Monoclonal antibodies against GFRα3 are efficacious against evoked hyperalgesic and allodynic responses in mouse join pain models but, one of these, REGN5069, was not effective against pain in a randomized, placebo-controlled clinical trial in patients with osteoarthritis pain.

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.

Humanization of T cell-mediated immunity in mice.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy.

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail.

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.

Inhibition of complement pathway activation with Pozelimab, a fully human antibody to complement component C5.

Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.

A Biparatopic Antibody That Modulates MET Trafficking Exhibits Enhanced Efficacy Compared with Parental Antibodies in MET-Driven Tumor Models.

PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

A novel bispecific antibody platform to direct complement activity for efficient lysis of target cells.

Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.

Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates.

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.

Pre- and postexposure efficacy of fully human antibodies against Spike protein in a novel humanized mouse model of MERS-CoV infection.

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.

Low back pain recovery.

A wide range of treatment is available for treating low back pain, depending on what is causing the pain and how long it lasts. Finding the imbalance, treating to correct imbalance, and instruction on proper movement patterns in a safe, proactive way are paramount to our success at Physical Therapy and Wellness (PTW) Institute. Most people find that their low back pain improves within a few weeks, Chances are good that the pain will go away soon with some basic self-care. At PTW, 121 new patients in 2008 with a diagnosis of acute low back pain initiated a program with our therapists at any one of our three sites; 33% were discharged completely rehabilitated, with 63% discharged improved. Overall, the patient satisfaction with this group was nearly 99%. This indicates to our staff that although we may not get patients completely restored in the short period of time they are with us, we interact well enough to exceed their expectations, improving our reputation in our community.

Creating a business backbone. A first person recount of the planning process involved in laying the foundation of a successful rehabilitation clinic.

Opening your own outpatient therapy center will likely seem an insurmountable task at times. Broken down into the stages of mission establishment, planning and goal setting, establishing financial viability, choosing equipment, promoting your product, and, finally, hiring staff, this task will become less onerous. Remember, it is your practice. True to your vision, and just like high school, be prepared for an emotional roller-coaster ride. Done correctly, this experience will be just as thrilling and ultimately much more gratifying.

SDP1 is a peroxisome-proliferator-activated receptor gamma 2 co-activator that binds through its SCAN domain.

Peroxisome-proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, play an important role in the regulation of lipid metabolism and energy homoeostasis. In a yeast two-hybrid experiment using the zinc-finger transcription factor ZNF202 as bait, we previously identified the SCAN-domain-containing protein SDP1. SDP1 shares a high degree of amino acid sequence identity with PGC-2, a previously identified PPAR gamma 2 co-activator from the mouse. Here we show that SDP1 and PGC-2 interact with PPAR gamma 2 through their SCAN domains, even though PPAR gamma 2 does not contain a SCAN domain. Similar to PGC-2, SDP1 enhanced PPAR gamma 2-dependent gene transcription in transiently transfected cells but did not alter the affinity of PPAR gamma 2 for agonists. Although the SCAN domain was necessary for binding to PPAR gamma 2, it was not sufficient for co-activation in cells, suggesting that other features of SDP1 are responsible for transcriptional co-activation. The ability of SDP1 to interact with two different transcription factors that regulate genes involved in lipid metabolism, ZNF202 and PPAR gamma 2, suggests that SDP1 may be an important co-regulator of such genes.

An mRNA splice variant of the AFX gene with altered transcriptional activity.

Several studies indicate that FKHR and AFX, mammalian homologues of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. Here we describe the discovery of a novel AFX isoform, which we designated AFX zeta, in which the first 16 amino acids of the forkhead domain are not present. PCR analysis showed that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFX zeta induced reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, and G6Pase promoters. AFX zeta-mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression resulted in phosphorylation of AFX zeta. Furthermore, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an AMP-activated protein kinase activator, repressed AFX zeta-dependent reporter activation. Taken together, these findings suggest that AFX zeta is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway.