Non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) account for about 9% of new cancer cases annually or 64,000 cases per year in the United States. Although the survival rate has significantly improved recently due to new combination therapy regimens, an unmet medical need remains for refractory or resistant patients. HCD122 is a fully human antagonistic anti-CD40 therapeutic monoclonal antibody (mAb) with a dual mechanism of action: blocking CD40 and CD40 ligand (CD40L) interactions and mediating antibody-dependent cellular cytotoxicity (ADCC). CD40 is expressed in all human B-cell malignancies, and the CD40/CD40L interaction is important for tumor cell proliferation and survival. Previously HCD122 was shown to potently inhibit CD40L-induced human B-cell and follicular NHL cell proliferation, mediate ADCC against CD40-positive human malignant B-cell lines and inhibit tumor growth in Burkitt's lymphoma and multiple myeloma xenograft models. In this study the antitumor activity of HCD122 was assessed in preclinical models of HD and other subtypes of human NHL, such as Mantle cell and Follicular lymphoma. CD40 was expressed in 5 of 7 established human HD and 11 of 12 NHL tumor cell lines tested, including Hs445, HDLM-2, KM-H2, L428, L1236, Jeko-1 and WSU-NHL. Using purified human NK cells as effector cells, HCD122 mediated potent ADCC against these cell lines in vitro with a picomolar EC50. When human macrophages were used as effector cells, HCD122 also induced antibody-dependent cellular phagocytosis (ADCP) against the NHL Daudi cell line and the HD cell line Hs445. The antitumor activity of HCD122 was further evaluated in vivo in EBV-negative NHL and HD xenograft models. When tested in a staged human Mantle cell lymphoma Jeko-1 s.c. xenograft model in which treatment was initiated when the mean tumor volume reached 100 mm3, HCD122 was highly efficacious and induced complete tumor regression in 70% (7/10) of treated animals when administered intraperitoneally at 1 mg/kg weekly for 4 weeks. In a staged human HD L428 s.c. xenograft model, which expresses CD20 as well as CD40, the antitumor activity of HCD122 was compared to rituximab. HCD122 was highly efficacious and induced a mean 74 % tumor growth inhibition (TGI) when administered at 0.1 mg/kg weekly for 3 weeks (p<0.001). At the same dose and schedule, rituximab achieved only 40% TGI (HCD122 vs. rituximab: p<0.001). These data combined with our previous studies in multiple myeloma and EBV-positive Burkitt's lymphoma models show that HCD122 is a potent anti-CD40 antibody with pronounced antitumor activity in both EBV-positive and EBV-negative malignant B cell preclinical models. HCD122 is currently in Phase I clinical trials in B-cell malignancies.

CD40 is expressed on most B cell malignancies including multiple myeloma and represents an attractive target for antibody therapy. We have generated a novel, highly potent, fully human antagonistic anti-CD40 monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc). The antibody can mediate anti-tumor activity potentially by at least two mechanisms: CHIR-12.12 can block CD40-ligand mediated survival signals and it can lyse tumor cells by antibody-dependent cellular cytotoxicity (ADCC). We have previously reported that CHIR-12.12 mediates stronger killing of CD40- and CD20-expressing lymphoma cells than rituximab by ADCC in vitro and significantly inhibits the growth of both rituximab-responsive and rituximab-resistant human lymphoma xenografts in vivo. In this study, we examined in vitro and in vivo efficacy of CHIR-12.12 against human multiple myeloma. The human MM cell line IM-9, which expresses both CD40 and CD20, the target antigen for CHIR-12.12 and rituximab respectively was used for the study. CHIR-12.12 induced lysis of target tumor cells by ADCC in a dose dependent manner reaching maximum cell lysis at 0.1ug/ml concentration. The maximum specific lysis of IM-9 cells by CHIR-12.12 was greater than the lysis induced by rituximab (64% vs 45 %, n=3, p<0.01). In addition, the EC50 of CHIR-12.12 was on average 5.9 picomolar, which was 10-fold lower than the EC50 of rituximab. Greater ADCC by CHIR-12.12 was not due to higher density of CD40 molecules on the target tumor cells compared to CD20 molecules. IM-9 cells expressed 35590 ±8858 CD40 molecules compared to 93783 ± 2247 CD20 molecules. The in vivo CHIR-12.12 efficacy was then evaluated in IM-9 xenograft model. In an un-staged conditional survival model, where treatment began one day after intravenous inoculation of IM-9 tumor cells, CHIR-12.12 significantly prolonged the survival of tumor-bearing mice in a dose-dependent manner with 60% survival in the 0.1 mg/kg CHIR-12.12 treated group and 80% survival in the 1 and 10 mg/kg groups respectively on day 56 (Log Rank Test: P<0.01 and P<0.001, respectively). All animals in the control IgG1 and bortezomib treated groups were terminated between day 18 and day 26 due to severe disease related to tumor development (i.e., hind limb paralysis and significant body weight loss). In a staged subcutaneous model, where treatment began once the tumor volume was 150–200mm3, CHIR-12.12 administered weekly at 0.1, 1 and 10 mg/kg significantly inhibited tumor growth with a tumor volume reduction of 17% (P>0.05), 34% (P<0.01) and 44% (P<0.001) respectively. Bortezomib, when tested at 0.5 mg/kg twice a week did not inhibit tumor growth. At the maximally tolerated dose (MTD) of 1 mg/kg twice a week, bortezomib inhibited tumor growth by 30% (P<0.01). Taken together, these data demonstrate that the anti-CD40 mAb CHIR-12.12 has potent activity against human multiple myeloma in vitro and xenograft models in vivo.

