Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinant adeno-associated virus-based gene loading of dendritic cells Yong Liu, 1 Maurizio Chiriva-Internati, 2 Changxuan You, 3 Rongcheng Luo, 3 Hong You, 4 C Krishna Prasad, 4 Fabio Grizzi, 5 Everardo Cobos, 2 V Suzanne Klimberg, 4 Helen Kay, 4 Jawahar L Mehta, 4 and Paul L Hermonat 1,4 1 Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 West Markham St., Little Rock, Arkansas 72205, USA; 2 Department of Microbiology and Immunology, Texas Tech University, Lubbock, Texas 79430, USA; 3 Department of Oncology, Nanfang Hospital, Guangzhou, Guangdong, China; 4 Department of Obstetrics and Gynecology, Slot 518, University of Arkansas for Medical Sciences, 4301 West Markham St., Little Rock, Arkansas 72205, USA; and 5 Scientific Direction, Instituto Clinico Humanitas, Rozzano, Milano, Italy. Antigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells but not in general hematopoietic cell populations. Moreover, it is much more difficult to generate CTLs against self-antigens. We wished to determine if the use of recombinant adeno-associated virus (rAAV) type 2 vectors for gene-loading of dendritic cells (DCs) could generate rapid, effective cytotoxic T lymphocytes (CTLs) against BA46. We were able to demonstrate that AAV/BA46/Neo-loading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46/Neo vector within DCs, (3) strong, rapid BA46-specific, MHC class I-restricted CTLs in only 1 week, (4) T-cell populations with significant interferon-g (IFN-g) expression but low IL-4 expression, (5) high CD80 and CD86 expression in DCs, and (6) high CD8:CD4 and CD8:CD56 T cell ratios. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens in addition to viral antigens and that the BA46 antigen is potentially appropriate for cell-mediated immunotherapeutic protocols addressing ductal breast cancer. Cancer Gene Therapy (2005) 12, 304–312. doi:10.1038/sj.cgt.7700785 Published online 26 November 2004 Keywords: adeno-associated virus; dendritic cell; BA46; breast cancer; cytotoxic T lymphocyte; immunology A ntigen-targeted immunotherapy has joined the ar- senal of chemotherapy and radiation therapy for the treatment of breast cancer. The search for useful breast cancer antigens has met with some success, identifying Her-2/neu and folate-binding protein as possible tar- gets. 1–5 However, these antigens are only found in a subset of cancer patients. BA46, also known as lactadher- in, is a membrane-associated glycoprotein that is ex- pressed in most breast cancer cells. Larocca et al 6 demonstrated its expression in seven of seven breast cell lines tested. BA46 is also a component of milk, commonly referred to as milk fat globular membrane. 6–9 We wished to determine if BA46 would be an appropriate anti-breast cancer antigen using recombinant adeno-associated virus (AAV)-based gene loading of dendritic cells (DCs). DCs are potent, professional antigen-presenting cells that are able to initiate a primary immune response to antigens by naive T cells. 10 Protocols for generating DCs in vitro from peripheral blood have been developed, and these new technologies permit the in vitro manipulation of DCs for clinical and laboratory immunotherapeutic studies. 11,12 These protocols include loading or incubating DCs with tumor lysates, tumor fragments, antigen peptides, specific tumor proteins, or with antigen genes by way of retrovirus or adenovirus vectors. 13–20 AAV type 2 has been shown in various studies to be an effective gene delivery vector for both immortalized tissue culture cells as well as primary hematopoietic cells. 21–24 We have shown that AAV type 2 can be used to transduce both cytokine and antigen genes into primary human monocytes (Mo) and derived DCs with high efficiency (70–90 þ %). 25–27 The use of AAV-based DC loading of the human papillomavirus E6 and E7 antigen genes Received November 15, 2003. Address correspondence and reprint requests to: Dr Paul L Hermonat, Department of Obstetrics and Gynecology, Slot 518, University of Arkansas for Medical Sciences, 4301 West Markham St., Little Rock, Arkansas 72205, USA. E-mail: plhermonat@uams.edu Cancer Gene Therapy (2005) 12, 304–312 r 2005 Nature Publishing Group All rights reserved 0929-1903/05 $30.00 www.nature.com/cgt