Efficacy Of mAbs Research Articles

Secretogranin III as a novel target for the therapy of choroidal neovascularization

Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV.

Development of Therapeutic Antibody Technology And Recent Perspectives For Leukemia-Lymphoma Treatment

Advancement of monoclonal antibodies (Mabs) to be accepted as a therapy has long history. This article describes the dramatic developmental trend of therapeutic antibody technology and their significant role for treatment of leukemia-lymphoma at the current time. The therapeutic Mab technology development from murine (mouse origin) to fully human resulted in low immunogenicity and high quality Mabs. Chemotherapy is standard care for leukemialymphoma treatments. However, it is associated with high toxicities (side effects), painful and relatively ineffective against certain leukemia-lymphoma subtypes. The remarkable contribution of Von Behring, Paul Ehrlich and the discovery of hybridoma technology marked the beginning of antibodies for therapeutic applications. Rituximab is the first Mab approved by Food and Drug Administration against B-cell malignancies. Today, a number of effective Mabs targeting leukemia and lymphoma were approved and successfully developed. The Mab therapeutic phenomena in the body (also called war in the blood) has various mechanism of cations. In recent years, Mab against leukemialymphoma achieved significantly therapeutic outcomes, eventually this led to traditional chemotherapy treatment free era. We also reviewed that, the therapeutic efficacy of Mabs against leukemia-lymphoma as compared antibody alone and antibody conjugated with potent chemotherapy or cytotoxic drugs. This article extends our understanding of advancements in therapeutic Mabs technology and provides new insights of Mab leukemia-lymphomas therapy. We also encourage further more studies for Mab based diagnostics, treatments of leukemia and lymphomas.

Basic Considerations for the Use of Monoclonal Antibodies in Migraine.

BackgroundThe current study aimed to describe the impact of parental migraine on adolescent children (aged 11–17) living at home with a parent with migraine.AimMany drug classes are prescribed for migraine treatment, but all have limitations. Recently, calcitonin gene‐related peptide (CGRP) activity has shown a significant promise as a target for preventive therapy. In this review, we provide an overview of the potential role of CGRP mAbs in migraine, with a focus on their design, pharmacokinetics, safety, and immunogenicity.ConclusionsThe CGRP mAbs are an innovative new therapy for migraine and address the need for effective and tolerable preventive options. MAbs, including those that target CGRP or its receptor, bind to a target with high specificity and affinity and lead to few off‐target adverse effects, although mechanism‐based adverse reactions may occur. Unlike other therapeutic antibodies used to treat neurologic disease, CGRP mAbs do not have a target within the immune system and have been designed to avoid altering the immune system. The safety and efficacy of mAbs against CGRP or its receptors are being investigated in clinical development programs, and the first of these therapies has received regulatory approval in the United States.

Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies.

Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs. Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [13C-1H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments. Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution. The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.

Antibody Inhibition of Properdin Prevents Complement-Mediated Intravascular and Extravascular Hemolysis.

Paroxysmal nocturnal hemoglobinuria (PNH) is a serious blood disorder characterized by dysregulated complement activation on blood cells. Eculizumab, the current standard therapy and a humanized anti-C5 mAb, relieves anemia and thrombosis symptoms of PNH patients by preventing complement-dependent intravascular hemolysis (IVH). However, up to 20% of PNH patients on long-term eculizumab treatment still suffer from significant anemia and are transfusion dependent because of extravascular hemolysis (EVH) of C3-opsonized PNH erythrocytes. In this study, we show that function-blocking anti-properdin (P) mAbs dose-dependently inhibited autologous, complement-mediated hemolysis induced by factor H dysfunction. Furthermore, anti-human P (hP) mAbs potently and dose-dependently inhibited acidified serum-induced hemolysis of PNH erythrocytes (Ham test). In contrast to erythrocytes rescued by anti-C5 mAb, nonlysed PNH erythrocytes rescued by anti-P mAb incurred no activated C3 fragment deposition on their surface. These results suggested that anti-P mAbs may prevent EVH as well as IVH of PNH erythrocytes. To test the in vivo efficacy of anti-hP mAbs in preventing EVH, we generated a P humanized mouse by transgenic expression of hP in P knockout mice (hP-Tg/P-/-). In a murine EVH model, complement-susceptible erythrocytes were completely eliminated within 3 d in control mAb-treated hP-Tg/P-/- mice, whereas such cells were protected and persisted in hP-Tg/P-/- mice treated with an anti-hP mAb. Collectively, these data suggest that anti-P mAbs can inhibit both IVH and EVH mediated by complement and may offer improved efficacy over eculizumab, the current standard therapy for PNH.

Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan.

Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold, and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine (N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), decreases the Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced the CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to that of afucosylated IgG1 Fc; however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan that impinges upon the linkage between the α-mannose(1-6)β-mannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted the IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution nuclear magnetic resonance spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to that of afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates the antibody affinity indirectly by reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineering to enhance intermolecular glycan-glycan contacts will likely provide limited improvement, and future designs should maximize the affinity by maintaining the CD16a Asn162-glycan conformational heterogeneity.

Anti-ADAM17 monoclonal antibody MEDI3622 increases IFNγ production by human NK cells in the presence of antibody-bound tumor cells.

Several clinically successful tumor-targeting mAbs induce NK cell effector functions. Human NK cells exclusively recognize tumor-bound IgG by the FcR CD16A (FcγRIIIA). Unlike other NK cell activating receptors, the cell surface density of CD16A can be rapidly downregulated in a cis manner by the metalloproteinase ADAM17 following NK cell stimulation in various manners. CD16A downregulation takes place in cancer patients and this may affect the efficacy of tumor-targeting mAbs. We examined the effects of MEDI3622, a human mAb and potent ADAM17 inhibitor, on NK cell activation by antibody-bound tumor cells. MEDI3622 effectively blocked ADAM17 function in NK cells and caused a marked increase in their production of IFNγ. This was observed for NK cells exposed to different tumor cell lines and therapeutic antibodies, and over a range of effector/target ratios. The augmented release of IFNγ by NK cells was reversed by a function-blocking CD16A mAb. In addition, NK92 cells, a human NK cell line that lacks endogenous FcγRs, expressing a recombinant non-cleavable version of CD16A released significantly higher levels of IFNγ than NK92 cells expressing equivalent levels of wildtype CD16A. Taken together, our data show that MEDI3622 enhances the release of IFNγ by NK cells engaging antibody-bound tumor cells by blocking the shedding of CD16A. These findings support ADAM17 as a dynamic inhibitory checkpoint of the potent activating receptor CD16A, which can be targeted by MEDI3622 to potentially increase the efficacy of anti-tumor therapeutic antibodies.

