Emmanuel Katsanis

Chaperone proteins are effective antitumor vaccines when purified from a tumor source, some of which are in clinical trials. Such vaccines culminate in tumor-specific T cell responses, implicating the role of adaptive immunity. We have developed a rapid and efficient procedure utilizing an isoelectric focusing technique to obtain vaccines from tumor or normal tissues called chaperone-rich cell lysate (CRCL). Tumor-associated peptides, the currency of T cell-mediated anticancer immunity, are believed to be purveyed by chaperone vaccines. Our purpose was to demonstrate our ability to manipulate the peptide antigen repertoire of CRCL vaccines as a novel anticancer strategy. Our methods allow us to prepare "designer" CRCL, utilizing the immunostimulation activity and the carrying capacity of CRCL to quantitatively acquire and deliver exogenous antigenic peptides (e.g., derived from the oncogenic BCR/ABL protein in chronic myelogenous leukemia). Using fluorescence-based and antigen-presentation assays, we determined that significant quantities of exogenously added peptide could accumulate in "designer" CRCL and could stimulate T cell activation. Further, we concluded that peptide-embedded CRCL, devoid of other antigens, could generate potent immunity against pre-established murine leukemia. Designer CRCL allows for the development of personalized vaccines against cancers expressing known antigens, by embedding antigens into CRCL derived from normal tissue.

Although primarily defined by their cardinal antigen-presenting function, dendritic cells (DCs) are also equipped with cytotoxic properties. We have recently reported that DCs activated by IFNγ-secreting Th-1 lymphocytes can kill cancer cells and subsequently present the acquired tumor-derived antigens to T lymphocytes both in vitro and in vivo.

We have documented previously that a multiple chaperone protein vaccine termed chaperone-rich cell lysate (CRCL) promotes tumor-specific T-cell responses leading to cancer regression in several mouse tumor models. We report here that CRCL vaccine generated from a mouse breast cancer (TUBO, HER2/neu positive) is also capable of eliciting humoral immunity. Administration of TUBO CRCL triggered anti-HER2/neu antibody production and delayed the progression of established tumors. This antitumor activity can be transferred through the serum isolated from TUBO CRCL-immunized animals and involved both B cells and CD4(+) T lymphocytes. Further evaluation of the mechanisms underlying TUBO CRCL-mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results suggest that tumor-derived CRCL vaccine has a wider applicability as a cancer vaccine because it can target both T-cell- and B-cell-specific responses and may represent a promising approach for the immunotherapy of cancer.

Mucolipidosis type II (MLII), or I-cell disease, is a rare but severe disorder affecting localization of enzymes to the lysosome, generally resulting in death before the 10th birthday. Although hematopoietic stem cell transplantation (HSCT) has been used to successfully treat some lysosomal storage diseases, only 2 cases have been reported on the use of HSCT to treat MLII. For the first time, we describe the combined international experience in the use of HSCT for MLII in 22 patients. Although 95% of the patients engrafted, overall survival was low, with only 6 patients (27%) alive at last follow-up. The most common cause of death post-transplant was cardiovascular complications, most likely due to disease progression. Survivors were globally delayed in development and often required complex medical support, such as gastrostomy tubes for nutrition and tracheostomy with mechanical ventilation. Although HSCT has demonstrated efficacy in treating some lysosomal storage disorders, the neurologic outcome and survival for patents with MLII were poor. Therefore, new medical and cellular therapies should be sought for these patients.

We have previously reported that stressed apoptotic tumor cells are more immunogenic in vivo than nonstressed ones. Using confocal microscopy we have confirmed our previous observation that heat-stressed apoptotic 12B1-D1 leukemia cells (BCR-ABL(+)) express HSP60 and HSP72 on their surface. To explore how the immune system distinguishes stressed from nonstressed apoptotic tumor cells, we analyzed the responses of dendritic cells to these 2 types of apoptotic cells. We found that nonstressed and heat-stressed apoptotic 12B1-D1 cells were taken up by dendritic cells in a comparable fashion. However, when stressed apoptotic 12B1-D1 cells were coincubated with immature dendritic cells for 24 hours, this resulted in greater up-regulation of costimulatory molecules (CD40, CD80, and CD86) on the surface of dendritic cells. Moreover, stressed apoptotic 12B1-D1 cells were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reactions. Furthermore, we demonstrated that immunization of mice with stressed apoptotic 12B1-D1 cells induced the secretion of T helper-1 (T(H)1) profile of cytokines by spleen cells. Splenocytes from mice immunized with stressed apoptotic cells, but not nonstressed ones, were capable of lysing 12B1-D1 and the parental 12B1 line, but not a B-cell leukemia line, A20. Our data indicate that stressed apoptotic tumor cells are capable of providing the necessary danger signals, likely through increased surface expression of heat shock proteins (HSPs), resulting in activation/maturation of dendritic cells and, ultimately, the generation of potent antitumor T-cell responses.