ABSTRACT
Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Hemorrhagic Fever, Ebola/drug therapy , Animals , Antibodies, Monoclonal/isolation & purification , Glycoproteins/immunology , Guinea Pigs , HEK293 Cells , Humans , Macaca mulatta , Male , MiceABSTRACT
Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and κ light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , Coronavirus Infections/therapy , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Coronavirus Infections/virology , Disease Models, Animal , HEK293 Cells , Humans , Mice , Middle East Respiratory Syndrome Coronavirus/immunologyABSTRACT
Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.
Subject(s)
Antibody Formation , Genes, Immunoglobulin , Alleles , Animals , B-Lymphocytes/immunology , Flow Cytometry , Humans , Mice , MutationABSTRACT
The artemin-GFRα3 signaling pathway has been implicated in various painful conditions including migraine, cold allodynia, hyperalgesia, inflammatory bone pain, and mouse knees contain GFRα3-immunoreactive nerve endings. We developed high affinity mouse (REGN1967) and human (REGN5069) GFRα3-blocking monoclonal antibodies and, following in vivo evaluations in mouse models of chronic joint pain (osteoarthritic-like and inflammatory), conducted a first-in-human phase 1 pharmacokinetics (PK) and safety trial of REGN5069 (NCT03645746) in healthy volunteers, and a phase 2 randomized placebo-controlled efficacy and safety trial of REGN5069 (NCT03956550) in patients with knee osteoarthritis (OA) pain. In three commonly used mouse models of chronic joint pain (destabilization of the medial meniscus, intra-articular monoiodoacetate, or Complete Freund's Adjuvant), REGN1967 and REGN5069 attenuated evoked behaviors including tactile allodynia and thermal hyperalgesia without discernably impacting joint pathology or inflammation, prompting us to further evaluate REGN5069 in humans. In the phase 1 study in healthy subjects, the safety profiles of single doses of REGN5069 up to 3000 mg (intravenous) or 600 mg (subcutaneous) were comparable to placebo; PK were consistent with a monoclonal antibody exhibiting target-mediated disposition. In the phase 2 study in patients with OA knee pain, two doses of REGN5069 (100 mg or 1000 mg intravenous every 4 weeks) for 8 weeks failed to achieve the 12-week primary and secondary efficacy endpoints relative to placebo. In addition to possible differences in GFRα3 biology between mice and humans, we highlight here differences in experimental parameters that could have contributed to a different profile of efficacy in mouse models versus human OA pain. Additional research is required to more fully evaluate any potential role of GFRα3 in human pain.
ABSTRACT
We evaluated interleukin-6 (IL-6) receptor-α subunit (IL-6Rα) signaling inhibition with sarilumab and tocilizumab, the association between IL-6Rα receptor occupancy (RO) and C-reactive protein (CRP), and the potential clinical relevance of any differences. For this, we measured IL-6Rα binding and signaling inhibition with sarilumab and tocilizumab in vitro, simulated soluble IL-6Rα RO over time for approved sarilumab subcutaneous (SC) and tocilizumab intravenous (IV) and SC doses, and assessed associations between calculated RO and CRP reduction, 28-joint Disease Activity Score based on CRP, and 20%/50%/70% improvement in American College of Rheumatology responses from clinical data. Sarilumab binds IL-6Rα in vitro with 15- to 22-fold higher affinity than tocilizumab, and inhibits IL-6-mediated classical and trans signaling via membrane-bound and soluble IL-6Rα. Sarilumab 200 and 150 mg SC every 2 weeks achieved >90% RO after first and second doses, respectively, maintained throughout the treatment period. At steady-state trough, RO was greater with sarilumab 200 mg (98%) and 150 mg SC every 2 weeks (94%), and tocilizumab 162 mg SC weekly (>99%) and 8 mg/kg IV every 4 weeks (99%), vs tocilizumab 162 mg SC every 2 weeks (84%) and 4 mg/kg IV every 4 weeks (60%). Higher RO was associated with greater CRP reduction and 28-joint Disease Activity Score based on CRP reduction, and more sarilumab patients achieving 20%/50%/70% improvement in American College of Rheumatology responses. The greatest reduction in CRP levels was observed with sarilumab (both doses) and tocilizumab 8 mg/kg IV every 4 weeks (reductions proportionally smaller with 4 mg/kg IV every 4 weeks). Higher IL-6Rα binding affinity translated into higher RO with sarilumab vs tocilizumab 4 mg/kg every 4 weeks or 162 mg every 2 weeks; tocilizumab required the higher dose or increased frequency to maintain the same degree of RO and CRP reduction. Higher RO was associated with clinical parameter improvements.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antirheumatic Agents/pharmacology , C-Reactive Protein/drug effects , Receptors, Interleukin-6/antagonists & inhibitors , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antirheumatic Agents/pharmacokinetics , Humans , Models, Biological , Protein BindingABSTRACT
Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.
