Danling Wang

Site-specific antibody-polymer conjugates for siRNA delivery.

We describe here the development of site-specific antibody-polymer conjugates (APCs) for the selective delivery of small interference RNAs (siRNAs) to target cells. APCs were synthesized in good yields by conjugating an aminooxy-derivatized cationic block copolymer to an anti-HER2 Fab or full-length IgG by means of genetically encoded p-acetyl phenylalanine (pAcF). The APCs all showed binding affinity comparable to that of HER2 as their native counterparts and no significant cellular cytotoxicity. Mutant S202-pAcF Fab and Q389-pAcF IgG polymer conjugates specifically delivered siRNAs to HER2(+) cells and mediated potent gene silencing at both the mRNA and protein levels. However, a mutant A121-pAcF IgG polymer conjugate, despite its high binding affinity to HER2 antigen, did not induce a significant RNA interference response in HER2(+) cells, presumably due to steric interference with antigen binding and internalization. These results highlight the importance of conjugation site on the activity of antibody-polymer-based therapeutics and suggest that such chemically defined APCs may afford a useful targeted delivery platform for siRNAs or other nucleic acid-based therapies.

A Small Molecule Promotes Mitochondrial Fusion in Mammalian Cells

Mitochondria are highly dynamic cellular organelles that continuously undergo fission and fusion. This dynamic nature plays a key role in regulating mitochondrial function, and also gives mitochondria their heterogeneous morphology. Disruption of the balance between mitochondrial fusion and fission, especially a shift towards fission, contributes to a variety of human disorders, including neurodegenerative disease, metabolic disease, and ischemia. In addition, fragmented mitochondria are early signs of activation of apoptosis, and fusion of mitochondria by genetic or chemical manipulation has been shown to have an anti-apoptotic effect. Thus, the identification of small molecules that modulate mitochondrial dynamics can provide useful tools to study mitochondrial function and may ultimately lead to new therapeutics. Here, we report the identification and preliminary biological characterization of the small molecule, M1, which significantly restores the mitochondrial tubular network in response to genetically or chemically induced fragmentation. Mitochondrial fusion is a two-step process in which the outer and inner mitochondrial membranes (OMM and IMM, respectively) fuse separately, but in an ordered fashion. The core components of the mitochondrial fusion machinery are the OMM proteins, mitofusin 1 and 2 (Mfn1 and Mfn2), and the IMM protein, optic atrophy 1 (Opa1). Unlike wild-type mouse embryonic fibroblasts (WT MEFs), which mainly have interconnected tubular mitochondria, Mfn1 Knockout (KO) MEFs exhibit severely and uniformly frag-

Functional Antibody CDR3 Fusion Proteins with Enhanced Pharmacological Properties

Most mammalian antibodies have complementarity determining region (CDR) loops of 8–16 residues, while some human antibodies have longer, protruding CDR loops that play a role in virus neutralization.[1] The bovine antibody repertoire features a subgroup of antibodies with exceptionally long heavy chain complementarity determining region 3 (CDR3H) loops. The lengths of CDR3H in these bovine antibodies range from 40 to 67 amino acid residues.[2] We recently solved the X-ray crystal structure of bovine antibody BLV1H12 which has a CDR3H of 61 residues (Figure 1).[3] The crystal structure revealed a novel CDR3H structure that forms a solvent exposed 2-stranded antiparallel sheet ~20 A (7 amino acids) in length (the stalk region). This β-sheet terminates in a folded protein domain that is stabilized by three pairs of disulfide bonds (the knob region). This knob region was shown to play a critical role in recognition of a viral antigen in the case of one such bovine antibody.[3] Herein we asked whether the knob region of BLV1H12 can be substituted with other native protein and peptide ligands to create chimeric antibodies with new or enhanced properties. For example, such fusion proteins may have improved serum half-lives relative to the native polypeptide,[4] improved binding affinity or specificity due to interactions of the cognate receptor with the other bovine CDRs (or simply through bivalent binding), or improved expression and/or stability in the context of the antibody framework. To begin to explore these possibilities, we first attempted to substitute bovine granulocyte colony-stimulating factor (bGCSF) for the knob region in CDR3H of antibody BLV1H12.[5] The resulting bovine antibody-bGCSF (Ab-bGCSF) fusion protein was stably expressed in mammalian cells, exhibits potency similar to bGCSF in proliferating GCSF-dependent cells, and can significantly increase and sustain neutrophil populations for over three weeks in rodents.

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

Significance Many therapeutic proteins suffer from short plasma half-lives and, as a consequence, require frequent injections to be therapeutically effective; this in turn can adversely affect patient compliance and quality of life. In contrast, therapeutic antibodies typically have half-lives of weeks in humans. Consequently, there is considerable interest in generating functional antibodies with agonist or antagonist activities. Based on the structure of a natural bovine antibody with an ultralong, well-folded heavy-chain complementarity-determining region, we have developed a strategy for the generation of functional human antibody–hormone chimeras with biological activities comparable to native hormones and significantly enhanced pharmacological properties. This approach likely provides a general, relatively straightforward platform for generating antibody agonists and antagonists for a range of therapeutic applications. On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

Small molecule-mediated inhibition of myofibroblast transdifferentiation for the treatment of fibrosis

