Combined therapy of an established, highly aggressive breast cancer in mice with paclitaxel and a unique DNA-based cell vaccine Amla Chopra 1 , Tae Sung Kim 2 , InSug O-Sullivan 1 , Don Martinez 1 and Edward P. Cohen 1 * 1 Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 2 School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea Here, we describe the enhanced benefits of treating a highly aggressive breast cancer in mice with a combination of paclitaxel and immunization with a unique DNA-based cell vaccine. An adenocarcinoma was isolated from a spontaneous neoplasm that arose in the mammary gland of a C3H/He mouse (H-2 k ) (SB5b cells). The vaccine was prepared by transfer of genomic DNA- fragments (25 kb) from the breast cancer cells into a mouse fibro- blast cell line (LM), modified to enhance its immunogenic proper- ties. As the transferred DNA is integrated, and replicated as the recipient cells divide, the vaccine could be prepared from rela- tively small numbers of cancer cells (10 7 5 4 mm tumor). SB5b cells were injected into the mammary fat pad of na € ıve C3H/He mice, which are highly susceptible to the growth of the cancer cells. When the tumors reached 3 mm, the mice were injected s.c. with a noncurative dose of paclitaxel. Six days later, when immune competence returned, the mice received the first of 3 weekly s.c. injections of the vaccine. The combined therapy induced robust cellular immunity to the breast cancer, mediated by CD81 and NK/LAK cells, which resulted in prolonged sur- vival. The immunity was specific, as immunization with a vaccine prepared by transfer of DNA from B16 melanoma cells into the fibroblasts failed to induce immunity to the breast cancer. This type of vaccine raises the possibility that an analogous strategy could be used in the treatment of breast cancer patients at an early stage of the disease. ' 2005 Wiley-Liss, Inc. Key words: immunotherapy; breast cancer; DNA-based cell vaccine; preclinical The potential benefits of immunotherapy as an adjunct to con- ventional forms of cancer treatment are under active investiga- tion. 1–4 Activated cytotoxic T lymphocytes (CTLs) capable of rec- ognizing and destroying cancer cells are generated in immunized mice and patients. The immunity is directed toward unique MHC class I restricted TAAs expressed by the malignant cells. 5–8 Although experimental immunotherapy protocols in mice are revealing the potential of this form of treatment, effective vaccina- tion strategies in cancer patients are wanting. One possible explan- ation is that even though the immune system can adversely affect diffuse and smaller tumors, it cannot effectively destroy large, established neoplasms. An immunotherapeutic strategy that would allow treatment at an early stage of the disease could have signifi- cant benefits. Tumor cells are the richest source of tumor antigens. Immuniza- tion with malignant cells modified to secrete immune-augmenting cytokines such as IL-2, 9–11 GM-CSF, 12 IL-4, 13 IL-6 14 and IL- 12 15,16 resulted in rejection of the cytokine-secreting cells and the induction of T cell mediated immunity toward the neoplastic cells. In some instances, the induced immunity was sufficient to prolong the lives of mice with established neoplasms. However, the direct modification of cancer cells from a primary neoplasm is tech- nically challenging. It requires the establishment of a tumor cell line, which cannot always be accomplished. This is especially the case for breast cancer in patients. It is notoriously difficult to establish breast cancer cell lines from primary neoplasms. In prior reports, 17–19 we described the results of studies in mice with breast cancer treated by immunization with a vaccine pre- pared by transfer of sheared genomic DNA fragments from vari- ous murine neoplasms, including adenocarcinoma of the breast, into a highly immunogenic, mouse fibroblast cell line. The ration- ale was that genes specifying TAA would be expressed in a highly immunogenic form by the transfected cells. As the transferred DNA is integrated into the genome of the recipient cells, and repli- cated as the cells divide, the vaccine could be prepared from DNA derived from relatively small numbers of cancer cells. Sufficient DNA could be recovered from as few as 10 million cancer cells. (A tumor of 4 mm contains an equivalent number of cells.) The vaccine was readily prepared from primary neoplasms. The estab- lishment of a tumor cell line was not required. Furthermore, as the DNA was not fractionated before transfer, it was likely that multiple mutant/dysregulated genes in the breast cancer cells spec- ifying an array of unidentified weakly immunogenic TAAs were expressed by the transfected cells. Like many other vaccination strategies, however, the DNA- based vaccine was not effective against larger and more aggres- sive tumors. The survival of mice with well-established breast neoplasms treated solely by immunization with the vaccine was not significantly different than that of untreated mice with breast cancer. In an attempt to improve the therapeutic outcome, we combined treatment with the vaccine with paclitaxel, a drug commonly used in breast cancer therapy. 20,21 C3H/He mice bearing an established, highly aggressive breast cancer (generation time 5 18.3 hr) de- rived from a neoplasm that arose spontaneously in a C3H/HeJ mouse were treated with noncurative amounts of paclitaxel, fol- lowed by immunization with the DNA-based cell vaccine. The results indicated that the survival of mice with established breast cancer receiving the combined therapy exceeded that of tumor- bearing mice receiving either form of treatment alone. To our knowledge, this report is the first indicating that combination ther- apy with paclitaxel along with immunization with a unique vac- cine prepared from microgram amounts of tumor tissue was capa- ble of prolonging the survival of mice with breast cancer. Material and methods Experimental animals and tumor cell lines Eight- to 10-week-old pathogen-free C3H/HeJ female mice were from the Jackson Laboratory (Bar Harbor, ME). The ani- mals, between 10 and 14 weeks old when used in the experiments, were maintained according to NIH Guidelines for the Care and Use of Laboratory Animals. SB5b cells were a short-term passage adenocarcinoma of the breast cell line derived from a breast neo- plasm that arose spontaneously in a C3H/He mouse in our animal colony. B16 cells, a melanoma cell line of C57BL/6 origin, were obtained originally from I. Fidler (M.D. Anderson, Houston, TX). The cells were maintained by serial passage in histocompatible The use of animals in these studies as reviewed and approved by the Animal Care Committee of the University of Illinois (Approval number 04-067, expires 7/07). Grant sponsor: NIDCR; Grant number: 1 RO1 DEO13970-O1A2. *Correspondence to: Department of Microbiology and Immunology (m/c 790), 835 South Wolcott Ave, Chicago, IL 60612, USA. Fax:1312-996-6415. E-mail: EPCohen@uic.edu Received 29 September 2005; Accepted after revision 2 November 2005 DOI 10.1002/ijc.21724 Published online 27 December 2005 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 118, 2888–2898 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer