Ian Foltz

Enhancing antibody Fc heterodimer formation through electrostatic steering effects: Applications to bispecific molecules and monovalent IgG

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.

Treating Diabetes and Obesity with an FGF21-Mimetic Antibody Activating the βKlotho/FGFR1c Receptor Complex

A monoclonal antibody mimic of FGF21 exerts beneficial metabolic effects in obese monkeys. A Metabolic Mimic Losing weight typically requires exercise and a healthy diet. Managing diabetes similarly relies on diet and exercise but also includes insulin therapy. Now, both diabetes and obesity could be treated together by targeting the fibroblast growth factor 21 (FGF21) pathway. Foltz and colleagues show that an antibody mimic of FGF21 works to regulate glucose and insulin homeostasis, leading to weight loss and glucose tolerance in monkeys. The authors first engineered the FGF21-mimetic monoclonal antibody, which they termed “mimAb1.” This antibody was able to activate human and monkey FGF receptor 1c (FGFR1c)/βKlotho signaling similar to its native counterpart, FGF21. In vivo in obese cynomolgus monkeys, mimAb1 treatment led to a decrease in body weight and body mass index (BMI)—a decrease that was maintained for 9 weeks after the second round of treatment. These beneficial effects on metabolism were seen only initially with FGF21, before animals regained weight. Animals treated with mimAb1 also showed a decrease in fasting and fed plasma insulin levels, suggesting an improvement in insulin sensitivity, as well as a reduction in plasma triglyceride and glucose levels. Native FGF21 is difficult to develop as a therapeutic for diabetes and obesity; efforts to date have fallen short. mimAb1 recreates all of the beneficial metabolic effects of FGF21 as measured but is easier to manufacture, has prolonged pharmacokinetics, and has been engineered with high specificity. This mimAb1 will need additional safety and toxicity testing for translation, but early efficacy data in nonhuman primates suggest that this antibody is on its way to helping treat patients with diet-induced obesity and diabetes. Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to βKlotho with high affinity and specifically activates signaling from the βKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on βKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.

Conatumumab, a fully human agonist antibody to death receptor 5, induces apoptosis via caspase activation in multiple tumor types

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG1) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122), and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the anti-tumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Identification and Characterization of MEDI4736, an Antagonistic Anti–PD-L1 Monoclonal Antibody

A human antibody to PD-L1, engineered to eliminate Fc effector functions, which potently inhibits PD-L1 function, is in phase III clinical trials. Its characterization here provides clinicians and researchers with a basis for understanding and interpreting clinical trial results. Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti–PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti–CTLA-4, anti–PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR. Cancer Immunol Res; 3(9); 1052–62. ©2015 AACR.

Evolution and Emergence of Therapeutic Monoclonal Antibodies What Cardiologists Need to Know

The concept of using antibodies for the treatment of disease dates back to the 1890s, when Emil Adolf von Behring discovered the ability of small doses of diphtheria or tetanus toxin to produce transferable immunity between animals via serum1 (later attributed to the presence of antibodies). However, it was not until the early 1960s that structural characteristics of antibodies were described,2,3 with the following decade marking the discovery of methods for producing monoclonal antibodies (mAbs), including the hybridoma technique.4,5 In 1986, the first mAb, muromonab-CD3, was approved5 for treating steroid-resistant acute allograft rejection in renal transplant recipients. The first fully human mAb (adalimumab) was approved for treating rheumatoid arthritis in 2002.5 As of March 2012, the number of US Food and Drug Administration–approved, actively marketed therapeutic mAbs for use in oncology/hematology, immunomodulatory settings, and other miscellaneous conditions is approaching 30 (Table 1). Most of the recently approved biologics are fully human mAbs, and cancer and immunologic disorders continue to be the focus of investigational therapeutic mAbs6 (estimated at >500 across various stages of development).7 View this table: Table 1. Marketed Therapeutic Monoclonal Antibodies With Food and Drug Administration–Approved Indications* (as of December 2012) This review provides an overview of the emergence and use of mAbs as therapeutics, including a discussion of their mechanism of action, delivery, clearance, and overall safety. Key differentiating aspects between mAbs and small-molecule therapeutics are also highlighted, along with a brief summary on the use of mAbs in the cardiology arena. ### Antibody Structure and Function Antibodies, also known as immunoglobulins (Igs), are B-cell–produced molecules composed of 4 polypeptide chains (2 heavy and 2 light chains) that come together to form their characteristic Y shape (Figure 1).8 They can circulate in soluble form or can be bound to the B-cell membrane as …

A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Background: Bispecific heterodimeric antibody consisting of two different heavy chains and two different light chains requires heterodimerization of heavy chains and cognate light-heavy chain pairings. Results: Cognate light-heavy chain pairing can be achieved by an antibody engineering approach. Conclusion: Bispecific hetero-IgG antibodies can be made in mammalian cells. Significance: The technology could be used in the production of bispecific antibodies for many biotechnological applications. Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.

MEDI0639: a novel therapeutic antibody targeting Dll4 modulates endothelial cell function and angiogenesis in vivo

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand–receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell–fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650–60. ©2012 AACR.

Asymmetrical Fc engineering greatly enhances antibody-dependent cellular cytotoxicity (ADCC) effector function and stability of the modified antibodies.

Background: Co-crystal structure of Fc-FcγRIII complex revealed that Fc binds to FcγRIII asymmetrically. Results: We identified a panel of novel Fc heterodimers with enhanced ADCC activity. Conclusion: Asymmetrical Fc engineering is an efficient approach for enhancing ADCC activity and stability of engineered antibodies. Significance: The discovery could be applied in therapeutic antibodies for the treatment of cancers and infectious diseases. Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

AMG 595, an Anti-EGFRvIII Antibody–Drug Conjugate, Induces Potent Antitumor Activity against EGFRvIII-Expressing Glioblastoma

Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 μg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients. Mol Cancer Ther; 14(7); 1614–24. ©2015 AACR.