The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

B-cell chronic lymphocytic leukemia (B-CLL IntroductionChronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States and is characterized by progressive accumulation of malignant B cells in the blood, bone marrow, and lymphoid organs. CLL has been considered a prime example of a malignancy involving defects in the regulation of cell death; the slow accumulation of B-cell CLL (B-CLL) cells is presumably the result of a low proliferative index coupled with an intrinsic defect in apoptosis. However, recent clinical data from B-CLL patients given deuterated water have shown that there is a considerably higher turnover of CLL cells than previously recognized, 1 and that rates of B-CLL cell proliferation, as well as cell death, can vary widely among the lymphatic and extralymphatic compartments. Observations from other studies suggest that there are 2 types of malignant cells: quiescent and apoptosis-resistant cells in the blood, and actively dividing cells found in lymphatic aggregates in the lymph nodes and bone marrow. 2 B-CLL cells in peripheral blood are arrested in the G0/G1 phase of the cell cycle 3 and express high levels of the cell-cycle inhibitor p27. 4 In contrast, Survivinand Ki67-positive B-CLL cells, 5 and those with low expression of p27, 6 have been identified in proliferation centers of the lymph nodes and bone marrow. Thus, the goal of developing therapeutics to treat and cure CLL is to disrupt the pathologic conditions that promote malignant cell growth while accelerating tumor cell death and clearance.Until recently, treatment of progressive CLL, with steroids and alkylating agents, was largely palliative, with no impact on the natural history of the disease. 7 Introduction of purine analogs as single-agent therapies (fludarabine [F]) 8 and in combination with alkylators (fludarabine/cyclophosphamide [FC]) 9 has improved clinical responses and complete remission rates. Purine analogues have a high specificity for lymphoid cells and can induce death in both proliferating and resting cells. As a result, these agents are as effective as single agents for treating bulky CLL disease, and substantially reduce tumor burden with little extramedullary toxicity. Severe myelosuppression and immunosuppression are, however, associated with this class of drugs, and despite improvements in clinical responses, an increase in median survival time has not been demonstrated. Addition of the monoclonal antibody rituximab Rituxan; Genentech [South San Francisco, CA] and IDEC Pharmaceuticals [San Diego, CA]) to FC regimens has resulted in significantly higher overall response rates (ORRs), complete responses (CRs), molecular remissions, and importantly, longer median overall survival. 10-12 Grades 3 to 4 myelosuppression, infection, and viral reactivation remain major morbidities.Monoclonal antibodies have demonstrated activity in CLL as single agents, fueling interest in targeted therapeutic agents that engage the immune effector system to kill tumor cells. Alemtuzumab (CamPath; Millenniu...

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CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.