Abstract 3831: Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Despite the recent advances in the treatment of NB, patients with high-risk NB still have poor prognosis. Immunotherapy using anti-GD2 monoclonal antibody (mAb), which binds to disialoganglioside GD2 on the surface of NB cells, has recently become a standard treatment for high-risk NB. However, the therapeutic efficacy of anti-GD2 mAb is insufficient in many clinical trials. Therefore, the enhancement of therapeutic potential of anti-GD2 mAb is needed to improve the clinical outcome of patients with high-risk NB. Near-infrared photoimmunotherapy (NIR-PIT) is a promising antitumor strategy to enhance the therapeutic potential of immunotherapy using an antibody-photoabsorber conjugate (APC). In this study, we investigated the therapeutic efficacy of anti-GD2-based NIR-PIT using anti-GD2 mAb conjugated to the photoabsorber IR700 (anti-GD2-IR700) in human NB cells. Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The expression of GD2 on the cell surface of NB cells was analyzed by flow cytometric analysis. Anti-GD2-IR700 was synthesized by incubating anti-GD2 mAb and IR700. The mixture was purified with a Sephadex G50 column. The antitumor effect of anti-GD2-IR700 was evaluated using XTT assay. NB cells were incubated with anti-GD2-IR700, anti-GD2 mAb or PBS and NIR light was irradiated at 2.0J/cm2. Results: IMR-32 and CHP-134 cells, but not SK-N-SH cells, expressed GD2 protein on the cell surface. In GD2-positive IMR-32 and CHP-134 cells, administration of anti-GD2-IR700 significantly suppressed the cell viability compared to anti-GD2 mAb or PBS when combined with NIR light irradiation. In contrast, in GD2-negative SK-N-SH cells, anti-GD2-IR700 showed no significant inhibition of cell viability in combination with NIR light irradiation. Conclusion: These results suggest that NIR-PIT using an anti-GD2-IR700 is a promising antitumor strategy to promote the therapeutic efficacy of anti-GD2 immunotherapy for high-risk NB. Citation Format: Hiroshi Nouso, Hiroshi Tazawa, Terutaka Tanimoto, Morimichi Tani, Takanori Oyama, Hiroaki Sato, Kazuhiro Noma, Shunsuke Kagawa, Hisataka Kobayashi, Takuo Noda, Toshiyoshi Fujiwara. Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3831.

Enhancing IgG distribution to lung mucosal tissue improves protective effect of anti–Pseudomonas aeruginosa antibodies

IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti-Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.

Vaccines Targeting PCSK9: A Promising Alternative to Passive Immunization with Monoclonal Antibodies in the Management of Hyperlipidaemia?

Hypercholesterolaemia is frequently observed in patients with cardiovascular diseases (CVD) and is associated with increased mortality. Statin treatment has been the standard of care for reducing low-density lipoprotein cholesterol (LDL-C) to improve cardiovascular outcomes. However, statins have limited effects in some patients and may be discontinued due to adverse effects resulting in LDL-C above target levels. The proprotein convertase subtilisin kexin type 9 (PCSK9) is a pivotal regulator in the LDL-C metabolism by degrading the LDL-C receptor on hepatocytes. Inhibition of PCSK9 by monoclonal antibodies (mAb) significantly lowers LDL-C levels and is considered to reduce the likelihood of adverse cardiac events. However, such treatment regimens are not cost-effective, and require frequent administrations at high doses that may be associated with side effects and poor drug adherence. Furthermore, it has been shown that these PCSK9 medicines may trigger the formation of antidrug antibodies followed by a significant attenuation of the LDL-C-lowering effect. Active vaccination inducing high-affinity antibodies against PCSK9 with less frequent administration intervals may be a novel promising therapeutic approach to overcome the drawback of passive immunization with PCSK9 mAb. However there is a paucity of available clinical safety and efficacy data. This article discusses challenges in the development of PCSK9 vaccines and their potential therapeutic benefits by reviewing clinical studies that evaluated the safety and efficacy of PCSK9 mAb.

CGRP Monoclonal Antibodies for the Preventative Treatment of Migraine.

CGRP is a key neuropeptide in migraine pathophysiology. The blockade of the CGRP pathway at the side of the CGRP receptor of the CGRP peptide leads to the interruption of trigeminal nerve system-mediated headache syndromes such as migraine. Monoclonal antibodies (mAbs) targeting the CGRP pathway have been developed and are currently under investigation for episodic (EM) and chronic migraine (CM) prevention. Here, we report data from these clinical trials. Placebo-controlled, randomized double-blind phase studies of CGRP mAbs in episodic and chronic migraine have shown that the specific blockade of the peptide or the CGRP receptor are both powerful mechanisms to reduce migraine frequency. Along with the reduction of acute migraine-specific medication intake, early onset of efficacy of mAbs has been demonstrated. Most common adverse events are injection sider reactions. Depending on the mAb, the administration mode is a monthly or even less frequently s.c. or I.V. formulation. Phase II studies in EM and CM demonstrate that CGRP mAbs are effective anti-migraine preventatives with a beneficial adverse event profile. Further detailed results from larger phase III clinical trials are expected soon.

Preparation and protection of monoclonal antibody against H7N9 influenza virus hemagglutinin

The first human infection with novel H7N9 influenza virus was reported in March 2013 in China. To date, the H7N9 influenza viruses possess a potential pandemic threat to public health worldwide and have caused severe infection and high mortality in humans in China. The latent period of human infection of H7N9 influenza virus is generally within 7 days. Patients usually show flu like symptoms, such as runny nose, sore throat, fever, cough, headache and muscle pain. Severe patients have rapid development, characterized by severe pneumonia and dyspnea. Antibodies play a major role in protective immunity against the influenza virus infection, and passive immunization with monoclonal antibodies (MAbs) specific to viral proteins might be a potential option for humans against lethal infection of the influenza viruses. It is known that hemagglutinin (HA), is the major viral antigenic protein on the influenza virus particle and the primary target of neutralizing antibodies against influenza virus. In this study, in order to obtain the HA MAbs of influenza virus H7N9 (A/Shanghai/2/2013), study their biology characteristics, and evaluate the prophylactic and therapeutic efficacy of the HA MAbs in mouse model. The BALB/c mice were immunized with inactivated H7N9 vaccine by subcutaneous administration for three times, and boosted with inactivated H7N9 vaccine by the tail vein three days before fusion. Finally, 28 MAbs were obtained and identified by ELISA and hemagglutination inhibition test (HI). Eight IgG subtype MAbs (1A8, 2A12, 2H3, 4A11, 4C8, 4G5, 6A3, 7G2) were selected to identify their biology characteristics. The ELISA titers of ascites of the eight MAbs were 102–106. The eight Mabs exhibited high affinity which can reach nmol/L level and meet the requirement of affinity for monoclonal antibody drug. The immunofluorescence assay indicated that the eight MAbs had excellent specificity for HA protein expressed in 293T cell and were able to recognize the H7N9 influenza virus particles. In addition, all the eight MAbs could inhibit HA activity and the MAbs 2H3, 4A11, 4G5 and 6A3 can also present neutralization activity against the H7N9 live virus. The highest neutralizing titer was up to 1:14125. At the same time, we evaluated the prophylactic and therapeutic efficacy of these MAbs against the homologous H7N9 strain. The activity against H7N9 virus was evaluated by clinical syndromes, survival rates and lung viral titers. The results showed that in the prophylactic and therapeutic experiments, MAb 6A3 can inhibit effectively H7N9 influenza virus infection, provided 100% protection and remarkably reduced the lung viral titers in mouse model. The HA monoclonal antibodies against influenza virus H7N9 were successfully obtained. The HA MAbs not only may be as a prophylactic and therapeutic measure to prevent H7N9 influenza virus infection, but also laid the foundation for us to research the H7N9 HA antigen epitope and develop diagnostic reagents.

Fast and Accurate Evaluation of Oxidation‐Induced Destabilization of mAbs

The applicability of monoclonal antibodies (mAbs) in therapeutic research continues to rise — they now account for almost 50% of protein‐based drugs. Monitoring their quality as well as binding properties are critical as these parameters provide insight into mAb functionality and efficacy as potential drugs.Here we study trastuzumab, a monoclonal antibody that has been used to successfully treat patients with certain forms of breast cancer. Trastuzumab acts by binding to and interfering with the HER2/neu receptor in cancer patients. Using two complementary technologies, we examine how targeted oxidation affects trastuzumab structure and therefore its binding capabilities to protein A. First, a rapid analysis of oxidation‐induced changes in mAb folding and stability was performed. The same samples were then analyzed to determine how oxidation compromises mAb interactions. Oxidational stress directly correlated with weaker binding affinities of the Fc region towards protein A, showcasing fast and accurate characterization of trastuzumab sample quality.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New insights in Type I and II CD20 antibody mechanisms-of-action with a panel of novel CD20 antibodies.

Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.

Development and Characterization of Neutralizing Antibodies Against Zaire Ebolavirus Glycoprotein and Protein 40

Background/Aims: Monoclonal antibodies (mAbs) are presently the most promising treatment against Ebola virus disease (EVD), and cocktail of two or more antibodies likely confers protection through complementary mechanisms. Zaire Ebolavirus (EBOV) glycoprotein (GP) and viral protein 40 (VP40) are targets for designing neutralizing antibodies. Currently, the antiviral therapeutics of mAb-cocktails are still limited solely to anti-GP antibodies，there is no Abs cocktail against Zaire EBOV GP and VP40, which both have important interactions with host cellular membrane. Methods: We used hybridoma technology to produce anti-Zaire EBOV GP mAb against GP receptor binding domain, and anti-Zaire EBOV VP40 mAbs against the N-terminal domain, the C-terminal domain, respectively; synthesized Zaire EBOV transcription and replication competent virus like particles (trVLPs), which model even all aspects of the EBOV life cycles in order to evaluate the anti-viral effect of mAbs. Then, we characterized the anti- Zaire EBOV trVLPs effect of anti-GP and VP40 mAbs in vitro by real time-PCR, immunofluorescence assay and western blot analysis. Results: Our results demonstrate that anti-GP or anti-VP40 mAbs effectively inhibit trVLPs replication. The cocktails of anti-GP and anti-VP40 mAbs, or between anti-VP40 mAbs, had synergistic anti-trVLPs effect. Meanwhile, the detailed DNA and amino acid sequences of the mAbs were checked. Conclusion: The study verifies neutralizing efficacy of anti-GP or anti-VP40 mAb, report promising cocktail of anti-GP and anti-VP40 mAb, or cocktail of two anti-VP40 mAbs. To our knowledge, this is the first account to report the important anti-viral effect of cocktails of anti-GP and anti-VP40 mAbs in vitro.

Differential Action of Anti-CD20 Monoclonal Antibodies: Role in Induction of Cell Death

Purpose: To study and assess to the sensitivity of Burkitt’s lymphoma cells harboring differential levels of cell surface CD20 for anti-CD20 monoclonal Antibodies. Material and methods: Burkitt’s lymphoma cell line ‘Daudi’ was used during present study. Cells were exposed to single dose of γ-radiation (0.5 Gy) and thereafter incubated with rituximab (Rtx) or tositumomab (Tst) (0.5 μg/ ml concentration each). The changes in expression of RelA, Akt and Bax/Bcl-2 ratio was measured to assess sensitivity of cells in term of cell death. Results: During the present investigation, we found that cells incubated with anti-CD20 mAbs at +20 hrs post radiation exposure showed higher levels of cell death in term of Bax/Bcl-2 ratio. In addition, we also found the significant changes in expression of pro-survival signaling proteins such as RelA and Akt pathways. Moreover, we found that tositumomab is a potent inducer of apoptotic cell death. Conclusion: These findings suggested that the efficacy of anti-CD20 mAbs depends on the number of CD20 molecules expressed on cell surface and type of antibody used. It may provide new treatment options for selection of anti-CD20 mAbs even in aggressive B-cell lymphoma, which harbors low levels of CD20 or even resistant to current therapies in vivo.

Monoclonal Antibody Therapy and Long-term Outcomes in Multiple Sclerosis – The Challenge of Treatment Optimisation

