ALBERT

Description: Key Points BAY 1143572, a novel and selective P-TEFb/CDK9 inhibitor, possessed significant antitumor activity against primary ATL cells in vitro. BAY 1143572 possessed significant antitumor activity in an ATL mouse model based on tumor cells from a patient.

Description: Abstract 423 Purpose: Engineering the Fc region of monoclonal antibodies (mAb) to enhance effector functions is likely to be a promising approach for next-generation mAb therapy. 113F, a complement-dependent cytotoxicity-(CDC)-enhancing variant of rituximab is such an antibody (Cancer Res 2008;68:3863-72). The first of the three major aims of the present study was to identify tumor-associated factors influencing tumor susceptibility to 113F-induced CDC, especially focusing on complement regulatory proteins (CRPs). The second aim of the present study was to compare 113F-induced CDC against primary lymphoma cells with rituximab in vitro. A current crucial problem in the field of human immunotherapy research, including antibody therapy, is the lack of suitable small animal models for in vivo preclinical testing. With respect to antibody-dependent cellular cytotoxicity (ADCC), we have established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients, in which human immune cells are engrafted and mediate ADCC (Cancer Immunol Immunother 2009;58:1195-206, J Immunol. 2009;183:4782-91). On the other hand, there is no mouse model in which human CDC can be evaluated. Thus, the third and final aim of the present study was to establish a mouse model in which it is human complement that mediates CDC against human tumor cells.Using this model, we assessed the therapeutic potential of 113F in comparison with rituximab. Experimental Design: Rituximab- and 113F-induced human CDC was compared in vitro, and in vivo using NOG mice with human complement. Result: First, we determined that tumor-associated factors influencing tumor susceptibility to 113F-induced CDC included the quantity of CRPs such as CD55 and CD59 on the cell surface, as observed in rituximab-induced CDC. Second, we found that 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells from patients, greater than rituximab. Finally, a novel human tumor-bearing mouse model has been developed in which human complement functions in CDC. NOG mouse serum is defective in its capacity to induce CDC against human cells, and endogenous immune cells from NOG mice are unable to mediate ADCC of therapeutic antibodies with an Fc region consisting of human IgG. Thus, we were able to evaluate purely human CDC without interference from endogenous mouse immune cells or complement-mediated mAb induced antitumor effects in this NOG mouse model. The present observation of significant therapeutic efficacy of rituximab together with pooled human serum (PHS) compared to rituximab with inactivated PHS indicated that human complement does function in rituximab-induced CDC in these mice in vivo. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes indicated that human CDC indeed contributed to the antitumor effect in this model. In addition, enhanced therapeutic efficacy of 113F together with PHS compared to rituximab with PHS in this mouse model was observed in vivo. In the cell proliferation assay, viability of the target cell line was not affected by rituximab or 113F alone, and thus no significant difference between these mAbs was observed in vitro. Therefore, the present in vivo observation emphasizes the concept of this type of CDC-enhancing antibody. Furthermore, the more abundant, denser signals of C1q at the tumor cell membrane in these human serum-bearing mice receiving 113F compared with rituximab, are consistent with the Fc region of the former having a much higher C1q binding affinity than the latter. These findings are concordant with the present observation of the greater therapeutic efficacy of 113F compared to rituximab in vivo. Conclusion: This animal model overcomes the limitations of preclinical in vivo investigations of CDC caused by species incompatibilities between humans and mice. This model also makes it possible to reconstitute the human complement system during mAb-based immunotherapy and to perform more appropriate preclinical evaluations of novel therapeutic mAb which mediate CDC. In the present study, highly enhanced human CDC mediated by this type of CDC-enhancing mAb was demonstrated both in vitro and in a humanized mouse model in vivo. In the near future, the efficacy of the type of CDC-enhancing antibody described here will be established in planned clinical trials in humans. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 726 There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities, and it is a current crucial problem in the field of human ADCC research. To overcome this, we have established “humanized mice,” in which human immune cells from healthy individuals function as ADCC effector cells against allogeneic tumor cell lines, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients. In this model, the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to highly enhance ADCC, showed potent antitumor activity by human ADCC against CCR4 expressing tumor cell lines. In addition, KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in the tumor bearing humanized mice. These observations indicate that KM2760 could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. Using this humanized mouse model, we now have the opportunity to perform more appropriate preclinical evaluation of many types of mAb based immunotherapy, although in the initial study, we could not completely exclude nonspecific allogeneic immune responses because target and effector cells were obtained from different individuals. In addition, susceptibility to immunotherapy is likely to be different in established cell lines and primary tumor cells isolated directly ex vivo from patients, with the latter certainly being more relevant for evaluation of immunotherapeutic agents. To overcome the subsequent problems, we have established a primary human tumor bearing NOG mouse model, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T-cell leukemia/lymphoma (ATLL) cells. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in mAb based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future. Disclosures: No relevant conflicts of interest to declare.