CD40 and CD20 are expressed in several B-cell malignancies and represent attractive targets for therapeutic intervention. The anti-CD20 monoclonal antibody, rituximab, is an approved drug for the treatment of non-Hodgkin’s lymphomas. however, the existence of patients with rituximab-resistant disease limits its clinical utility. We have previously reported that the novel, highly potent, fully human antagonistic anti-CD40 monoclonal antibody, CHIR-12.12, generated from XenoMouse® mice (Abgenix, Inc) has greater anti-tumor activity than rituximab in both rituximab-responsive and rituximab-resistant human NHL models. In this study, we evaluated the potential therapeutic application of combining CHIR-12.12 and rituximab for the treatment of NHL. Namalwa is a Burkitt’s lymphoma cell line that gives rise to aggressive rituximab-resistant tumors when implanted in nude mice. Direct treatment of these tumor cells with CHIR-12.12 or rituximab in culture does not affect cell growth when compared to treatment with an isotype control antibody. Although Namalwa cells express more CD20 than CD40 (average of 10,059 CD20 and 3,138 CD40 molecules per cell respectively, P=0.05), when tested for in vitro ADCC killing using human NK cells as effectors, CHIR-12.12 mediated stronger target cell lysis than rituximab (31.43% vs. 14.15%, P<0.0001). Adding CHIR-12.12 and rituximab together did not enhance the in vitro ADCC killing. When CHIR-12.12 and rituximab were tested in a subcutaneous Namalwa xenograft model, CHIR-12.12 alone caused 60% tumor growth inhibition (P=0.028) whereas rituximab alone at 10 and 20 mg/kg did not inhibit Namalwa tumor growth. When tumor-bearing mice were administered rituximab at 10 mg/kg plus CHIR-12.12 at 5 or 10 mg/kg, synergistic anti-tumor activity was observed in a CHIR-12.12 dose-dependent manner. The mean tumor volume reduction in combination groups is 77% with CHIR-12.12 at 5 mg/kg (P=0.0037) and 83% with CHIR-12.12 at 10 mg/kg (P=0.0018), respectively. The potential interaction between CD40 and CD20 molecules was evaluated in vitro by treating the tumor cells with CHIR-12.12 and assessing the change in CD20 expression and vice versa. The result showed no augmentation of one antigen expression by treating the tumor cells with the other antibody. The mechanism of anti-tumor synergy observed in this combination is under evaluation. Taken together, these data suggest that the combination therapy with anti-CD40 CHIR-12.12 and rituximab has the potential to improve patient outcome in B-cell malignancies co-expressing CD20 and CD40 antigens and support the further development of CHIR-12.12 antibody for treatment of B-cell malignancies.

B-cell chronic lymphocytic leukemia (CLL) is characterized by in vivo accumulation of long-lived CD5 + B cells. However when cultured in vitro CLL cells die quickly by apoptosis. Protection from apoptosis in vivo is believed to result from supply of survival signals provided by cells in the microenvironment. We and others have previously reported that CLL cells express CD40 receptor, and that CD40 stimulation of CLL cells may rescue CLL cells from spontaneous and drug-induced apoptosis in vitro . These observations suggested that blocking CD40-CD40L pathway might deprive CLL cells from survival signals and induce apoptosis. To test this hypothesis, we have generated a fully human anti-CD40 blocking monoclonal antibody in XenoMousemice (Abgenix, Inc.). The antibody CHIR-12.12 was first evaluated for its effect on normal human lymphocytes. Lymphocytes from all 10 healthy blood donors did not proliferate in response to CHIR-12.12 at any concentration tested (0.0001 mg/ml to 10 mg/ml range). In contrast, activating CD40 on normal B-lymphocytes by CD40L induced their proliferation in vitro . Importantly, CHIR-12.12 inhibited CD40L- induced proliferation in a dose dependent manner with an average IC50 of 51 ± 26 pM (n=10 blood donors). The antagonistic activity of CHIR-12.12 was then tested in primary CLL samples from 9 patients. CHIR-12.12 alone did not induce CLL cell proliferation. In contrast, primary CLL cells incubated with CD40L, either resisted spontaneous cell death or proliferated. This effect was reversed by co-incubation with CHIR-12.12 antibody, restoring CLL cell death (n=9). CHIR-12.12 was then examined for its ability to lyse CLL cell line EHEB by antibody dependent cell mediated cytotoxicity (ADCC). Freshly isolated human NK cells from normal volunteer blood donors were used as effector cells. CHIR-12.12 showed lysis activity in a dose dependent manner and produced maximum lysis levels at 0.1 mg/ml. When compared with rituximab, CHIR-12.12 mediated greater maximum specific lysis (27.2 % Vs 16.2 %, p= 0.007). The greater ADCC by CHIR-12.12 was not due to higher density of CD40 molecules on CLL cell line compared to CD20 molecules. The CLL target cells expressed 509053 ±13560 CD20 molecules compared to 48416 ± 584 CD40 molecules. Collectively, these preclinical data suggest that CHIR-12.12 monoclonal antibody may have a therapeutic role in patients with CLL.