Breast and ovarian cancer are two of the leading causes of cancer deaths among women in the United States. Overexpression of the HER2/neu oncoprotein has been reported in patients affected with breast and ovarian cancers, and is associated with poor prognosis. To develop a novel targeted therapy for HER2/neu expressing tumors, we have constructed a fully human IgE with the variable regions of the scFv C6MH3-B1 specific for HER2/neu. This antibody was expressed in murine myeloma cells and was properly assembled and secreted. The Fc region of this antibody triggers in vitro degranulation of rat basophilic cells expressing human FcεRI (RBL SX-38) in the presence of murine mammary carcinoma cells that express human HER2/neu (D2F2/E2), but not the shed (soluble) antigen (ECDHER2) alone. This IgE is also capable of inducing passive cutaneous anaphylaxis in a human FcεRIα transgenic mouse model, in the presence of a cross-linking antibody, but not in the presence of soluble ECDHER2. Additionally, IgE enhances antigen presentation in human dendritic cells and facilitates cross-priming, suggesting that the antibody is able to stimulate a secondary T-cell antitumor response. Furthermore, we show that this IgE significantly prolongs survival of human FcεRIα transgenic mice bearing D2F2/E2 tumors. We also report that the anti-HER2/neu IgE is well tolerated in a preliminary study conducted in Macaca fascicularis (cynomolgus) monkeys. In summary, our results suggest that this IgE should be further explored as a potential therapeutic against HER2/ neu overexpressing tumors, such as breast and ovarian cancers.

Summary We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc γ receptors and the complement component C1q, suggesting that it is capable of eliciting Fcmediated effector functions such as antibody-dependent cellmediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

BackgroundProstate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa.MethodsBinding characteristics of the antibody were determined by ELISA and flow cytometry. In vitro degranulation was determined by the release of β-hexosaminidase from effector cells. In vivo degranulation was monitored in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. These mice were also used for a vaccination study to determine the in vivo anti-cancer effects of this antibody. Significant differences in survival were determined using the Log Rank test. In vitro T-cell activation was studied using human dendritic cells and autologous T cells.ResultsThe anti-PSA IgE, expressed in murine myeloma cells, is properly assembled and secreted, and binds the antigen and FcεRI. In addition, this antibody is capable of triggering effector cell degranulation in vitro and in vivo when artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis. Importantly, the anti-PSA IgE combined with PSA also triggers immune activation in vitro and in vivo and significantly prolongs the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors in a prophylactic vaccination setting.ConclusionsThe anti-PSA IgE exhibits the expected biological properties and is capable of triggering immune activation and anti-tumor protection. Further studies on this antibody as a potential PCa therapy are warranted.

Prostate cancer (PCa) is the second leading cause of cancer deaths among men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal is to take advantage of the unique properties of the IgE molecule in order to trigger immune activation against PCa. The properly assembled and secreted anti-PSA IgE binds the antigen and the Fc epsilon RI (FcεRI) as demonstrated by ELISA and flow cytometry, respectively. This novel IgE antibody is capable of triggering effector cell degranulation in vitro as determined by the release of β-hexosaminidase from rat basophil leukemic effector cells that express human FcεRI (RBL SX-38). Furthermore, the IgE triggered degranulation in vivo in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. Degranulation was only observed when the IgE was artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis in patients. Importantly, when incubated in vitro with human dendritic cells the anti-PSA IgE complexed with the antigen (PSA) triggered autologous CD4 and CD8 T-cell activation, suggesting enhancement of antigen presentation. Immune activation was also observed in vivo where a prophylactic vaccination with the anti-PSA IgE complexed to PSA significantly prolonged the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors, under conditions in which its chimeric mouse/human anti-PSA IgG1 counterpart failed to confer protection. Our results provide initial proof-of principle that the anti-PSA IgE can stimulate immune activation against tumor cells expressing PSA, and further studies on the anti-tumor activity of this antibody are warranted. These studies belong to the new field of AllergoOncology that aims to reveal the function of IgE-mediated immune responses against cancer and develop novel IgE-based cancer therapies. Citation Format: Tracy R. Daniels-Wells, Gustavo Helguera, Richard K. Leuchter, Rafaela Quintero, Maggie Kozman, Jose A. Rodríguez, Elizabeth Ortiz-Sánchez, Otoniel Martínez-Maza, Birgit C. Schultes, Christopher F. Nicodemus, Manuel L. Penichet. IgE-mediated immune activation targeting the prostate specific antigen: a potential prostate cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2848. doi:10.1158/1538-7445.AM2013-2848

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, CD71) that demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is due to its ability to decrease TfR1 levels leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc gamma receptors and the complement component C1q, suggesting that is capable of eliciting Fc-mediated effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). Additionally, in two disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant anti-tumor activity including long-term survival. Interestingly, the parental antibody without avidin (ch128.1) also shows remarkable in vivo anti-cancer activity despite its lack of in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the Long-Term Cell-Initiating Culture (LTC-IC) assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2011-3622