RESUMEN
Repulsive guidance molecule A (RGMa) is an inhibitor of neuronal growth and survival which is upregulated in the damaged central nervous system following acute spinal cord injury (SCI), traumatic brain injury, acute ischemic stroke (AIS), and other neuropathological conditions. Neutralization of RGMa is neuroprotective and promotes neuroplasticity in several preclinical models of neurodegeneration and injury including multiple sclerosis, AIS, and SCI. Given the limitations of current treatments for AIS due to narrow time windows to intervention (TTI), and restrictive patient selection criteria, there is significant unmet need for therapeutic agents that enable tissue survival and repair following acute ischemic damage for a broader population of stroke patients. In this preclinical study, we evaluated whether elezanumab, a human anti-RGMa monoclonal antibody, could improve neuromotor function and modulate neuroinflammatory cell activation following AIS with delayed intervention times up to 24 h using a rabbit embolic permanent middle cerebral artery occlusion model (pMCAO). In two replicate 28-day pMCAO studies, weekly intravenous infusions of elezanumab, over a range of doses and TTIs of 6 and 24 h after stroke, significantly improved neuromotor function in both pMCAO studies when first administered 6 h after stroke. All elezanumab treatment groups, including the 24 h TTI group, had significantly less neuroinflammation as assessed by microglial and astrocyte activation. The novel mechanism of action and potential for expanding TTI in human AIS make elezanumab distinct from current acute reperfusion therapies, and support evaluation in clinical trials of acute CNS damage to determine optimal dose and TTI in humans. A: Ramified/resting astrocytes and microglia in a normal, uninjured rabbit brain. B: Rabbit pMCAO brain illustrating lesion on right side of brain (red), surrounded by penumbra (pink) during acute phase post stroke, with minimal injury to left brain hemisphere. Penumbra characterized by activated astrocytes and microglia (region in crosshair within circle), with upregulation of free and bound RGMa. C: Elezanumab binds to both free and bound RGMa, preventing full activation of astrocytes and microglia. D: Elezanumab is efficacious in rabbit pMCAO with a 4 × larger TTI window vs. tPA (6 vs. 1.5 h, respectively). In human AIS, tPA is approved for a TTI of 3-4.5 h. Elezanumab is currently being evaluated in a clinical Ph2 study of AIS to determine the optimal dose and TTI (NCT04309474).
RESUMEN
Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Proteínas Ligadas a GPI , Regeneración Nerviosa , Proteínas del Tejido Nervioso , Neuroprotección , Traumatismos del Nervio Óptico , Nervio Óptico , Neuritis Óptica , Recuperación de la Función , Retina , Animales , Ratones , Cuprizona/toxicidad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/fisiopatología , Proteínas Ligadas a GPI/antagonistas & inhibidores , Inhibidores de la Monoaminooxidasa/toxicidad , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuroprotección/efectos de los fármacos , Nervio Óptico/efectos de los fármacos , Nervio Óptico/fisiología , Traumatismos del Nervio Óptico/fisiopatología , Neuritis Óptica/fisiopatología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Retina/efectos de los fármacos , Resonancia por Plasmón de SuperficieRESUMEN
Traumatic spinal cord injury (SCI) causes a cascade of degenerative events including cell death, axonal damage, and the upregulation of inhibitory molecules which prevent regeneration and limit recovery. Repulsive guidance molecule A (RGMa) is a potent neurite growth inhibitor in the central nervous system, exerting its repulsive activity by binding the Neogenin receptor. Here, we show for the first time that inhibitory RGMa is markedly upregulated in multiple cell types after clinically relevant impact-compression SCI in rats, and importantly, also in the injured human spinal cord. To neutralize inhibitory RGMa, clinically relevant human monoclonal antibodies were systemically administered after acute SCI, and were detected in serum, cerebrospinal fluid, and in the injured tissue. Rats treated with RGMa blocking antibodies showed significantly improved recovery of motor function and gait. Furthermore, RGMa blocking antibodies promoted neuronal survival, and enhanced the plasticity of descending serotonergic pathways and corticospinal tract axonal regeneration. RGMa antibody also attenuated neuropathic pain responses, which was associated with fewer activated microglia and reduced CGRP expression in the dorsal horn caudal to the lesion. These results show the therapeutic potential of the first human RGMa antibody for SCI and uncovers a new role for the RGMa/Neogenin pathway on neuropathic pain.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Neuralgia/terapia , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Células Cultivadas , Femenino , Proteínas Ligadas a GPI , Humanos , Ratones , Plasticidad Neuronal , Ratas , Ratas WistarRESUMEN
