Steven Coats

Dual IGF-I/II-neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth.

Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation, and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). The role of the IGF-1R pathway in promoting tumor growth and survival is well documented. Overexpression of IGF-II and IR-A is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy. MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. Here, we show that MEDI-573 blocks the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF-induced signaling pathways and cell proliferation. MEDI-573 significantly inhibited the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF-1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity is mediated via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in IGF-driven tumor models, highlighting the potential utility of (18)F-FDG-PET as a noninvasive pharmacodynamic readout for evaluating the use of MEDI-573 in the clinic. Taken together, these results demonstrate that the inhibition of IGF-I and IGF-II ligands by MEDI-573 results in potent antitumor activity and offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways.

A Human Antibody–Drug Conjugate Targeting EphA2 Inhibits Tumor Growth In vivo

The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1-maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC(50) values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors.

A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy.

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA.

Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells.

Immunoglobulin E (IgE) plays a key role in allergic asthma and is a clinically validated target for monoclonal antibodies. Therapeutic anti-IgE antibodies block the interaction between IgE and the Fc epsilon (Fcε) receptor, which eliminates or minimizes the allergic phenotype but does not typically curtail the ongoing production of IgE by B cells. We generated high-affinity anti-IgE antibodies (MEDI4212) that have the potential to both neutralize soluble IgE and eliminate IgE-expressing B-cells through antibody-dependent cell-mediated cytotoxicity. MEDI4212 variants were generated that contain mutations in the Fc region of the antibody or alterations in fucosylation in order to enhance the antibodys affinity for FcγRIIIa. All MEDI4212 variants bound to human IgE with affinities comparable to the wild-type (WT) antibody. Each variant was shown to inhibit the interaction between IgE and FcεRI, which translated into potent inhibition of FcγRI-mediated function responses. Importantly, all variants bound similarly to IgE at the surface of membrane IgE expressing cells. However, MEDI4212 variants demonstrated enhanced affinity for FcγRIIIa including the polymorphic variants at position 158. The improvement in FcγRIIIa binding led to increased effector function in cell based assays using both engineered cell lines and class switched human IgE B cells. Through its superior suppression of IgE, we anticipate that effector function enhanced MEDI4212 may be able to neutralize high levels of soluble IgE and provide increased long-term benefit by eliminating the IgE expressing B cells before they differentiate and become IgE secreting plasma cells.

9th Annual European Antibody Congress, November 11–13, 2013, Geneva, Switzerland

The annual European Antibody Congress (EAC) has traditionally been the key event for updates on critical scientific advances in the antibody field, and 2013 was no exception. Organized by Terrapinn, the well-attended meeting featured presentations on considerations for developing antibodies and antibody-like therapeutics, with separate tracks for antibody-drug conjugates, naked antibodies, and multispecific antibodies or protein scaffolds. The overall focus of the EAC was current approaches to enhance the functionality of therapeutic antibodies or other targeted proteins, with the ultimate goal being improvement of the safety and efficacy of the molecules as treatments for cancer, immune-mediated disorders and other diseases. Roundtable discussion sessions gave participants opportunities to engage in group discussions with industry leaders from companies such as Genmab, Glenmark Pharmaceuticals, MedImmune, Merrimack Pharmaceuticals, and Pierre Fabre. As the 2013 EAC was co-located with the World Biosimilar Congress, participants also received an update on European Medicines Agency guidelines and thoughts on the future direction and development of biosimilar antibodies in the European Union.

Abstract 5625: In vitro and in vivo pharmacology of MEDI-565 (MT111), a novel CEA/CD3-bispecific single-chain BiTE antibody in development for the treatment of gastrointestinal adenocarcinomas

MEDI-565 (MT111) is a novel bispecific single-chain antibody of the BiTE (Bispecific T cell engager) class that transiently links carcinoembryonic antigen (CEA) on cancer cells with human CD3 on T cells. MEDI-565 specifically binds to human CEA with an affinity of 8.5 nM but not to any other member of the CEACAM family. In targeted expression studies on cryopreserved and formalin-fixed paraffin-embedded sections, MEDI-565 demonstrated membrane-localized binding specifically to epithelial cells in human cancerous tissues. The highest prevalence of MEDI-565 binding (∼90%) was in adenocarcinomas of gastrointestinal origin. In in vitro killing assays, MEDI-565 recruited T cells via the CD3 antigen (affinity of 310 nM) in a process that required concomitant binding to CEA positive tumor cells for T cell activation. As a consequence, T cells expanded, increased cell surface expression of the activation markers CD69 and CD25, and released perforin and granzymes. The release of cytotoxic granule content led to a Ca2+-dependent activation of pro-caspases and subsequent apoptosis of CEA-expressing tumor cells. Efficient target cell lysis by MEDI-565 was predominantly mediated by CD8+ T cells and occurred within a wide range of effector-to-target ratios (80:1 to 5:1) for T cells derived from various human donors. BiTE mediated killing was not affected by the presence of soluble CEA (≤5 µg/mL). The in vivo activity of MEDI-565 was investigated in subcutaneous xenograft models using immunocompromised SCID mice inoculated with mixtures of human T cells and human cancer cell lines. The antibody was eliminated from the serum with a half-life of a few hours following a single intravenous (IV) or subcutaneous (SC) injection. Despite a relatively short serum half-life, daily IV or SC bolus treatments over 5 days provided sufficient levels of exposure to inhibit the growth of CEA-positive tumors in a dose-dependent manner. Inhibition of growth was observed for tumors of different tissue origins and was dependent on the presence of human T cells and CEA expression by tumor cells. There were no MEDI-565-related in-life observations following treatment. These studies demonstrated that MEDI-565 has potent and selective anti-cancer activity in vitro and in vivo and provides evidence that MEDI-565 may be an effective monotherapy to treat CEA-expressing malignancies. 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 5625.

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

Abstract 2970: MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potent cytotoxicity of small molecule drugs and have shown to provide therapeutic options for various cancers. We report herein the discovery of a HER2-targeting ADC MEDI4276 that showed potent cell killing activity in vitro in cancer cell lines that express the HER2 receptor. The observed in vitro activity translated into in vivo tumor growth inhibition in various xenograft mouse models. MEDI4276 is a homogeneous molecule with precise control of drug loading following site specific conjugation of a cytotoxic drug. The drug in MEDI4276 is MMETA, a fully synthetic analog of the tubulysin family that showed pM potency in a panel of cancer cell lines. MMETA was conjugated to the antibody via engineered cysteines with a maleimide-bearing mc-Lys protease cleavable linker. The antibody in MEDI4276 is a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 that leads to antibody-receptor clustering following binding and thus promoting internalization, lysosomal trafficking and degradation. The combination of enhanced internalization and potent cytotoxic drug allows for this ADC to kill tumor cell populations with a broader range of HER2 expression. Preclinical studies showed that MEDI4276 induced tumor regression in HER2-positive tumor models that had developed acquired resistance to T-DM1 and in a number of models with lower HER2 expression that are refractory to T-DM1 treatment. Overall, our findings underscore the potential application of MEDI4276 to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies. MEDI4276 is currently being investigated in a Phase I clinical trial. Citation Format: John Li, Dorin Toader, Samuel R. Perry, Vanessa Muniz-Medina, Leslie Wetzel, Marlon C. Rebelatto, Mary Jane Masson Hinrichs, Ryan Fleming, Binyam Bezabeh, Pamela Thompson, Nazzareno Dimasi, Brandon Lam, Xian-Qing Yu, Changshou Gao, Rakesh Dixit, Steven Coats, Jane Osbourn, Herren Wu. MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies. [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 2970.

Abstract LB-295: MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies

MEDI7247 is a first in class ADC consisting of a human anti-ASCT2 monoclonal antibody site specifically conjugated to DNA cross-linking pyrrolobenzodiazepine (PBD) dimers. ASCT2 (SLC1A5) is a multi-pass, Na+-dependent neutral amino acid transporter that mediates the uptake of amino acids required for tumor growth and progression. ASCT2 is highly overexpressed in many hematologic cancers, most notably Multiple Myeloma (MM - 100% positive), Acute Myeloid Leukemia (AML - 100% positive) and Diffuse Large B cell lymphoma (DLBCL - 95% positive). ASCT2 expression is low in normal tissues. MEDI7247 (Q1Wx4) demonstrated a significant survival advantage in 3 disseminated AML cell line models, TF1α(ASCT2-High), MOLM-13(ASCT2-low) and M.V.411(ASCT2-High), when compared to the untreated control at the lowest dose levels examined: 0.05, 0.1 and 0.1 mg/kg, respectively. Further exemplifying the activity of MEDI7247, both the TF1α and MOLM-13 models did not reach 50% survival by the end of the study, with 80% survival at >200 days for TF1α and 70% survival at >180 days for MOLM-13. Similarly, a single dose of MEDI7247 in the TF1α model resulted in a 60% survival at >200 days at 0.05 mg/kg. MEDI7247 was also tested in a disseminated AML PDX(ASCT2-low) model at 0.05, 0.1 and 0.4 mg/kg. A significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by >80 days. MEDI7247 activity was further confirmed by monitoring peripheral blood CD33+ve cells, which initially receded, with the timing of reappearance preempting survival. Multiple Myeloma is another indication that exhibits a high level of ASCT2 expression. MEDI7247 (Q1Wx4) efficacy was examined in 3 disseminated MM cell line models, NCI-H929(ASCT2-High), MM.1S(ASCT2-medium) and OPM2(ASCT2-Medium), with a significant improvement in survival from control at the lowest dose levels examined: 0.1, 0.1 and 0.05 mg/kg, respectively. The activity of MEDI7247 (Q1Wx4) was also examined in the subcutaneous DLBCL model KARPAS 422(ASCT2-High). Tumor regressions were observed at all dose levels tested (0.1, 0.2, 0.3 and 0.4 mg/kg), with the higher two dose levels resulting in complete tumor regression without regrowth beyond 150 days. Additionally, MEDI7247 (Q1Wx4) is efficacious against the disseminated 697(ASCT2-Low) (Acute Lymphoblastic Leukemia - ALL) and RAJI(ASCT2-High) (Burkitt9s lymphoma) models. A significant survival advantage was seen in both tumor models at the lowest dose examined of 0.05 mg/kg. In conclusion, MEDI7247 demonstrates antitumor efficacy across all tumor indications tested and varying levels of ASCT2 expression. These data support the use of MEDI7247 in ASCT2 positive hematological malignancies. MEDI7247 is currently in Phase 1 clinical trials. Citation Format: Noel R. Monks, Kevin P. Schifferli, Ravinder Tammali, M. Jack Borrok, Steven R. Coats, Ronald Herbst, David A. Tice, Nabendu Pore. MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-295.