Abstract The prolactin receptor (PRLR) is a class I cytokine receptor frequently expressed in breast and prostate cancer. The polypeptide hormone prolactin (PRL) has been demonstrated to induce PRLR signaling through the Jak/Stat, PI3-kinase/AKT and MAPK pathways, leading to cell proliferation and survival. Breast- and prostate-specific overexpression of PRL in transgenic mice leads to a higher incidence of mammary and prostate tumors, respectively. In addition, the PRLR locus is the site of frequent viral integrations in MMTV-derived mammary tumors. Elevated serum PRL levels in humans have been correlated with an increased risk for breast cancer, and an analysis of more than 3000 breast tumor specimens indicates that PRLR is expressed with high prevalence (60-70% of tumors) across all breast cancer subtypes. In prostate cancer specimens, the presence of prolactin and phosphorylated Stat5 have been reported to be associated with high-grade tumors and poor clinical outcomes, suggesting a role of the PRL/PRLR signaling pathway in the pathology of this disease as well. All of these lines of evidence support the hypothesis that targeting the PRL/PRLR axis may be a new approach for addressing unmet medical need in these tumor types. LFA102 is a Human Engineered™ anti-PRLR antibody of the IgG1 isotype that neutralizes the function of PRLR through a nonligand competitive binding interaction. LFA102 blocks PRL-induced signaling and proliferation in T47D and MCF7 human breast cancer cells in vitro, and abolishes PRL-induced phospho-Stat5 signaling in T47D xenograft tumors in vivo. This antibody also cross-reacts with and neutralizes rat PRLR and is capable of potently regressing PRL-dependent Nb2-C11 pre-T cell lymphoma tumors in vivo. In vitro studies have shown that LFA102 can mediate antibody-dependent cellular cytotoxicity (ADCC) and inhibit the PRL-dependent release of the proangiogenic factor VEGF from breast cancer cells. Thus, there are multiple potential mechanisms through which LFA102 could show antitumor activity in vivo. Preclinical toxicological studies of LFA102 indicate that this therapeutic is well tolerated and exhibits a normal pharmacokinetic profile in relevant animal species. The safety and pharmacokinetics of LFA102 in humans are currently being evaluated in a phase I healthy volunteer trial. A phase 1b trial in breast and prostate cancer is planned to evaluate the efficacy of this antibody in patient populations predicted to have the highest probability of benefiting from an anti-PRLR therapeutic. This presentation will provide a summary of the preclinical data supporting the clinical development of LFA102. 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 DDT02-02. doi:10.1158/1538-7445.AM2011-DDT02-02

B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-alpha, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

HCD122 (formerly known as CHIR-12.12), is a fully human anti-CD40 monoclonal antibody (mAb) currently in Phase I clinical trials for treatment of chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). An IgG1 antibody selected for its potency as an antagonist of the CD40 signaling pathway, HCD122 both inhibits CD40/CD40L-stimulated growth of lymphoma cells ex vivo, and mediates highly effective Antibody Dependent Cell-mediated Cytotoxicity (ADCC) in vitro. As a single agent, HCD122 exhibits potent anti-tumor activity in vivo, in preclinical models of MM, Hodgkin's lymphoma, Burkitt's lymphoma, mantle cell lymphoma and diffused large B-cell lymphoma (DLBCL). Although several therapeutic antibodies approved for treatment of Non-Hodgkin's Lymphoma have clinical activity as single agents, combining these antibodies with standard-of-care chemotherapeutic regimens such as CHOP (cytoxan, vincristine, doxorubicin and prednisone) is proving optimal for both increasing response rates and extending survival, and antibodies currently in clinical development are likely to be used in combination therapies in the future. Therefore the studies reported here examine the effects of combining HCD122 with CHOP, the standard for treatment of high grade NHL, in in vitro and in vivo models of DLBCL. In the xenograft RL model of DLBCL, HCD122 administered intraperitoneally weekly at 1 mg/kg as a single agent, or in combination with CHOP (H-CHOP), and CHOP alone all significantly reduced tumor growth at day 25 when compared to treatment with huIgG1 control antibody (P<0.001). However, tumor growth delay (time to reach tumor size of 500 mm3) was significantly longer for H-CHOP (17.5 days), than for CHOP (8 days) or HCD122 (6 days) (p < 0.001). No toxicity was observed with the H-CHOP combination. Interestingly, at the end of the study (day 35), reduction in tumor growth was significantly greater in the treatment group that received H-CHOP than the groups that received either 10 mg/kg Rituxan plus CHOP (R-CHOP) (p < 0.05) or CHOP alone (p < 0.001). These data show that in this model, treatment with the combination H-CHOP results in greater anti-tumor efficacy than with either modality alone or R-CHOP. We have observed that in vitro, exposure to CD40 Ligand (CD40L) results in aggregation of DLBCL cells, and postulate that interfering with the ability of cancer cells to adhere and interact with each other and their microenvironment may potentiate the effect of chemotherapeutics. To elucidate the mechanism by which the combination of HCD122 and CHOP enhanced efficacy in vivo, we developed an in vitro system to examine the effects of HCD122 on the expression of adhesion molecules in the RL and SU-DHL-4 cell lines. In these studies, HCD122 inhibited CD40L-induced expression of CD54, CD86 and CD95 in both cell lines, as well as aggregation of SU-DHL-4 cells. The combined effect of each of the components of CHOP with HCD122 in three-dimensional spheroid cultures is currently under investigation. These data provide a therapeutic rationale for combination of HCD122 with CHOP in DLBCL clinical trials.

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.

