Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Development of a Cell-Based Assay to Assess Binding of the proNGF Prodomain to Sortilin.

Sortilin was first identified based on its activity as part of intracellular protein sorting machinery. Recently, it was discovered that sortilin also acts as a cell surface receptor for the propeptide form of nerve growth factor (proNGF), progranulin, and neurotensin. The interaction of sortilin to these neurotrophic ligands is linked to diseases of the nervous system that lead to neurodegeneration and neuropathic pain. Blocking of the interaction of sortilin to these ligands may prevent or slow the progress of these nervous system disorders. In vitro screening assays for blocking compounds or peptides are part of the standard set of tools for drug discovery. However, assays for sortilin biology are not readily available to determine if the selected blocking agent inhibits sortilin activity on the surface of cells. We have developed a sortilin specific cell based assay to identify compounds that specifically block interaction between sortilin and proNGF prodomain. The assay system records both the presence of sortilin on the cell surface and the interaction with the pro domain of NGF. Fluorescent images of the sortilin expressing cells are analyzed for the presence of pro domain of NGF. Sortilin-positive and sortilin-negative cells within one well are concomitantly and automatically analyzed. Sortilin-pro domain interaction can be blocked dose dependently by neurotensin and synthetic compounds. The assay will facilitate the discovery of entities interfering with the binding of sortilin to the NGF pro domain. This assay can be modified to screen for inhibitors of the binding of ligands to other complex cell surface receptors.

Comparing the efficacy and neuroinflammatory potential of three anti-abeta antibodies.

Immunotherapy is a promising strategy for the treatment of Alzheimer's disease (AD). Antibodies directed against Amyloid Beta (Aß) are able to successfully clear plaques and reverse cognitive deficits in mouse models. Excitement towards this approach has been tempered by high profile failures in the clinic, one key issue has been the development of inflammatory side effects in the brain (ARIAs). New antibodies are entering the clinic for Alzheimer's disease; therefore, it is important to learn all we can from the current generation. In this study, we directly compared 3 clinical candidates in the same pre-clinical model, with the same effector function, for their ability to clear plaques and induce inflammation in the brain. We produced murine versions of the antibodies: Bapineuzumab (3D6), Crenezumab (mC2) and Gantenerumab (chGantenerumab) with an IgG2a constant region. 18-month transgenic APP mice (Tg2576) were injected bilaterally into the hippocampus with 2 µg of each antibody or control. After 7 days, the mice tissue was analysed for clearance of plaques and neuroinflammation by histology and biochemical analysis. 3D6 was the best binder to plaques and in vitro, whilst mC2 bound the least strongly. This translated into 3D6 effectively clearing plaques and reducing the levels of insoluble Aß, whilst chGantenerumab and mC2 did not. 3D6 caused a significant increase in the levels of pro-inflammatory cytokines IL-1ß and TNFα, and an associated increase in microglial expression of CD11B and CD68. chGantenerumab increased pro-inflammatory cytokines and microglial activation, but minimal changes in CD68, as an indicator of phagocytosis. Injection of mC2 did not cause any significant inflammatory changes. Our results demonstrate that the ability of an antibody to clear plaques and induce inflammation is dependent on the epitope and affinity of the antibody.

The identification of AF38469: an orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin.

The identification of the novel, selective, orally bioavailable Sortilin inhibitor AF38469 is described. Structure-activity relationships and syntheses are reported, along with an X-ray crystal structure of the sortilin-AF38469 protein-inhibitor complex.

Longitudinal Imaging of the Skull Base Synchondroses Demonstrate Prevention of a Premature Ossification After Recifercept Treatment in Mouse Model of Achondroplasia.

