Susanne Mangold

T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer cells

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.

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies

BackgroundEpithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety.MethodsWe recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation.ResultsING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells.ConclusionA moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.

Antitumor activity of an EpCAM/CD3-bispecific BiTE antibody during long-term treatment of mice in the absence of T-cell anergy and sustained cytokine release.

muS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. MT110, its human-specific analog, is in a clinical phase 1 trial for treatment of patients with adenocarcinoma of the lung or gastrointestinal tract. Recent studies have shown a therapeutic window for muS110, have explored single-dose toxicity of muS110, and have found that a 1-week low-dose treatment dramatically increased the tolerability of mice to very high doses of muS110 (Cancer Immunol. Immunother. 2009;58:95–109). Here we analyzed the impact of long-term, high-dose treatment of mice with muS110 on antitumor activity and functionality of T cells. After an initial self-limiting cytokine release, the 1-week adaptation period effectively blunted further cytokine production in response to a subsequent high-dose treatment with muS110. The much-increased tolerability of mice adapted to muS110 was not because of anergy of T cells. T cells isolated from chronically muS110-treated mice fully retained their cytotoxic potential, proliferative capacity, and responsiveness to stimulation by either muS110 or anti-CD3/anti-CD28/interleukin-2 when compared with T cells from control mice. Unimpaired T-cell performance was also evident from the effective prevention of orthotopic 4T1 breast tumor outgrowth in mice treated long term with escalating doses of muS110. Finally, we show that muS110 and MT110 recognize orthologous epitopes on mouse and human EpCAM proteins, suggesting that the target-related safety profile of muS110 in mice may be predictive for MT110 in humans.

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

NKG2D is a surface receptor expressed on NK cells but also on CD8+ T cells, γδ T cells, and auto-reactive CD4+/CD28- T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 ± 1.4 x 10-11 M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after 7 days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.

Mining the human autoantibody repertoire: Isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.

Abstract 2429: Evidence of a therapeutic window with a T cell-engaging BiTE antibody based on monoclonal antibody cetuximab

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.

Abstract 2435: Validation of Cynomolgus monkeys as relevant species for safety assessment of a novel human BiTE antibody platform for cancer therapy

Nonclinical safety assessment of biopharmaceuticals for human use relies on their cross-reactivity with the respective target in other species, the absence of which precludes testing in commonly used animal models. This is the case for a first-generation of T cell-engaging BiTE antibodies, which solely cross-react with T cells of chimpanzee. BiTE antibodies are designed to transiently connect the CD3 subunit of T cell receptors on T cells with an antigen on target cells. This can very effectively activate T cells, leading to a potent redirected lysis of target cells. Here, we have generated a series of novel human anti-CD3 antibodies that cross-react with non-human primate species, including Cynomolgus monkeys (Macaca fascicularis) and marmoset (Callithrix jacchus). The variable domains of a selected anti-CD3 antibody were used to generate next-generation BiTE antibodies specific for (1) CD33, a target antigen for treatment of acute myelogenous leukemia, (2) melanoma-associated chondroitin sulfate proteoglycan (MCSP), a potential target for treatment of melanoma, and (3) epidermal growth factor receptor 1 (EGFR), a validated target for treatment of colorectal cancer. In vitro, CD33-, MCSP- and EGFR-specific BiTE antibodies showed comparable binding to CD3 on human and macaque T cells, and to CHO cells expressing CD33, MCSP and EGFR of human or macaque origin. In human and macaque cell-based assays, bispecific binding triggered redirected target cell lysis as well as conditional T cell activation, as previously described for first-generation BiTE antibodies. When continuously infused for one week to Cynomolgus monkeys, the CD33-specific BiTE antibody caused depletion of CD33-expressing peripheral blood cells at tolerated doses, and reached a maximum tolerated dose (MTD) at 120 µg/m2/day. The EGFR-specific BiTE antibody reached an MTD at 150 µg/m2/kg. In contrast, the MCSP-specific BiTE antibody was well-tolerated up to a dose of 5,000 µg/m2/day. Our primate data suggest that the tolerability and toxicity of CD33-, MCSP- and EGFR-specific BiTE antibodies were apparently determined by target cell-dependent bispecific binding, and not by monovalent binding to CD3 on macaque T cells. Our data validate the Cynomolgus monkey as a relevant species for assessing the safety and tolerability of next-generation BiTE antibodies that use a more broadly cross-reactive anti-CD3 human single-chain antibody. 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 2435.

Abstract 5339: Inhibition of breast cancer cell proliferation and unique epitope recognition by EpCAM-specific human monoclonal antibody adecatumumab

Epithelial cell adhesion molecule (EpCAM; CD326) is frequently and highly expressed on a wide variety of human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients for safety and anti-tumor activity. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. We have recombinantly produced all antibodies but murine edrecolomab in Chinese hamster ovary cells and investigated all five antibodies for their binding affinity, EpCAM epitope recognition, antibody-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC), and inhibition of breast cancer cell proliferation. ING-1 and 3622W94 bound to recombinant EpCAM with much higher affinity than adecatumumab and edrecolomab, which was largely determined by slower off-rates. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded, N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing human cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities for recombinant EpCAM. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC with human effector cells than the murine IgG2a version of the antibody. Of the five antibodies, only adecatumumab showed a significant inhibition of MCF7 breast cancer cell proliferation in the absence of complement and immune cells. A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis. Adecatumumab emerges as an anti-EpCAM antibody with unique characteristics and biological activity. 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 5339.

Abstract 2434: Novel primate-crossreactive BiTE antibodies that eliminate cancer cells expressing cMet, IGFR-1, FAP-alpha, PSCA, Endosialin, CAIX or Her2/neu

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-Hodgkin9s 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.