Systemic lupus erythematosus (lupus) is characterized by autoantibody-mediated organ injury. Follicular Th (Tfh) cells orchestrate physiological germinal center (GC) B cell responses, whereas in lupus they promote aberrant GC responses with autoreactive memory B cell development and plasma cell-derived autoantibody production. IL-21, a Tfh cell-derived cytokine, provides instructional cues for GC B cell maturation, with disruption of IL-21 signaling representing a potential therapeutic strategy for autoantibody-driven diseases such as systemic lupus erythematosus. We used blockade of IL-21 to dissect the mechanisms by which this cytokine promotes autoimmunity in murine lupus. Treatment of lupus-prone B6.Sle1.Yaa mice with an anti-IL-21 blocking Ab reduced titers of autoantibodies, delayed progression of glomerulonephritis and diminished renal-infiltrating Tfh and Th1 cells, and improved overall survival. Therapy inhibited excessive accumulation of Tfh cells coexpressing IL-21 and IFN-γ, and suppressed their production of the latter cytokine, albeit while not affecting their frequency. Anti-IL-21 treatment also led to a reduction in GC B cells, CD138hi plasmablasts, IFN-γ-dependent IgG2c production, and autoantibodies, indicating that Tfh cell-derived IL-21 is critical for pathological B cell cues in lupus. Normalization of GC responses was, in part, caused by uncoupling of Tfh-B cell interactions, as evidenced by reduced expression of CD40L on Tfh cells and reduced B cell proliferation in treated mice. Our work provides mechanistic insight into the contribution of IL-21 to the pathogenesis of murine lupus, while revealing the importance of T-B cellular cross-talk in mediating autoimmunity, demonstrating that its interruption impacts both cell types leading to disease amelioration.
Asunto(s)
Linfocitos B/inmunología , Interleucinas/inmunología , Lupus Eritematoso Sistémico/inmunología , Receptor Cross-Talk/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Autoinmunidad/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Masculino , Ratones , Ratones MutantesRESUMEN
Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity.
Asunto(s)
Anticuerpos Monoclonales/química , Ácido Cítrico/química , Aminas/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Mapeo Peptídico , Péptidos/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-frame intein gene. A single mRNA and subsequent polypeptide are produced upon transient and stable transfection into HEK293 and CHO cells, respectively. Heavy and light chains are separated by the autocatalytic action of the intein and antibody processing proceeds to produce active, secreted antibody. Here, we report advances in sORF technology toward establishment of a viable manufacturing platform for therapeutic antibodies in CHO cells. Increasing expression levels and improving antibody processing by intein and signal peptide selection are discussed.
Asunto(s)
Expresión Génica , Vectores Genéticos/genética , Inteínas , Sistemas de Lectura Abierta , Anticuerpos de Cadena Única , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/genéticaRESUMEN
We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method.
Asunto(s)
Anticuerpos/metabolismo , Expresión Génica , Sistemas de Lectura Abierta , Poliproteínas/metabolismo , Ingeniería de Proteínas/métodos , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Línea Celular , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Inteínas , Datos de Secuencia Molecular , Poliproteínas/química , Poliproteínas/genética , Pyrococcus horikoshii/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Preclinical animal studies have shown that Ab12.6, an agonistic human Ab targeting the erythropoietin receptor (EPOR), exhibits several potential dosing and safety features that make it an attractive clinical candidate for the treatment of anemia. Ab12.6 was derived by yeast display affinity maturation of parental Ab12, a strategy initially intended to improve off-rate and affinity for EPOR, thereby enhancing erythropoietic activity. Analysis of full-length IgGs derived from yeast clones identified sequences within Ab12 CDRH2 that independently influenced both affinity and potency. The Ab12.6 derivative displayed improved in vitro potency and in vivo efficacy, although its binding affinity to the EPOR was lower than that of the parent Ab12. Additional Ab12 derivatives also exhibited an inverse correlation between affinity and potency. These results suggest that for this class of agonistic Abs, faster off-rates may permit continuous receptor stimulation and more efficient erythropoiesis.
Asunto(s)
Anticuerpos/inmunología , Afinidad de Anticuerpos , Eritropoyesis , Eritropoyetina/metabolismo , Receptores de Eritropoyetina/agonistas , Anticuerpos/metabolismo , Eritropoyetina/inmunología , Humanos , Imitación Molecular , Receptores de Eritropoyetina/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.