Significance The treatment of fibrosis remains a critically important unmet medical need, as nearly 45% of all natural deaths in the Western world are attributed to chronic fibroproliferative disease complications. Fibrosis is characterized by the excessive deposition of extracellular matrix proteins by resident fibroblast-derived myofibroblasts. From an imaging-based screen, we identified the antifungal drug itraconazole as an inhibitor of myofibroblast transdifferentiation from multiple resident fibroblast populations. A derivative of this drug was found to inhibit fibrotic disease progression in mouse models of lung, liver, and skin fibrosis, demonstrating that inhibiting differentiation to the myofibroblast cell state is a practical strategy to treat a wide range of fibrosis-related diseases. Fibrosis, a disease in which excessive amounts of connective tissue accumulate in response to physical damage and/or inflammatory insult, affects nearly every tissue in the body and can progress to a state of organ malfunction and death. A hallmark of fibrotic disease is the excessive accumulation of extracellular matrix-secreting activated myofibroblasts (MFBs) in place of functional parenchymal cells. As such, the identification of agents that selectively inhibit the transdifferentiation process leading to the formation of MFBs represents an attractive approach for the treatment of diverse fibrosis-related diseases. Herein we report the development of a high throughput image-based screen using primary hepatic stellate cells that identified the antifungal drug itraconazole (ITA) as an inhibitor of MFB cell fate in resident fibroblasts derived from multiple murine and human tissues (i.e., lung, liver, heart, and skin). Chemical optimization of ITA led to a molecule (CBR-096-4) devoid of antifungal and human cytochrome P450 inhibitory activity with excellent pharmacokinetics, safety, and efficacy in rodent models of lung, liver, and skin fibrosis. These findings may serve to provide a strategy for the safe and effective treatment of a broad range of fibrosis-related diseases.

Multifunctional Antibody Agonists Targeting Glucagon‐like Peptide‐1, Glucagon, and Glucose‐Dependent Insulinotropic Polypeptide Receptors

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), glucagon (GCG) receptor (GCGR), and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor (GIPR), are three metabolically related peptide hormone receptors. A novel approach to the generation of multifunctional antibody agonists that activate these receptors has been developed. Native or engineered peptide agonists for GLP-1R, GCGR, and GIPR were fused to the N-terminus of the heavy chain or light chain of an antibody, either alone or in pairwise combinations. The fusion proteins have similar in vitro biological activities on the cognate receptors as the corresponding peptides, but circa 100-fold longer plasma half-lives. The GLP-1R mono agonist and GLP-1R/GCGR dual agonist antibodies both exhibit potent effects on glucose control and body weight reduction in mice, with the dual agonist antibody showing enhanced activity in the latter.

Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases

Liver X receptor (LXR), a nuclear hormone receptor, is an essential regulator of immune responses. Activation of LXR‐mediated transcription by synthetic agonists, such as T0901317 and GW3965, attenuates progression of inflammatory disease in animal models. However, the adverse effects of these conventional LXR agonists in elevating liver lipids have impeded exploitation of this intriguing mechanism for chronic therapy. Here, we explore the ability of a series of sterol‐based LXR agonists to alleviate inflammatory conditions in mice without hepatotoxicity. We show that oral treatment with sterol‐based LXR agonists in mice significantly reduces dextran sulfate sodium colitis‐induced body weight loss, which is accompanied by reduced expression of inflammatory markers in the large intestine. The anti‐inflammatory property of these agonists is recapitulated in vitro in mouse lamina propria mononuclear cells, human colonic epithelial cells, and human peripheral blood mononuclear cells. In addition, treatment with LXR agonists dramatically suppresses inflammatory cytokine expression in a model of traumatic brain injury. Importantly, in both disease models, the sterol‐based agonists do not affect the liver, and the conventional agonist T0901317 results in significant liver lipid accumulation and injury. Overall, these results provide evidence for the development of sterol‐based LXR agonists as novel therapeutics for chronic inflammatory diseases.—Yu, S., Li, S., Henke, A., Muse, E. D., Cheng, B., Welzel, G., Chatterjee, A. K., Wang, D., Roland, J., Glass, C. K., Tremblay, M. Dissociated sterol‐based liver X receptor agonists as therapeutics for chronic inflammatory diseases. FASEB J. 30, 2570‐2579 (2016). www.fasebj.org

An Epitope‐Specific Respiratory Syncytial Virus Vaccine Based on an Antibody Scaffold

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope-specific RSV vaccine by grafting a neutralizing epitope (F-epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B-cell epitopes.

Targeted Delivery of an Anti-inflammatory PDE4 Inhibitor to Immune Cells via an Antibody-drug Conjugate.

Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody-drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.

Stapled, Long-Acting Glucagon-like Peptide 2 Analog with Efficacy in Dextran Sodium Sulfate Induced Mouse Colitis Models

Glucagon-like peptide 2 (GLP-2) is a hormone that has been shown to stimulate intestinal growth and attenuate intestinal inflammation. Despite being efficacious in a variety of animal models of disease, its therapeutic potential is hampered by the short half-life in vivo. We now describe a highly potent, stapled long-acting GLP-2 analog, peptide 10, that has a more than 10-fold longer half-life than teduglutide and improved intestinotrophic and anti-inflammatory effects in mouse models of DSS-induced colitis.