Anthony Gerard Doyle

Anti-TNFα domain antibody construct CEP-37247: Full antibody functionality at half the size.

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 – 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising VL dAb, truncated CH1, hinge, CH2 and CH3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact – CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.

A novel class of anti-IL-12p40 antibodies: potent neutralization via inhibition of IL-12-IL-12Rβ2 and IL-23-IL-23R.

While current therapeutic antibodies bind to IL-12 and IL-23 and inhibit their binding to IL-12Rβ1, we describe a novel antibody, termed 6F6, that binds to IL-12 and IL-23 and inhibits the interaction of IL-12 and IL-23 with their cognate signalling receptors IL-12Rβ2 and IL23R. This antibody does not affect the natural inhibition of the IL-12/23 pathway by the antagonists monomeric IL-12p40 and IL-12p80, which suggests that a dual antagonist system is possible. We have mapped the epitope of 6F6 to domain 3 of the p40 chain common to IL-12 and IL-23 and demonstrate that an antibody bound to this epitope is sufficient to inhibit engagement of the signalling receptors. Antibodies with this unique mechanism of inhibition are potent inhibitors of IL-12 induced IFN-γ production and IL-23 induced IL-17 production in vitro, and in an in vivo model of psoriasis, treatment with a humanized variant of this antibody, h6F6, reduced the inflammatory response, resulting in decreased epidermal hyperplasia. We believe that this new class of IL-12/23 neutralising antibodies has the potential to provide improved potency and efficacy as anti-inflammatory agents, particularly in diseases characterized by an overproduction of IL-12.

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model.

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques

Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are needed. To facilitate these efforts, we developed a gluten-sensitive (GS) rhesus macaque model to study the effects of novel therapies. Recently reported results from phase one of this project suggest that partial improvement—but not remission—of gluten-induced disease can be accomplished by 100-fold reduction of dietary gluten, i.e., 200 ppm—by replacement of conventional dietary sources of gluten with a mutant, reduced gluten (RG) barley (lys3a)-derived source. The main focus of this (phase two) study was to determine if the inflammatory effects of the residual gluten in lys3a mutant barley grain could be further reduced by oral supplementation with a prolylendopeptidase (PE). Results reveal that PE supplementation of RG barley diet induces more complete immunological, histopathological and clinical remission than RG barley diet alone. The combined effects of RG barley diet and PE supplementation resulted in a further decrease of inflammatory mediators IFN-γ and TNF secretion by peripheral lymphocytes, as well as decreased plasma anti-gliadin and anti-intestinal tissue transglutaminase (TG2) antibodies, diminished active caspase production in small intestinal mucosa, and eliminated clinical diarrhea—all comparable with a gluten-free diet induced remission. In summary, the beneficial results of a combined RG barley and PE administration in GS macaques may warrant the investigation of similar synergistic approaches.

Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity.

Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα’s TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity.

An anti-TL1A antibody for the treatment of asthma and inflammatory bowel disease

ABSTRACT TL1A is an attractive therapeutic target for the treatment of mucosal inflammation associated with inflammatory bowel disease (IBD) and asthma. Blockade of the TL1A pathway has been shown to reduce inflammatory responses while leaving baseline immunity intact, and to be beneficial in animal models of colitis and asthma. Given the therapeutic potential of blocking this pathway in IBD and asthma, we developed C03V, a human antibody that binds with high affinity to soluble and membrane-bound TL1A. In an assay measuring apoptosis induced by exogenous TL1A, C03V was 43-fold more potent than the next most potent anti-TL1A antibody analyzed. C03V also potently inhibited endogenous TL1A activity in a primary cell-based assay. This potency was linked to the C03V-binding epitope on TL1A, encompassing the residue R32. This residue is critical for the binding of TL1A to its signaling receptor DR3 but not to its decoy receptor DcR3, and explains why C03V inhibited TL1A-DR3 binding to a much greater extent than TL1A-DcR3 binding. This characteristic may be advantageous to preserve some of the homeostatic functions of DcR3, such as TL1A antagonism. In colitis models, C03V significantly ameliorated microscopic, macroscopic and clinical aspects of disease pathology, and in an asthma model it significantly reduced airways inflammation. Notable in both types of disease model was the reduction in fibrosis observed after C03V treatment. C03V has the potential to address unmet medical needs in asthma and IBD.

Beneficial Effects of Human Anti-Interleukin-15 Antibody in Gluten-Sensitive Rhesus Macaques with Celiac Disease

Overexpression of interleukin-15 (IL-15) is linked with immunopathology of several autoimmune disorders including celiac disease. Here, we utilized an anti-human IL-15 antibody 04H04 (anti-IL-15) to reverse immunopathogenesis of celiac disease. Anti-IL-15 was administered to six gluten-sensitive rhesus macaques with celiac disease characteristics including gluten-sensitive enteropathy (GSE), and the following celiac-related metrics were evaluated: morphology (villous height/crypt depth ratio) of small intestine, counts of intestinal intraepithelial lymphocytes, IFN-γ-producing CD8+ and CD4+ T cells, plasma levels of anti-gliadin and anti-intestinal tissue transglutaminase IgG antibodies, as well as peripheral effector memory (CD3+CD28−CD95+) T cells. Anti-IL-15 treatment reversed the clinically relevant disease endpoints, intraepithelial lymphocyte counts, and villous height/crypt depth ratios within jejunal biopsies to normal levels (P < 0.001). Additionally, intestinal CD8+ and CD4+ T cell IFN-γ production was reduced (P < 0.05). Extra-intestinally, anti-IL-15 treatment reduced peripheral NK cell counts (P < 0.001), but otherwise, non-NK peripheral lymphocytes including effector memory T cells and serum blood chemistry were unaffected. Overall, providing the beneficial disease-modulatory and immunomodulatory effects observed, anti-IL-15 treatment might be considered as a novel therapy to normalize intestinal lymphocyte function in celiac disease patients with GSE.

Abstract A13: Anti-CD38-attenukine: A myeloma-targeting immunocytokine containing an engineered IFNα that provides >10,000-fold enhanced tumor-specific activity compared to native IFNα.

Multiple myeloma (MM) remains an incurable disease despite recent approvals of novel therapies. Treatments which utilize alkylating agents, corticosteroids, proteasome inhibitors and immunomodulatory drugs have provided significant survival benefits; however, tumor relapse eventually occurs with the use of these agents. The cytokine, interferon-α (IFNα), has been used clinically to treat MM for over 30 years. IFNα exerts good anti-myeloma tumor activity but durable responses are rarely achieved due to dose limiting toxicities. Tumor specificity can be modestly increased by attaching IFNα to an anti-CD38 antibody, thus targeting the cytokine to MM cells. However, by introducing an attenuating mutation to the IFNα portion of this fusion protein, the activity of the cytokine is dramatically reduced on CD38 negative cells while exhibiting a similar potency to native IFNα on CD38+ MM cells. As a result, the anti-CD38-attenuated IFNα, referred to as anti-CD38-attenukine™, has over 10,000-fold greater myeloma-specificity than native IFNα in vitro. In vivo, even though the off-target activity is decreased, anti-CD38-attenukine™ has profound anti-tumor effects in murine MM and NHL xenograft models. Treatment with this fusion protein can cure mice with large (>700mm3) tumors—an effect not observed with any other MM agents tested. Here we report that in a Velcade (bortezomib) refractory, IFNα-insensitive primary MM xenograft model, the combination of Velcade with anti-CD38-attenukine™ has potent anti-tumor activity, resulting in tumour elimination and curing of mice. Our findings indicate that anti-CD38-attenukine™ may be a well tolerated, potent anti-MM treatment that may be utilized alone or in combination with Velcade. Citation Format: Sarah Pogue, Tetsuya Taura, Mingying Bi, Glen Mikesell, Yong Yun, Angela Sho, Eric Sanchez, Haiming Chen, James Berenson, Collette Behrens, Maxwell Stevens, Teresa Domagala, Maya Sokolovsky, Hussein Hallak, Moti Rosenstock, Anthony Doyle, David Wilson. Anti-CD38-attenukine: A myeloma-targeting immunocytokine containing an engineered IFNα that provides >10,000-fold enhanced tumor-specific activity compared to native IFNα. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A13.

Abstract 2660: Targeting an attenuated cytokine to tumor cells via antibody fusion results in enhanced tumor killing with significantly reduced off-target activity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Many cytokines have potential therapeutic applications and several, including interferon (IFN)α and IFNβ, have been approved for the treatment of cancer and/or autoimmune disease. While these agents are effective, they are associated with significant dose-limiting toxicities that prevent their use at levels sufficient to promote optimal therapeutic benefit. The toxicity of type I interferons, like other cytokines, is likely mediated via cognate receptor activation on cells other than those that mediate their therapeutic effects. For example, IFNα2b, which is indicated for the treatment of multiple myeloma (MM), has a narrow therapeutic index due to significant systemic toxicity. Approaches that seek to minimize dose-limiting systemic toxicity while maximizing tumor-specific cytokine exposure could greatly enhance the therapeutic index and effectiveness of type I IFNs and other cytokines. Other groups have previously shown that a moderate degree of tumor-specificity can be achieved by attaching a cytokine such as IFNα to a tumor-targeting antibody; such first generation antibody-targeted cytokines are highly active but show only moderate tumor-specificity since the cytokine can still potently stimulate its receptor on antigen-negative cells. We sought to improve the therapeutic index of antibody-targeted cytokines by mutating the cytokine portion to significantly reduce affinity for its receptor, thereby making it more dependent on antibody-based cell-targeting. Here we demonstrate that such fusion proteins, consisting of mutant or attenuated cytokines (Attenukines™) attached to tumor-targeting antibodies, are 1,000 to 100,000-fold more potent on target-positive cells compared to target-negative (normal) cells. This is shown for antibody-Attenukine™ fusion proteins based on multiple tumor antigens (CD20, CD38, CD138, HMW-MAA, HLA) and multiple attenuated mutants of IFNα, IFNβ, IL-4 and IL-6. Furthermore, we have evaluated an anti-CD38-attenuated IFNα molecule (anti-CD38-Attenukine™) in various CD38+ MM xenograft models and found that this molecule retains potent tumor-targeting activity and anti-tumor efficacy. Moreover, in non-human primates, we have confirmed that the attenuating mutation in IFNα indeed decreases non-targeted IFNα biomarker responses by greater than 100-fold. Taken together, our findings suggest that the administration of antibody-attenuated cytokine fusion proteins to cancer patients may promote robust cytokine-dependent tumor-killing while minimizing systemic toxicity. Citation Format: Sarah L. Pogue, Tetsuya Taura, Mingying Bi, Glen Mikesell, Yong Yun, Angela Sho, Collette Behrens, Maxwell Stevens, Teresa Domagala, Maya Sokolovsky, Hussein Hallak, Moti Rosenstock, Anthony Doyle, David S. Wilson. Targeting an attenuated cytokine to tumor cells via antibody fusion results in enhanced tumor killing with significantly reduced off-target activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2660. doi:10.1158/1538-7445.AM2014-2660