Achondroplasia is the most common form of short-limb dwarfism. In this disorder, endochondral ossification is impaired due to gain-of-function mutation in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene. In addition to short limbs, cranial base bones are also affected leading to shortening of the skull base and to serious neurological complications associated with foramen magnum stenosis. These complications are thought to be due to the delay or premature arrest of skull base growth, caused by an accelerated ossification of the sphenooccipital (SOS) and the intraoccipital (IOS) synchondroses. Skull synchondroses consist of two opposite growth plates sharing a common reserve zone of chondrocytes. In this study, we first characterized the skull base synchondroses ossification in a mouse model of achondroplasia carrying the human G380R mutation (Fgfr3 ach/+ ). We then addressed whether Recifercept, a soluble FGFR3, could prevent premature closure of these synchondroses. Postnatal radiological observations revealed the presence of bony bridge structures in one or more synchondroses in Fgfr3 ach/+ mice as early as postnatal day 3 in the most severe cases. The presence of early ossification correlated with the severity of the disease as it was associated with an arrest of the cranial base bone growth. Histological analyses indicated changes in the synchondroses structure and matrix proteoglycan contents confirming a process of ossification. Treatment of Fgfr3 ach/+ mice with Recifercept compared with vehicle prevented premature synchondrosis ossification and the transition to bone, resulting in improved skull shape and cranium ratio. Given the impact of Recifercept on synchondrosis inactivation, it is possible that it could prevent one of the most severe complication of achondroplasia if used early enough during bone development. These data support the clinical development of Recifercept for achondroplasia, and suggests that early treatment may be required to best address impaired endochondral bone growth. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties.

INTRODUCTION: Response to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A. METHODS: MGAH22 was compared to an identical anti-HER2 mAb except for a wild type Fc domain. Antibody-dependent cell cytotoxicity (ADCC) assays were performed with HER2-expressing cancer cells as targets and human PBMC or purified NK cells as effectors. Xenograft studies were conducted in mice with wild type murine FcγRs; in mice lacking murine CD16; or in mice lacking murine CD16 but transgenic for human CD16A-158F, the low-binding variant. The latter model reproduces the differential binding between wild type and the Fc-optimized mAb for human CD16A. The JIMT-1 human breast tumor line, derived from a patient that progressed on trastuzumab therapy, was used in these studies. Single and repeat dose toxicology studies with MGAH22 administered intravenously at high dose were conducted in cynomolgus monkeys. RESULTS: The optimized Fc domain confers enhanced ADCC against all HER2-positive tumor cells tested, including cells resistant to trastuzumab's anti-proliferative activity or expressing low HER2 levels. The greatest improvement occurs with effector cells isolated from donors homozygous or heterozygous for CD16A-158F, the low-binding allele. MGAH22 demonstrates increased activity against HER2-expressing tumors in mice transgenic for human CD16A-158F. In single and repeat-dose toxicology studies in cynomolgus monkeys, a species with a HER2 expression pattern comparable to that in humans and Fcγ receptors that exhibit enhanced binding to the optimized Fc domain, MGAH22 was well tolerated at all doses tested (15-150 mg/kg) and exhibited pharmacokinetic parameters similar to that of other anti-HER2 antibodies. Induction of cytokine release by MGAH22 in vivo or in vitro was similar to that induced by the corresponding wild type mAb or trastuzumab. CONCLUSIONS: The data support the clinical development of MGAH22, which may have utility in patients with low HER2 expressing tumors or carrying the CD16A low-binding allele.

In vitro and in vivo characterization of Recifercept, a soluble fibroblast growth factor receptor 3, as treatment for achondroplasia.

Achondroplasia is a rare genetic disorder caused by mutations in the Fibroblast Growth Factor receptor 3 (FGFR3). These mutations lead to aberrant increase of inhibitory signaling in proliferating chondrocytes at the growth plate. Recifercept is a potential treatment for this disease using a decoy approach to sequester FGFR3 ligands subsequently normalizing activation of the mutated FGFR3 receptor. Recifercept binds to FGF isoforms in vitro and in cellular model systems and reduces FGFR3 signaling. In addition, in a transgenic mouse model of achondroplasia, Recifercept restores reduced body weight and long bone growth in these mice. These data suggest that Recifercept treatment could lead to clinical benefits in children treated with this molecule.

Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life.

Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma receptors.

Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkin's lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to FcgammaRIV, a unique mouse activating FcgammaR that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory FcgammaR, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158(phe) on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating FcgammaR present on the surface of tumor-infiltrating effector cell populations.

Antibody-mediated clearance of tau in primary mouse microglial cultures requires Fcγ-receptor binding and functional lysosomes.

