Patricia A. Culp

Antibodies to TWEAK Receptor Inhibit Human Tumor Growth through Dual Mechanisms

Purpose: Targeted therapeutics have significantly changed the outcome for patients diagnosed with cancer. Still, effective therapeutic intervention does not exist for many cancers and much remains to be done. The objective of this study was to identify novel genes that potentially regulate tumor growth, to target these gene products with monoclonal antibodies, and to examine the therapeutic potential of these antibodies. Experimental Design: Using cDNA microarray analysis, we identified genes overexpressed in several solid malignancies. We generated a mouse monoclonal antibody, 19.2.1, and its humanized counterpart, PDL192, to one such target, TweakR (TWEAK receptor, Fn14, TNFRSF12A, CD266), and characterized the antitumor activities in vitro and in mouse xenograft models. Results: Both 19.2.1 (mouse IgG2a) and PDL192 (human IgG1), like TWEAK, the natural ligand of TweakR, inhibited the growth of several TweakR-expressing cancer cell lines in anchorage-dependent and anchorage-independent assays in vitro. Both antibodies showed significant antitumor activity in multiple mouse xenograft models. PDL192 and 19.2.1 also induced antibody-dependent cellular cytotoxicity (ADCC) of cancer cell lines in vitro. A chimeric version of 19.2.1 containing the mouse IgG1 Fc region (19.2.1×G1) exhibited significantly less ADCC than 19.2.1. However, 19.2.1×G1 showed differential activity in vivo, with activity equivalent to 19.2.1 in one model, but significantly less efficacy than 19.2.1 in a second model. These results indicate that PDL192 and 19.2.1 mediate their antitumor effects by signaling through TweakR, resulting in reduced tumor cell proliferation, and by ADCC. Clin Cancer Res; 16(2); 497–508

Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb

BackgroundThe receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer.MethodsExpression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR.ResultsOverexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis.ConclusionsTweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.

Predictive gene signature of response to the anti-TweakR mAb PDL192 in patient-derived breast cancer xenografts.

Purpose (1) To determine TweakR expression in human breast cancers (BC), (2) evaluate the antitumor effect of the anti-TweakR antibody PDL192, used alone or after chemotherapy-induced complete remission (CR), on patient-derived BC xenografts (PDX) and (3) define predictive markers of response. Experimental Design TweakR expression was analyzed by IHC on patients and PDXs BC samples. In vivo antitumor effect of PDL192 was evaluated on eight TweakR-positive BC PDXs alone or after complete remission induced by a combination of doxorubicin and cyclophosphamide. Using both responding and resistant PDX tumors after PDL192 administration, RT-QPCR were performed on a wide list of selected candidate genes to identify predictive markers of response. Results TweakR protein was expressed in about half of human BC samples. In vivo PDL192 treatment had significantly anti-tumor activity in 4 of 8 TweakR-positive BC PDXs, but no correlation between the expression level of the Tweak receptor and response to therapy was observed. PDL192 also significantly delayed tumor relapse after CR. Finally, an 8 gene signature was defined from sensitive and resistant PDXs. Conclusions PDL192 was highly efficient in some BC PDXs. We found 8 genes that were differentially expressed in responding and resistant tumors and could constitute a gene expression signature which would need to be extended to other xenograft models for confirmation. These data confirm the therapeutic potential of TweakR targeting in BC and the possibility of prospectively selecting patients who might benefit from therapy.

Abstract 2722: Active recruitment of immune effector cells mediates in vivo tumor growth inhibition by enavatuzumab, an antibody to human TWEAK receptor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Enavatuzumab (PDL192) is a humanized anti-TWEAK receptor antibody currently under clinical evaluation for the treatment of solid malignancies. Enavatuzumab is postulated to exert its potent anti-tumor activity in xenograft models through two distinct mechanisms of action: direct inhibition of tumor cell growth and Fc-mediated function. The goal of this study was to elucidate the role of immune effector cells in Fc-mediated function. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models which responded to enavatuzumab but did not respond to a version of enavatuzumab containing a mutation in the FcαRIII binding region. Subsequent analysis of circulating effector cells showed increased expression of activation markers DX5 and CD27 on monocyte-like cells (CD45+CD11bHigh) and DX5 on NK-like cells (CD45+CD11bLow) after enavatuzumab treatment in xenograft models dependent on Fc-FcR interactions for enavatuzumab efficacy. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in non-responders. Consistent with the in vivo xenograft studies, human effector cells migrated towards enavatuzumab-treated tumor cells in vitro. Effector cells preferentially migrated toward in vivo responsive tumor cells, and the majority of migratory cells were monocytes and NK cells. Conditioned media from enavatuzumab-treated tumor cells also induced migration, and contained elevated levels of cytokines/chemokines, including IL8 and MCP-1. The presence of an anti-MCP-1 neutralizing antibody in enavatuzumab-treated tumor cell conditioned medium significantly blocked effector cell migration, suggesting that MCP-1 might be responsible for effector cell migration triggered by enavatuzumab. We also observed elevated levels of human MCP-1 in the serum of both enavatuzumab-treated xenograft mouse models and in a subset of patients from the enavatuzumab Phase I clinical study. In summary, in vivo and in vitro studies suggest that enavatuzumab exerts its potent anti-tumor activity, in part, by actively recruiting effector cells and activating effectors to kill tumor cells. In addition, enavatuzumab-induced chemokines may be potential pharmacodynamic biomarkers and warrant further evaluation in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2722. doi:1538-7445.AM2012-2722

Phase I Study of Enavatuzumab, a First-in-Class Humanized Monoclonal Antibody Targeting the TWEAK Receptor, in Patients with Advanced Solid Tumors

This phase I study evaluates the safety, MTD, pharmacokinetics (PK), pharmacodynamics, and preliminary anticancer activity of enavatuzumab, a humanized IgG1 antibody to the TWEAK receptor, in patients with advanced solid malignancies. Patients received escalating doses of enavatuzumab given intravenously over 60 minutes every 2 weeks. Blood was obtained for PK and biomarker assessment. Three patients were enrolled per dose level in a standard 3+3 design with response assessment by RECIST version 1.0, every 8 weeks. Thirty patients were enrolled at 6 dose levels ranging from 0.1 to 1.5 mg/kg. Dose-limiting toxicities included grade 4 (G4) lipase, G3 bilirubin, and G4 amylase elevations. There was no apparent correlation of liver or pancreatic enzyme elevation with drug exposure or the presence of liver metastases. Enavatuzumab exhibited a two-compartment linear PK model. Estimated systemic clearance was 23 to 33 mL/h with an elimination half-life of 7 to 18 days. The predicted target efficacious peak and trough concentrations occurred at 1.0 mg/kg following the second dose. There were no objective responses; 4 patients had stable disease. The MTD of enavatuzumab is 1.0 mg/kg i.v. every 2 weeks. Higher doses were not tolerated due to hepatopancreatic lab abnormalities. Further evaluation of the mechanisms of the liver and pancreatic enzyme toxicities is needed before embarking on further single-agent or combination strategies. Mol Cancer Ther; 17(1); 215–21. ©2017 AACR.

Abstract C18: A phase I study of enavatuzumab (PDL192, ABT-361), a first-in-class human monoclonal antibody targeting TWEAK (tumor necrosis factor-like inducer of apoptosis) receptor, in patients (Pts) with advanced solid tumors.

Background: TWEAK receptor (TweakR, TNFRSF12A, CD266) is a member of the TNF receptor superfamily and is expressed on a range of solid tumors. Enavatuzumab is a humanized IgG1 antibody to TweakR that exhibits anti-tumor activity in preclinical models through two mechanisms: direct signaling through TweakR and antibody-dependent cellular cytotoxicity. Methods: This phase I, multicenter study was designed to determine the maximum tolerated dose (MTD) and to evaluate the safety and pharmacokinetic (PK) profiles of enavatuzumab, given intravenously over 60 minutes on days 1 and 15 of each 28-day cycle. Pts with advanced solid tumors; ECOG ≤1; adequate hematologic, renal, and hepatic function; no brain metastasis; no history of cirrhosis or pancreatitis; no recent history of acute cholecystitis; and not on immunosuppressive drugs were enrolled. Cohorts of 3–6 pts were treated at escalating dose levels of enavatuzumab ranging from 0.1 mg/kg to 1.5 mg/kg. Dose limiting toxicity (DLT) was defined as grade (G) 3 AST or ALT lasting >14 days or associated with clinical hepatitis and/or bilirubin ≥G2; G3 amylase or lipase lasting >14 days; ≥G3 bilirubin; any G4 AST, ALT, amylase, or lipase; acute pancreatitis; ≥G3 hematologic and non-hematologic toxicities; G4 cytokine release syndrome (CRS) or G3 CRS despite premedication. MTD was defined as the highest dose level with 0/3 or ≤1 of 6 pts experiencing a first-cycle DLT. PK sampling was performed on cycle 1 day 1 (C1D1), C1D2, C1D8, Day 15 of all cycles, C1D16, C2D1, and C3D1. Response was assessed by RECIST every 8 weeks. Results: Thirty pts [12 male, 18 female; median age 64.5 (range 36–82)] were enrolled at 6 dose levels: 0.1, 0.3, 0.5, 0.7, 1.0, and 1.5 mg/kg. Tumor types included colorectal (15); pancreas (4); ovarian (4); and tongue, cervical, prostate, endometrial, breast, hepatocellular, and thyroid (1 each). Sixteen pts had liver metastases. DLTs included G4 lipase and G3 bilirubin in 1 of 6 pts at the 1.0 mg/kg level and G4 lipase and G4 amylase in 1 pt at the 1.5 mg/kg level. Grade 3/4 toxicities in ≥5% of subjects included fatigue, pneumonia, hyponatremia, hypoxia, dyspnea, and elevated AST, ALT, GGT, lipase, and amylase. There was no apparent correlation of liver or pancreatic enzyme elevation with tumor type or presence of liver metastases. Of the disease evaluable pts, 11 had disease progression as their best response and 2 had stable disease (2 and 4 month duration). Enavatuzumab followed a two-compartment linear PK model. The estimated clearance was 23–33 mL/hr and elimination half-life was 7–18 days. The mean Cmax after the first dose was 2.0 mcg/mL and 28.1 mcg/mL for the 0.1 mg/kg and 1.0 mg/kg cohorts, respectively. Mean Css was reached following the second dose and was within the target efficacious peak and trough levels for the 1.0 mg/kg cohort. Conclusions: The MTD for this first-in-class drug was reached at 1.0 mg/kg. Six of 7 pts at 1.0 mg/kg and 1 of 1 pts at 1.5 mg/kg had ≥G3 liver and/or pancreatic enzyme elevations; therefore higher doses are not being tested. The mechanisms of liver and pancreatic enzyme toxicity are currently being investigated and preclinical studies are ongoing to assess potential combination strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C18.

Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

Abstract 1770: Enavatuzumab (PDL192), a humanized monoclonal antibody to TweakR, displays broad anticancer activity which is dependent on NFκB signaling

TWEAK (TNFSF12) is a multifunctional cytokine that binds to the cell surface receptor TweakR (Fn14, TNFRSR12A). TweakR is overexpressed in many solid tumor types, including cancers of breast, pancreatic, and ovarian origin. The initial functional description of TWEAK described it as a weak inducer of apoptosis in certain cancer cell lines. TWEAK is also known to function in other diverse biological processes such as inflammation, tissue repair, angiogenesis, cell migration, and growth inhibition. The growth inhibitory activity of TWEAK observed in some cancer cell lines and the overexpression of its receptor in cancer led to investigation of targeting TweakR as a therapeutic modality. Enavatuzumab (PDL192), a humanized IgG1 mAb to TweakR, exhibits some of the same functional activities of TWEAK and is currently in clinical investigation for the treatment of solid tumors due to its significant in vitro and in vivo growth inhibitory activity in models of multiple solid tumor types. The purpose of the current study was to provide insight into the mechanism of action of enavatuzumab and to further our understanding of the biology of TweakR in tumor cell lines. A panel of 106 cancer cell lines was tested for the ability of enavatuzumab to inhibit in vitro cell growth to identify characteristics of response. Of the 106 cell lines tested, 33 cell lines had >25% growth inhibition in response to the drug. Using luciferase transcriptional reporter constructs, it was revealed that the NFκB pathway was activated in response to enavatuzumab treatment in responder cells. Both classical (p50/p65) and non-classical NFκB (p52/RelB) pathways were subsequently shown to be induced by enavatuzumab treatment using Western blot and functional ELISA in responder cell lines. Using Affymetrix™ microarrays, we observed that cell lines sensitive to enavatuzumab had a higher number of transcriptional changes compared with resistant cells and included many genes known to be regulated by NFκB. To confirm a role for NFκB in enavatuzumab-mediated signaling, we inhibited NFκB pathway induction using siRNAs to the individual NFκB subunits p50, p65, p52, and RelB as well as the upstream kinases IKKα and IKKβ. The siRNAs were able to block enavatuzumab9s growth inhibitory activity in multiple sensitive cell lines. Further confirmation of the importance of NFκB was obtained using a small molecule inhibitor of IKKα/β (IKK16) which significantly reduced the drug-induced growth inhibition. The finding that NFκB drives the growth inhibitory activity of enavatuzumab is a compelling function for a family of transcription factors that are more frequently associated with cancer growth and survival than inhibition of cell proliferation. 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 1770. doi:10.1158/1538-7445.AM2011-1770

Abstract 1772: PDL192, a humanized anti-Tweak receptor monoclonal antibody, mediates antitumor effects in primary human breast carcinoma xenografts

Background: PDL192 is a humanized IgG1 monoclonal antibody that binds the human TWEAK receptor (TweakR). TweakR, a member of the TNFR (Tumor Necrosis Factor Receptor) superfamily, is overexpressed in several human cancers including breast cancer (BC). In BC, it may also play a role in the invasive and metastatic potential of the disease (Willis et al, Mol Cancer Res 2008). In TweakR-expressing cancer cell lines or mouse xenograft models, PDL192 has a potent antitumor effect (Culp et al., CCR 2010). All these data therefore suggest that anti-TweakR targeting could be a promising new therapeutic approach for human BC patients. Material and methods: TweakR expression was assessed by IHC (immunohistochemistry) on 3 Tissue-Micro-Array (TMA) banks of BC samples (basal-like, ERBB2, and luminal A/B), and 25 primary human BC xenografts (HBCx). The cut-off of positivity was defined as at least 25% cells with membraneous or cytoplasmic staining or by a combined score of percentage of positive staining cells x intensity > 50. The in vivo antitumor effect of PDL192 was then assessed on 7 TweakR-positive models (10 mg/kg thrice a week for 3 weeks by intraperitoneal route) in which one in combination with chemotherapy as maintenance therapy, as previsouly reported (Marangoni et al., BJC 2009). Results: TMA analyses showed that TweakR was expressed in 16/37 basal like BC (43%), 23/37 ERBB2-positive BC (62%), and 38/71 luminal BC (54%). A high TweakR expression was correlated with double estrogen receptor- and Her2-positive tumors. Moreover, 13/25 xenografts have been found to be TweakR-positive (52%). Nine human BC models have been treated with PDL192, with 4 models (44%) showing a tumor growth inhibition (TGI) ranging between 59% and 91%. No correlation has been observed between TweakR expression and in vivo TGI. Moreover, when PDL192 was administered in complete remission after chemotherapy (doxorubicin + cyclophosphamide), we observed a highly significant delay of relapse greater than 2 months. Conclusions: TweakR is expressed in 77/145 human BC samples (53%). In in vivo experiments, PDL192 showed potent TGI in 4/9 models, and significantly delayed tumor relapses after chemotherapy-induced complete remission. All these data therefore support the use of anti-TWEAK receptor monoclonal antibodies in the treatment of TweakR-positive BC patients. 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 1772. doi:10.1158/1538-7445.AM2011-1772

Abstract B17: PDL192, a humanized antibody to TweakR exhibits potent antitumor activity in pancreatic cancer models

Approximately 42,000 new cases of pancreatic cancer were diagnosed in the United States in 2009, and the 5-year survival rate for this disease is exceedingly low (approximately 5%). Given the lack of therapeutic success with current standards of care, new therapies are clearly needed to treat this disease. We have generated PDL192, a humanized IgG1 antibody to TweakR (Fn14, TNFRSF12A, CD266), a cell surface protein and member of the TNF receptor superfamily. PDL192 has been shown to exhibit antitumor activity in xenograft models on a range of solid tumor types via both direct tumor cell growth inhibition and by antibody-dependent cellular cytotoxicity. In this study, we explored the activity of PDL192 in pancreatic cancer models. Approximately 60% of primary pancreatic cancers express TweakR protein at high levels, as determined by immunohistochemistry. PDL192 exhibited potent anti-tumor activity in two cell line xenograft models, Panc1 and MiaPaCa2. PDL192 also exhibited significant anti-tumor activity against 3 of 6 primary tumor xenograft models derived from pancreatic patients. PDL192 was also tested in combination with gemcitabine, the most common treatment for pancreatic cancer patients. In the Panc1 and MiaPaCa2 xenograft models, the combination of PDL192 and gemcitabine exhibited more potent activity than either agent alone. In three of five primary tumor xenograft models tested, combining PDL192 and gemcitabine also resulted in greater anti-tumor activity than either agent alone. In one of the primary tumor models, where neither PDL192 nor gemcitabine alone exhibited significant anti-tumor activity, the combination completely inhibited tumor growth. The activity of PDL192 in multiple pancreatic xenograft models using both cell lines and primary tumors is supportive of the use of PDL192 for the treatment of pancreatic cancer. PDL192 is currently being evaluated in a phase1 safety study in patients with solid tumors. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B17.