PDF file - 83KB, Supplementary Figure S1. Characteristics of purified AMG 330.

Abstract Bispecific T cell engager (BiTE®) antibody constructs redirect T cells to induce lysis of tumor cells. The anti-CD19/CD3 BiTE® Blincyto® can deplete target cells in both the blood and tissue compartment in B cell malignancies, suggesting that the BiTE® mechanism of action will also be effective against solid tumors. The tumor differentiation antigen mesothelin (MSLN) is an attractive target for the BiTE® approach. MSLN is highly expressed in >80% of ovarian and pancreatic tumors and mesothelioma. Expression of MSLN in normal tissues appears restricted to mesothelial cell surfaces such as the pleural, pericardial, and peritoneal layer. Here, we report the preclinical characterization of an anti-MSLN/CD3 BiTE® antibody construct with extended half-life. The BiTE® antibody construct binds MSLN and CD3 with low nM affinity and has low pM cytotoxic activity against MSLN-positive ovarian, pancreatic and lung cancer cell lines in vitro. Activity of the anti-MSLN/CD3 BiTE® antibody construct is observed at low effector to target cell ratios, and in cancer cells that express low levels of MSLN. This BiTE® antibody construct is also cytotoxic to cell lines that are resistant to chemotherapy. In mice, significant tumor growth inhibition of an established ovarian tumor xenograft model was achieved by IV dosing of the anti-MSLN/CD3 BiTE® antibody construct every 5 days. Pharmacokinetic evaluation of the anti-MSLN/CD3 BiTE® antibody construct in non-human primates demonstrated a half-life of 4-10 days. The potency and specificity of the anti-MSLN/CD3 BiTE® antibody construct, together with projected ability to dose once a week IV in humans, supports development of this BiTE® for treatment of MSLN-positive tumors. Citation Format: Alexander Sternjak, Fei Lee, Joachim Wahl, Dan Rock, Oliver Thomas, Sabine Stienen, Ralf Lutterbuese, Patrick Hoffmann, Tobias Raum, Peter Kufer, Benno Rattel, Angela Coxon, Matthias Friedrich, Julie Bailis. Preclinical evaluation of a BiTE® antibody construct with extended half-life that targets the tumor differentiation marker mesothelin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3630. doi:10.1158/1538-7445.AM2017-3630

T cell engaging bispecific antibody constructs (BiTE®), such as blinatumomab which targets CD19-positive cells, have shown great promise for treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain Fv (scFv) that binds CD19 and a scFv that targets the T cell CD3 protein. The molecular weight of this “canonical” BiTE® is ~ 55 kDa, making it susceptible to kidney-mediated clearance and resulting in a short serum half-life (~ 4 hours). To maintain effective serum concentrations, canonical BiTE® antibody constructs must be administered by continuous IV (cIV) infusion. While there are many advantages associated with cIV administration (e.g., safety and uniform PK profile), patient convenience could be enhanced if the BiTE® antibody construct were compatible with once-weekly administration. To achieve this, the serum half-life of the BiTE® antibody construct would need to be extended. A canonical BiTE® targeting CD33 (AMG 330) is currently being evaluated in a phase I clinical trial. Like blinatumomab, AMG 330 is dosed cIV. To extend the serum half-life of AMG 330 and enable once-weekly dosing, several approaches were evaluated including fusion of AMG 330 to human albumin and Fc-containing moieties. Each of these half-life extended (HLE) constructs was evaluated in vitro, in mouse xenograft models and in non-human primates. In vitro assays evaluated 1) binding to both human and cynomolgus CD33 and CD3 proteins, and 2) cytotoxicity using human and cynomolgus target and effector cells. In each of these assays the canonical and HLE BiTE® antibody constructs demonstrated similar activity: single-digit nM binding and single digit pM cytotoxicity. Canonical and HLE BiTE® antibody constructs were subsequently evaluated in an orthotopic mouse model in which MOLM13 cells were administered IV and activated human T cells were administered IP two days later. The Fc-based HLE BiTE® antibody constructs provided a similar survival advantage when administered Q4D or Q5D as the canonical BiTE® when administered QD. However, the albumin fusion–based HLE BiTE® was less efficacious when administered Q4D than the QD- administered canonical BiTE®. Lastly, the PK/PD relationship was evaluated for each of the constructs in non-human primates. The serum half-lives varied from 6 hours for the canonical BiTE® to 44-167 hours for the HLE BiTE® antibody constructs. Each of the HLE BiTE® antibody constructs showed on-target depletion of CD33-positive monocytes and neutrophils in the blood and depletion of CD33-positive cells in the bone marrow. These data demonstrate that half-life extended BiTE® antibody constructs can be generated that retain comparable in vitro and in vivo activity as a canonical BiTE® and achieve a serum half-life compatible with once weekly dosing. Citation Format: Tara L. Arvedson, Mercedesz Balazs, Pamela Bogner, Kurt Black, Kevin Graham, Anja Henn, Matthias Friedrich, Patrick Hoffmann, Roman Kischel, Peter Kufer, Ralf Lutterbuese, Markus Muenz, Tobias Raum, Benno Rattel, Karen Rex, Dan Rock, Oliver Thomas, Joachim Wahl, Andreas Wolf, Angela Coxon. Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 55. doi:10.1158/1538-7445.AM2017-55