Neurodegenerative diseases such as Alzheimer's disease are characterized by the progressive spreading and accumulation of hyper-phosphorylated tau protein in the brain. Anti-tau antibodies have been shown to reduce tau pathology in in vivo models and antibody-mediated clearance of tau exerted by microglia has been proposed as a contributing factor. By subjecting primary microglia cultured in vitro to anti-phospho-tau antibodies in complex with pathological tau, we show that microglia internalise and degrade tau in a manner that is dependent on FcγR interaction and functional lysosomes. It has recently been discussed if anti-tau antibody effector-functions are required for induction of tau clearance. Using antibodies with compromised FcγR binding and non-compromised control antibodies we show that antibody effector functions are required for induction of microglial clearance of tau. Understanding the inflammatory consequences of targeting microglia using therapeutic antibodies is important when developing these molecules for clinical use. Using RNA sequencing, we show that treatment with anti-tau antibodies increases transcription of mRNA encoding pro-inflammatory markers, but that the mRNA expression profile of antibody-treated cells differ from the profile of LPS activated microglia. We further demonstrate that microglia activation alone is not sufficient to induce significant tau clearance.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau.

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

New roles for Fc receptors in neurodegeneration-the impact on Immunotherapy for Alzheimer's Disease.

There are an estimated 18 million Alzheimer's disease (AD) sufferers worldwide and with no disease modifying treatment currently available, development of new therapies represents an enormous unmet clinical need. AD is characterized by episodic memory loss followed by severe cognitive decline and is associated with many neuropathological changes. AD is characterized by deposits of amyloid beta (Aß), neurofibrillary tangles, and neuroinflammation. Active immunization or passive immunization against Aß leads to the clearance of deposits in transgenic mice expressing human Aß. This clearance is associated with reversal of associated cognitive deficits, but these results have not translated to humans, with both active and passive immunotherapy failing to improve memory loss. One explanation for these observations is that certain anti-Aß antibodies mediate damage to the cerebral vasculature limiting the top dose and potentially reducing efficacy. Fc gamma receptors (FcγR) are a family of immunoglobulin-like receptors which bind to the Fc portion of IgG, and mediate the response of effector cells to immune complexes. Data from both mouse and human studies suggest that cross-linking FcγR by therapeutic antibodies and the subsequent pro-inflammatory response mediates the vascular side effects seen following immunotherapy. Increasing evidence is emerging that FcγR expression on CNS resident cells, including microglia and neurons, is increased during aging and functionally involved in the pathogenesis of age-related neurodegenerative diseases. Therefore, we propose that increased expression and ligation of FcγR in the CNS, either by endogenous IgG or therapeutic antibodies, has the potential to induce vascular damage and exacerbate neurodegeneration. To produce safe and effective immunotherapies for AD and other neurodegenerative diseases it will be vital to understand the role of FcγR in the healthy and diseased brain. Here we review the literature on FcγR expression, function and proposed roles in multiple age-related neurological diseases. Lessons can be learnt from therapeutic antibodies used for the treatment of cancer where antibodies have been engineered for optimal efficacy.

Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.

CD32B, the human inhibitory Fc-gamma receptor IIB, as a target for monoclonal antibody therapy of B-cell lymphoma.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory receptor for IgG, is the predominant Fc receptor (FcR) present on B cells. Immunohistochemical and expression studies have identified CD32B expression in a variety of B-cell malignancies, suggesting that CD32B is a potential immunotherapeutic target for B-cell malignancies. A high-affinity monoclonal antibody (mAb 2B6), from a novel panel of anti-human CD32B-specific mAbs, was chimerized (ch2B6) and humanized (hu2B6-3.5). Both ch2B6 and hu2B6-3.5 were capable of directing cytotoxicity by peripheral blood mononuclear cells and monocyte-derived macrophages against B-lymphoma lines in vitro. In a human B-cell lymphoma mouse xenograft model, administration of ch2B6 or hu2B6-3.5 reduced tumor growth rate and improved tumor-free survival. Both the in vitro and in vivo activities of 2B6 required an intact Fc, suggesting an FcR-mediated mechanism of action. These data support the hypothesis that CD32B is a viable target for mAb treatment of B-cell lymphoproliferative disorders.