The therapeutic landscape of multiple sclerosis (MS) has been transformed by the advent of several new monoclonal antibody (MAb) therapies that can potentially lead to full stabilisation of detectable disease activity. Natalizumab, alemtuzumab and ocrelizumab are currently licensed MAbs for the treatment of MS. Daclizumab was licensed for the treatment of MS, although it has been recently withdrawn from the market by the manufacturer. Most patients are initially managed with first-line treatments, and, if disease breakthrough occurs, are escalated to a stronger compound, yet the available evidence indicates an early window of therapeutic opportunity for MAbs to exert most of their efficacy. It is important to balance the superior efficacy of MAbs compared with injectable treatments against more serious side effects, although these are well recognised and can be monitored where indicated and treated. In particular, the risk of progressive multifocal leucoencephalopathy with natalizumab can be managed by screening potential patients for the John Cunningham virus. The MAbs also have the benefit of convenience to patients compared with daily or weekly treatments since they are given via less frequent administration. The cost of these treatments, compared with other therapies, may be an important issue in many countries where healthcare budgets are under pressure. The complex decision of choosing the best treatment for an individual should be made jointly between the doctor and the patient after careful consideration of the many factors to be weighed.

Monoclonal Antibodies for Treatment of Eosinophilic Esophagitis

Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus affecting both children and adults, with debilitating and progressive symptoms. EoE has shown an explosive epidemiological rise in the past few decades. Many patients experience a poor level of disease control despite maximal use of available guideline-based therapies, which seriously hampers their quality of life. Diet restrictions and systemic and topical corticosteroids are the current mainstays of EoE therapy, but are associated with significant efficacy, treatment compliance, and safety issues such as oral or esophageal candidiasis, growth retardation, osteopenia, osteoporosis, glucose intolerance, and cataract formation. As EoE is a chronic inflammatory disease, immune cells and cytokines are responsible for the inflammatory response and symptoms. Monoclonal antibodies specifically targeting these pathophysiologic effectors offer more potent relief of histologic and clinical disease features while keeping off-target adverse effects to a minimum. Herein, we have reviewed the current evidence regarding efficacy and safety of monoclonal antibodies including mepolizumab (anti-IL-5), reslizumab (anti-IL-5), QAX576 (anti-IL-13), omalizumab (anti-immunoglobulin-E), and infliximab (anti-TNF-α) in treatment of EoE. Our review indicates that although the use of monoclonal antibodies for EoE treatment is safe with limited and reversible adverse events, however, it is not yet possible to reach a final verdict on the efficacy of mAbs in EoE. Future well-designed studies are needed to clarify the exact role of mAbs in EoE.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Co-administration of RANKL and CTLA4 Antibodies Enhances Lymphocyte-Mediated Antitumor Immunity in Mice.

Purpose: Novel partners for established immune checkpoint inhibitors in the treatment of cancer are needed to address the problems of primary and acquired resistance. The efficacy of combination RANKL and CTLA4 blockade in antitumor immunity has been suggested by recent case reports in melanoma. Here, we provide a rationale for this combination in mouse models of cancer.Experimental Design: The efficacy and mechanism of a combination of RANKL and CTLA4 blockade was examined by tumor-infiltrating lymphocyte analysis, tumor growth, and metastasis using a variety of neutralizing antibodies and gene-targeted mice.Results: RANKL blockade improved the efficacy of anti-CTLA4 mAbs against solid tumors and experimental metastases, with regulatory T-cell (Treg)-depleting anti-CTLA4 mAbs of the mouse IgG2a isotype showing greatest combinatorial activity. The optimal combination depended on the presence of activating Fc receptors and lymphocytes (NK cells for metastatic disease and predominantly CD8+ T cells for subcutaneous tumor control), whereas anti-RANKL alone did not require FcR. The significantly higher T-cell infiltration into solid tumors post anti-RANKL and anti-CTLA4 was accompanied by increased T-cell effector function (cytokine polyfunctionality), and anti-RANKL activity occurred independently of Treg depletion. The majority of RANKL expression in tumors was on T cells whereas RANK-expressing cells were mostly tumor-associated macrophages (TAM), with some expression also observed on dendritic cells (DC) and myeloid-derived suppressor cells (MDSC).Conclusions: These results provide a rationale for the further investigation of RANKL-RANK interactions in tumor immunity and a basis for development of translational markers of interest in human clinical trials. Clin Cancer Res; 23(19); 5789-801. ©2017 AACR.