Subject(s)
Dermatitis/etiology , Dermatitis/metabolism , Gastroenteritis/etiology , Gastroenteritis/metabolism , Receptors, Interleukin-1/metabolism , Signal Transduction , Animals , Biomarkers , Dermatitis/pathology , Disease Models, Animal , Disease Susceptibility , Gastroenteritis/pathology , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mice , Skin/metabolism , Skin/pathologyABSTRACT
Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Antibody Affinity , Antibody-Dependent Cell Cytotoxicity , Betacoronavirus/chemistry , Binding Sites, Antibody , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/immunology , COVID-19 , Cell Line , Coronavirus Infections/therapy , Cytophagocytosis , Epitopes , Humans , Immunization, Passive , Mice , Middle Aged , Models, Molecular , Neutralization Tests , Pandemics , Peptidyl-Dipeptidase A/metabolism , Protein Interaction Domains and Motifs , Receptors, Coronavirus , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Young Adult , COVID-19 SerotherapyABSTRACT
Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.
Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Bispecific/therapeutic use , CA-125 Antigen/immunology , CA-125 Antigen/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/immunology , T-Lymphocytes/metabolism , Animals , CD13 Antigens/immunology , CD13 Antigens/metabolism , Female , Flow Cytometry , Humans , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Jurkat Cells , Macaca fascicularis , Mice , Ovarian Neoplasms/metabolism , T-Lymphocytes/immunologyABSTRACT
The Notch ligand delta-like 4 (Dll4) has been identified as a promising target in tumor angiogenesis in preclinical studies, and Dll4 inhibitors have recently entered clinical trials for solid tumors, including ovarian cancers. In this study, we report the development of REGN421 (enoticumab), a fully human IgG1 monoclonal antibody that binds human Dll4 with sub-nanomolar affinity and inhibits Notch signaling. Administering REGN421 to immunodeficient mice engineered to express human Dll4 inhibited the growth of several human tumor xenografts in association with the formation of nonfunctional tumor blood vessels. In ovarian tumor xenograft models, Dll4 was expressed specifically by the tumor endothelium, and Dll4 blockade by human-specific or mouse-specific Dll4 antibodies exerted potent antitumor activity, which relied entirely on targeting Dll4 expressed by tumor stromal cells but not by the tumor cells themselves. However, Dll4 blockade reduced Notch signaling in both blood vessels and tumor cells surrounding the blood vessels, suggesting that endothelial-expressed Dll4 might induce Notch signaling in adjacent ovarian tumor cells. The antitumor effects of targeting Dll4 were augmented significantly by simultaneous inhibition of VEGF signaling, whereas this combined blockade reversed normal organ vascular changes induced by Dll4 blockade alone. Overall, our findings deepen the rationale for antibody-based strategies to target Dll4 in ovarian cancers, especially in combination with VEGF blockade.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Intercellular Signaling Peptides and Proteins/physiology , Molecular Targeted Therapy , Neoplasm Proteins/antagonists & inhibitors , Neovascularization, Pathologic/drug therapy , Ovarian Neoplasms/drug therapy , Adaptor Proteins, Signal Transducing , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacology , Calcium-Binding Proteins , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/immunology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/immunology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, SCID , Neoplasm Proteins/physiology , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/metabolism , Receptors, Notch/physiology , Signal Transduction/drug effects , Species Specificity , Stromal Cells/drug effects , Stromal Cells/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
Murine cytomegalovirus (MCMV) m137 null mutants, Deltam137A and Deltam137B, were generated by inserting a gpt cassette into a deleted region of the open reading frame. A polyclonal antiserum produced to an Escherichia coli expressed gst-m137 fusion protein was used to show that a 38 kDa polypeptide corresponding to the predicted m137 gene product was present in NIH 3T3 fibroblasts infected with wild-type MCMV but was not detected in Deltam137 infected cells. The protein did not fractionate with infected cell membranes and was not detectable in purified wild-type virions. Plaque size, plaque morphology, and viral yield did not differ significantly between Deltam137 and wild-type MCMV infected 3T3 fibroblasts. The results showed that deletion of the 38 kDa protein did not negatively effect viral growth in 3T3 fibroblasts indicating that the m137 gene product is not essential for replication in these cells. In vivo analysis revealed that two independently isolated m137 mutants showed a significant delay in time until death but ultimately killed 100% of the mice in a SCID mouse model of virulence.
Subject(s)
Herpesviridae Infections/virology , Muromegalovirus/metabolism , Viral Proteins/physiology , 3T3 Cells , Animals , Herpesviridae Infections/mortality , Mice , Mice, SCID , Muromegalovirus/genetics , Muromegalovirus/physiology , Mutagenesis , Viral Plaque Assay , Viral Proteins/genetics , Virus ReplicationABSTRACT
EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.
Subject(s)
Colorectal Neoplasms/metabolism , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , Head and Neck Neoplasms/metabolism , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Disease Models, Animal , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Humans , Mice , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-3/antagonists & inhibitors , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The preparation of alpha-methylbenzyl thioureas and their biological activity against varicella zoster virus is described. Several analogs demonstrated IC50s<0.1 microM and their SAR are discussed. These compounds represent a novel class of potent and selective nonnucleoside inhibitors of varicella zoster virus.
Subject(s)
Antiviral Agents/pharmacology , Herpesvirus 3, Human/drug effects , Thiourea/pharmacology , Antiviral Agents/chemistry , Microbial Sensitivity Tests , Structure-Activity Relationship , Thiourea/chemistryABSTRACT
A series of nonnucleoside, N-alpha-methylbenzyl-N'-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.