We have generated a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc.) and have previously demonstrated that it inhibits normal human B cell proliferation and survival and has potent ADCC against primary CLL and NHL cells. CHIR-12.12 and the anti-CD20 monoclonal antibody rituximab were compared for their relative ADCC activity against a variety of malignant human B-cell lines expressing both CD40 and CD20 antigens, including two lymphoma cell lines (Daudi, Namalwa), two multiple myeloma cell lines (ARH77, IM-9), a B-ALL cell line (CCRF-SB), and a B-CLL cell line (EHEB). All cell lines expressed both CD20 and CD40 antigens, and the number of cell surface CD20 molecules per cell were 2.6- to 30.8-fold higher than CD40. For all target cell lines, despite the greater number of CD20 receptors, CHIR-12.12 showed greater maximum cell lysis and a lower ED50 than rituximab. ADCC activity of rituximab is known to correlate with the FcγRIIIa genotype of the effector cells. The homozygous valine (V/V) or heterozygous valine/phenylalanine (V/F) polymorphisms at aa158 are associated with greater cell lysis than is the homozygous F/F polymorphism. The role of the FcγRIIIa aa158 genotype as it relates to CHIR-12.12 activity was explored using Daudi lymphoma target cells and effector NK cells purified from human donors expressing the three polymorphisms. CHIR-12.12 induced potent ADCC with NK cells of all three genotypes (ED50s of 4, 2, and 0.4 pM for F/F, V/F, and V/V, respectively). The rituximab ED50s were 53, 21, and 9 pM for F/F, V/F, and V/V, respectively. Comparison of affinity of the FcγRIIIa F and V alleles for CHIR-12.12 and rituximab using Biacore® analysis showed that CHIR-12.12 bound the F allele with a 4.6-fold higher affinity than rituximab (2.8 μM versus 13 μM, respectively). These data demonstrate that CHIR-12.12 is a more potent ADCC mediator than rituximab, even with human NK cells of the aa158 F/F genotype. CHIR-12.12 is currently in Phase I clinical trials for B-cell malignancies.

Monoclonal antibodies (mAb) directed against lineage-specific B-cell antigens have provided clinical benefit for patients with hematologic malignancies, but to date no antibody-mediated immunotherapy is available for multiple myeloma. In the present study, we assessed the efficacy of a fully human anti-CD40 mAb CHIR-12.12 against human multiple myeloma cells. CHIR-12.12, generated in XenoMouse mice, binds to CD138-expressing multiple myeloma lines and freshly purified CD138-expressing cells from >80% multiple myeloma patients, as assessed by flow cytometry. Importantly, CHIR-12.12 abrogates CD40L-induced growth and survival of CD40-expressing patient multiple myeloma cells in the presence or absence of bone marrow stromal cells (BMSC), without altering constitutive multiple myeloma cell proliferation. Immunoblotting analysis specifically showed that PI3-K/AKT, nuclear factor-kappaB (NF-kappaB), and extracellular signal-regulated kinase activation induced by CD40L (5 mug/mL) was inhibited by CHIR-12.12 (5 mug/mL). Because CD40 activation induces multiple myeloma cell adhesion to both fibronectin and BMSCs, we next determined whether CHIR-12.12 inhibits this process. CHIR-12.12 decreased CD40L-induced multiple myeloma cell adhesion to fibronectin and BMSCs, whereas control human IgG1 did not. Adhesion of multiple myeloma cells to BMSCs induces interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion, and treatment of multiple myeloma cells with CD40L further enhanced adhesion-induced cytokine secretion; conversely, CHIR-12.12 blocks CD40L-enhanced IL-6 and VEGF secretion in cocultures of multiple myeloma cells with BMSCs. Finally, CHIR-12.12 triggered lysis of multiple myeloma cells via antibody-dependent cellular cytotoxicity (ADCC) but did not induce ADCC against CD40-negative multiple myeloma cells, confirming specificity against CD40-expressing multiple myeloma cells. These results provide the preclinical rationale for clinical trials of CHIR-12.12 to improve patient outcome in multiple myeloma.

CD40 and CD40 ligand (CD40L) interaction is a key regulator of B-chronic lymphocytic leukemia (CLL) survival. CD40 activation leads to binding with tumor necrosis factor receptor-associated factors (TRAFs) and the subsequent activation of multiple downstream signaling pathways involved in cellular proliferation and survival. We have generated a novel fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc). CHIR-12.12 blocks CD40L binding to CD40 and inhibits CD40L-induced proliferation/survival of normal human B cells, primary CLL cells, and primary non-Hodgkin's lymphoma (NHL) cells. We have also demonstrated that it has highly potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin's lymphoma cells. We have now investigated its effects on primary CLL cell survival. Soluble human CD40L prolongs primary CLL cell survival in culture, and treatment with CHIR-12.12 inhibits this survival when measured 48–72 hours after addition of CHIR-12.12. CD40L-mediated survival is associated with activation and phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases a and b. Additionally, the anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP are induced, and markers of apoptosis (cleaved PARP and Caspase-3) are reduced. In contrast, CHIR-12.12 treatment of CD40L-stimulated primary CLL cells ex vivo inhibited downstream phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases (IKK) a and b. Additionally, CHIR-12.12 treatment resulted in induction of cleaved caspase-3 and PARP, and reduction of XIAP, Mcl-1, and Bcl-xl expression, ultimately leading to CLL cell apoptosis. These results demonstrate that CHIR-12.12 inhibits CD40L-mediated signaling pathways and cell survival and could be a potential therapeutic treatment for CLL. CHIR-12.12 is currently in a Phase I clinical study for CLL.