens make excellent vaccine candidates as their expression is limited to only cancerous cells and normal, tes- tis germ cells. 6 However, testis germ cells are MHC class 1 negative 7 and will not elicit a cytotoxic response. To this end, NY-ESO-1 vaccines supplemented with the ISCOMA- TRIX adjuvant have been successful in melanoma vaccine trials. 8 NY-ESO-1 has also been shown to be a prognostic factor for malignant melanoma (MM). It was additionally demonstrated that MM expressing NY-ESO-1 was associated with thicker primary lesions, and had a higher frequency metastatic disease. 9 NY-ESO-1 vaccines were shown to be even more successful when combined with an anti-CTLA-4 antibody treatment, as CTLA-4 has been shown to downreg- ulate T-cell signaling and activity. 10 Because of the protein’s success in initial experiments, a critical need is growing for material used in limited phase-I clinical trials. Pilot-scale production of vaccine antigens, under current good manufac- turing practices (cGMP), fills an important role where indus- trial scale production may not be economically viable. Here, we have developed a process suitable for the pilot- scale production of cGMP grade His-tagged NY-ESO-1. Because of its extremely high hydrophobicity, the protein presented complex solubility and endotoxin removal Additional Supporting Information may be found in the online ver- sion of this article. Current address of Kyle A. Anderson: Tetragenetics, Inc., Ithaca, NY 14850. Current address of Chung-Jr Huang: DeLisa Laboratory, Department of Chemical Engineering, Cornell University. Ithaca, NY 14853. Current address of Leonardo M. Teixeira: Graduate School, Faculdade de Tecnologia e Cie ˆncias, Salvador, Brazil. Current address of Leonardo M. Damasceno: Graduate School, Faculdade de Technologia e Ciencias, Salvador, Brazil. Correspondence concerning this article should be addressed to A. J. Lowe at ajl248@cornell.edu. V V C 2011 American Institute of Chemical Engineers 435