Abstract BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model. Citation Format: Olivier Nolan-Stevaux, Flordeliza Fajardo, Lily Liu, Suzanne Coberly, Patricia McElroy, Aaron Nazarian, Franziska Bott, Elisabeth Nahrwold, Tobias Raum, Roman Kischel, Ralf Lutterbuese, Patrick Hoffmann, Peter Kufer. Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 585.

Abstract There is high demand for novel therapeutic options for patients with acute myelogenous leukemia (AML). One possible approach is the bispecific T-cell–engaging (BiTE, a registered trademark of Amgen) antibody AMG 330 with dual specificity for CD3 and the sialic acid–binding lectin CD33 (SIGLEC-3), which is frequently expressed on the surface of AML blasts and leukemic stem cells. AMG 330 binds with low nanomolar affinity to CD33 and CD3ϵ of both human and cynomolgus monkey origin. Eleven human AML cell lines expressing between 14,400 and 56,700 CD33 molecules per cell were all potently lysed with EC50 values ranging between 0.4 pmol/L and 3 pmol/L (18–149 pg/mL) by previously resting, AMG 330–redirected T cells. Complete lysis was achieved after 40 hours of incubation. In the presence of AML cells, AMG 330 specifically induced expression of CD69 and CD25 as well as release of IFN-γ, TNF, interleukin (IL)-2, IL-10, and IL-6. Ex vivo, AMG 330 mediated autologous depletion of CD33-positive cells from cynomolgous monkey bone marrow aspirates. Soluble CD33 at concentrations found in bone marrow of patients with AML did not significantly affect activities of AMG 330. Neoexpression of CD33 on newly activated T cells was negligible as it was limited to 6% of T cells in only three out of ten human donors tested. Daily intravenous administration with as low as 0.002 mg/kg AMG 330 significantly prolonged survival of immunodeficient mice adoptively transferred with human MOLM-13 AML cells and human T cells. AMG 330 warrants further development as a potential therapy for AML. Mol Cancer Ther; 13(6); 1549–57. ©2014 AACR.

For treatment of patients with prostate cancer (PCa), we developed a novel T cell-engaging (BiTE) antibody designated AMG 212 or BAY2010112 that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. AMG 212/BAY2010112 induced target cell-dependent activation and cytokine release of T cells, and efficiently redirected T cells for lysis of target cells. In addition to Chinese hamster ovary cells stably expressing human or cynomolgus monkey PSMA, T cells redirected by AMG 212/BAY2010112 also lysed human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC-3-huPSMA, and LnCaP at half maximal BiTE concentrations between 0.1 and 4 ng/mL (1.8-72 pmol/L). No lysis of PSMA-negative human PCa cell lines PC-3 and DU145 was observed. The subcutaneous (s.c.) formation of tumors from PC-3-huPSMA cells in NOD/SCID mice was significantly prevented by once daily intravenous (i.v.) injection of AMG 212/BAY2010112 at a dose level as low as 0.005 mg/kg/d. Rapid tumor shrinkage with complete remissions were observed in NOD/SCID mice bearing established s.c. 22Rv1 xenografts after repeated daily treatment with AMG 212/BAY2010112 by either the i.v. or s.c. route. Of note, 22Rv1 tumors were grown in the absence of human T cells followed by intraperitoneal injection of T cells 3 days before BiTE treatment. No effects on tumor growth were observed in the absence of human T cells or AMG 212/BAY2010112. On the basis of these preclinical results, AMG 212/BAY2010112 appears as a promising new BiTE antibody for the treatment of patients with PSMA-expressing PCa.

We investigate thin films of diindenoperylene deposited on Au(100) single crystals by using real time in situlow energy electron microscopy (LEEM) in synergy with X-ray photoelectron spectroscopy (XPS), and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. We find evidence for Stranski–Krastanov growth, with island nucleation prevalently at step bunches, and a lower probability of island nucleation at terraces. We also directly observe the effect of the Ehrlich–Schwoebel barrier at the substrate and in the film morphology, in particular inside the islands that show a non-homogeneous thickness due to a rough front growth, because of the additional energy required for the diffusing molecules to surmount a downward step.

Abstract Prostate-specific membrane antigen (PSMA) has been frequently selected as target antigen for antibody-based therapy of prostate cancer. Here, we recombinantly constructed a PSMA/CD3-bispecific BiTE antibody, called MT112/BAY 2010112. The BiTE antibody was produced in Chinese hamster ovary cells as a secreted protein of 55 kDa, showing high serum and thermal stability. MT112/BAY 2010112, purified as homogenous monomeric protein, bound at low nanomolar concentrations to defined epitopes on PSMA and CD3 antigens of human and macaque origin, respectively. In cell culture studies bispecific binding of MT112/BAY 2010112 selectively redirected human T cells against several PSMA-positive human prostate cancer cell lines as well as PSMA cDNA-transfected cell lines and potently induced specific target cell lysis with EC50 values ranging from 1 to 50 pM using non-stimulated human peripheral blood mononuclear cells as effector cells. EC50 values correlated with the number of PSMA molecules on the cell surface ranging from 37,000-500,000 molecules per cell among the cell lines analyzed. The anti-tumor activity of MT112/BAY 2010112 was assessed in several SCID mouse models bearing subcutaneous xenografts derived from the human prostate cancer cell lines LNCaP, PC3 and 22Rv1. The BiTE antibody completely inhibited growth of tumors at doses as low as 0.005 mg/kg administered daily intravenously when human T cells and tumor cells were co-inoculated. Transient regression of tumors were observed in a model where human T cells were adoptively transferred into the peritoneal space of mice with subcutaneously established tumors. These data suggest that the PSMA/CD3-bispecific antibody MT112/BAY 2010112 has high therapeutic potential for treatment of prostate cancer. Its cross-reactivity with human and macaque PSMA and CD3 antigens will greatly facilitate assessment of pharmacology, pharmacokinetics and safety during further pre-clinical development. 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 4561. doi:10.1158/1538-7445.AM2011-4561

In collaborative environment, providing flexible interoperation between heterogeneous knowledge-based software applications is critical for efficiency reasons, particularly in the context of unanticipated business collaborations. It is an objective pursued by ongoing research efforts on semantic interoperability: one expects that describing information within ontologies and reconciling these ontologies is sufficient to reach seamless information exchange. However, limits of ontology model and development result in lack of reliability of agreement between ontologies, thus reconciliation of ontologies is often pragmatically inconsistent. This paper proposes a methodology to determine what is contextual information, how to model and use it. Then, this methodology is applied to the reconciliation of ontologies, in the situation of unanticipated collaborations across organizations and between collaborators. The outcome is a context-based system that provides an evaluation of the pertinence of data associated with a concept, based on three distinct kinds of contextual information: (1) user’s domains and tasks, to personalize the search interface according to user’s needs; (2) perspectives used to annotate ontology concepts, as means for disambiguation of the pragmatic meaning of concepts; (3) the task for which the data is intended to, as a way to evaluate data relevance to fill the interoperability need.

Abstract BiTE antibodies are designed to transiently connect target cells with T cells of patients for induction of potent redirected lysis of cancer cells. Clinical proof-of-concept has been obtained with CD19/CD3-bispecific BiTE antibody blinatumomab (MT103), which showed a high response rate in patients with relapsed/refractory non-Hodgkin's lymphoma and acute B-lymphocytic leukemia. An EpCAM/CD3-bispecific BiTE antibody called MT110 currently is in a dose-escalating phase 1 study in patients with lung or gastrointestinal cancers, and BiTE antibodies targeting carcinoembryonal antigen (CEA), EGFR, CD33 or melanoma chondroitin sulfate proteoglycan (MCSP) are in earlier stages of development. Here, we have generated a series of novel BiTE antibodies for cancer therapy that are based on our novel BiTE antibody platform, which is human in sequence and cross-reactive between human and non-human primate antigens. One set of target antigens selected for new BiTE generation is also targeted by conventional monoclonal antibodies that are in various stages of clinical development. These are mesenchymal-epithelial transition factor (cMet), insulin-like growth factor receptor type I (IGFR-1), prostate-specific stem cell antigen (PSCA), carboanhydrase type IX (CAIX) and Her2/neu. These targets are all expressed by cancer cells of solid tumors, and some also by their so-called cancer stem cells. By targeting fibroblast activating protein alpha (FAP-alpha), a first BiTE antibody was generated that not only targets sarcoma cells but also stromal fibroblasts, which constitute the stromal tissue of many human cancers. By targeting endosialin (CD248), a first BiTE antibody was generated targeting cancer blood vasculature. We show that various single-chain antibodies specific for the seven target antigens cMet, IGFR-1, FAP-alpha, PSCA, CAIX, Her2/neu and endosialin could in each case be used to construct novel BiTE antibodies. All BiTE antibodies showed bispecific binding, cross-reactivity between human and Cynomolgus monkey antigens, and, most importantly, potent redirected lysis of CHO cells expressing the respective target antigen. This shows that new T cell-engaging BiTE antibodies can be made specific for a great variety of potential tumor-associated and also for stromal antigens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2434.

Abstract Treatment with chimeric monoclonal antibody cetuximab has become the standard of care for patients with advanced colorectal cancer (CRC). Cetuximab and other EGFR-specific monoclonal antibodies predominantly inhibit cancer growth by interfering with receptor signalling. Recent analyses have shown that CRC patients with mutated KRAS and BRAF oncogenes do not profit from treatment with such antibodies. Here we have used the binding domains of cetuximab to construct a BiTE antibody that can directly engage and activate T cells by transiently connecting T cells via the CD3 subunit with EGFR-expressing target cells. The cetuximab-based BiTE antibody (C-BiTE) showed potent redirected lysis of KRAS- and BRAF-mutated human CRC lines HCT116 and HT-29, respectively, by unstimulated peripheral human T cells at half maximal concentrations of 10-40 pg/ml (ca. 0.2-0.8 picomolar). The BiTE antibody also completely prevented the outgrowth of tumors from HCT116 and HT-29 xenografts in mice at doses as low as 0.005 mg/kg when given daily i.v. for 10 days. The monoclonal antibody cetuximab was not effective in these models even at 50 mg/kg given twice weekly for 4 weeks. As a prerequisite for nonclinical safety assessment of C-BiTE in non-human primates, crossreactive binding of C-BiTE to CD3 and EGFR antigens of human and Cynomolgus monkey (Macaca fascicularis) origin was demonstrated. This binding translated into a comparable potency of redirected lysis of human EGFR-expressing cancer cells by stimulated Cynomolgus and human PBMC. Continuous i.v. infusion of 6.2 or 12.4 µg/kg C-BiTE to Cynomolgus monkeys for 3 weeks was well tolerated and led to relatively constant serum concentrations of C-BiTE between 1 and 10 ng/ml. In vitro, these serum levels would support a >90% lysis of EGFR-overexpressing CRC cells within 24 h by both Cynomolgus and human effector cells. Of note, no skin toxicity was observed during the 3-week exposure to C-BiTE. The highest doses of 31 and 154 µg/kg caused lethal damage to EGFR-expressing cells in vital organs. Inflammatory cell infiltration into the EGFR-positive tissues was observed at all dose levels but its incidence and severity showed no correlation with clinical signs. Our data suggest that C-BiTE can be safely administered to a relevant non-human primate species at dose levels similar to those of CD19-specific BiTE antibody blinatumumab, which has shown partial and complete responses in lymphoma patients. We anticipate that C-BiTE has a therapeutic window in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2429.

Epidermal growth factor receptor (EGFR)-specific monoclonal antibodies predominantly inhibit colorectal cancer (CRC) growth by interfering with receptor signaling. Recent analyses have shown that patients with CRC with mutated KRAS and BRAF oncogenes do not profit from treatment with such antibodies. Here we have used the binding domains of cetuximab and pantitumumab for constructing T cell-engaging BiTE antibodies. Both EGFR-specific BiTE antibodies mediated potent redirected lysis of KRAS- and BRAF-mutated CRC lines by human T cells at subpicomolar concentrations. The cetuximab-based BiTE antibody also prevented at very low doses growth of tumors from KRAS- and BRAF-mutated human CRC xenografts, whereas cetuximab was not effective. In nonhuman primates, no significant adverse events were observed during treatment for 3 wk at BiTE serum concentrations inducing, within 1 d, complete lysis of EGFR-overexpressing cancer cells. EGFR-specific BiTE antibodies may have potential to treat CRC that does not respond to conventional antibodies.