Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7

BACKGROUND AND PURPOSE

The mGlu₇ receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu₇ monoclonal antibody, MAB1/28, that triggers receptor internalization.

EXPERIMENTAL APPROACH

MAB1/28''s activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [³⁵S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa.

KEY RESULTS

mGlu₇/mGlu₆ chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu₇. MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gα_i protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu₇ dimers.

CONCLUSIONS AND IMPLICATIONS

We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu₇ function and selective activation of its intracellular trafficking.     

抗人死亡受体5单链抗体的构建表达纯化及活性分析

目的构建、表达、纯化抗人死亡受体5(DR5)单链抗体,并检测其诱导肿瘤细胞凋亡的活性。方法采用RT-PCR获取鼠源性抗人DR5单克隆抗体重链和轻链可变区基因序列,以一柔性连接肽连接二者,转入表达载体,以大肠杆菌表达融合蛋白,亲和色谱纯化后用MTT实验和凋亡检测试剂盒检测其凋亡诱导活性。结果获得的序列经比对为抗体重链和轻链可变区基因,表达纯化后的重组蛋白具有接近完整抗体的肿瘤细胞凋亡诱导活性。结论抗人DR5 scFv可作为诱导肿瘤细胞凋亡的候选药物,为肿瘤免疫学研究提供材料。     

Synthesis and biological evaluation of a radioiodinated spiropiperidine ligand as a potential σ1 receptor imaging agent

We report the synthesis and evaluation of 1′‐(4‐[¹²⁵I]iodobenzyl)‐3H‐spiro[isobenzofuran‐1,4′‐piperidine] ([¹²⁵I]Spiro‐I) as a potential SPECT tracer for imaging of σ₁ receptors. [¹²⁵I]Spiro‐I was prepared in 55–65% isolated radiochemical yield, with radiochemical purity of >99%, via iododestannylation of the corresponding tributyltin precursor. In receptor binding studies, Spiro‐I displayed low nanomolar affinity for σ₁ receptors (σ₁: K_i=2.75±0.12 nM; σ₂: K_i=340 nM) and high subtype selectivity (σ₂/σ₁=124). Biodistribution in mice demonstrated relatively high concentration of radioactivity in organs known to contain σ₁ receptors, including the lung, kidney, heart, spleen, and brain. Administration of haloperidol 5 min prior to injection of [¹²⁵I]Spiro‐I significantly reduced the concentration of radioactivity in the above‐mentioned organs. These findings suggest that the binding of [¹²⁵I]Spiro‐I to σ₁ receptors in vivo is specific. Copyright © 2010 John Wiley & Sons, Ltd.     

Targeting apoptosis: preclinical and early clinical experience with mapatumumab,an agonist monoclonal antibody targeting TRAIL-R1

In spite of the advances in survival with chemotherapy and radiotherapy, many cancer patients continue to experience failure with treatments. Advances in molecular oncology and the development of numerous targeted therapies, used by themselves or in combination with at present available treatments such as chemotherapy and radiation, will hopefully improve the fate of these patients. It has been well understood for many years now that deregulation of apoptosis is a major hallmark of cancer cells. Mapatumumab, a fully human agonistic monoclonal antibody to TNF-related apoptosis-inducing ligand receptor 1, has been developed to induce apoptosis in cancer cells although having minimal effects on normal cells. This paper reviews the preclinical and early clinical data of this exciting new agent and discusses options for future development of mapatumumab, mostly in combinations with other therapies.     

FasL、TRAIL及其受体DcR1在宫颈癌中的表达

目的：探讨FasL、TRAIL及其受体DcR1在宫颈癌中的表达及其临床意义。方法：应用免疫组化SP法对10例正常宫颈组织、18例宫颈上皮内瘤变（CIN）组织和40例宫颈癌组织中FasL、TRAIL及DcR1的表达情况进行检测,并结合患者年龄、肿瘤分化程度、组织类型、有无淋巴结转移等临床病理因素进行综合分析。结果：FasL在正常宫颈、CIN和宫颈癌组织中的表达呈递增趋势;宫颈癌组织中,FasL阳性表达随组织学分级的降低而升高;有淋巴结转移者FasL阳性表达明显高于无淋巴结转移者;TRAIL及DcR1在正常宫颈组织中的表达高于宫颈癌组织,宫颈癌组织中,TRAIL阳性表达随组织学分级降低而降低。结论：FasL．TRAIL及DcR1的异常表达在宫颈癌变过程中起到了一定的作用。     

ZCH-2B8a,an antibody targeting actin-binding protein coronin-1a,is a potential therapeutic agent for B-lineage malignancies

Abstract

Background: Monoclonal antibody (mAb)-based targeted therapy is one of the most promising strategies to cure cancers. MAb ZCH-2B8a (2B8a) was a novel antibody generated in our laboratory, which presented potential to be a therapeutic agent for hematologic malignancies.

Methods: We investigated the reactivity profile of 2B8a mAb, identified the targeting antigen by proteomic and genetic approaches and evaluated its potential to exert tumor cell killing.

Results: 2B8a antigen was strictly expressed on lymph tissues and hematopoietic cells (mainly leukocytes), and was highly expressed on B-lineage leukemia cell lines and acute lymphoblastic leukemia (ALL) cells from patients. 2B8a antibody was quickly internalized into the target cells once binding to the antigen, but was capable of killing tumor cells through complement dependent cytotoxicity. To identify the 2B8a antigen, the proteins of Raji cells were immunoprecipitated with 2B8a antibody and analyzed by mass spectrometry, which indicated that coronin-1a was a potential candidate. Then, coronin-1a gene was cloned from Raji cells, inserted into plasmid pcDNA3.1 (+), and transfected into CHO cells. The intracellular 2B8a antigen level was significantly increased in the coronin-1a transfectant cell line.

Conclusion: 2B8a mAb is a novel antibody targeting coronin-1a, which has the potential to be a therapeutic agent for B-lineage malignancies.     

Development of a multidose formulation for a humanized monoclonal antibody using experimental design techniques

The purpose of this study was to identify optimal preservatives for a multidose formulation of a humanized monoclonal antibody using experimental design techniques. The effect of antimicrobial parenteral preservatives (benzyl alcohol, chlorobutanol, methyl paraben, propylparaben, phenol, and m-cresol) on protein stability was assessed using size-exclusion chromatography, differential scanning calorimetry, right-angle light scattering, UV spectroscopy, and potency testing using a cell-based fluorescence-activated cell sorting method. A quick, cost-effective preservative screening test was designed. Combinations of preservatives were examined using an I-optimal experimental design. The protein was most stable in the presence of methylparaben and propylparaben, and was compatible with benzyl alcohol and chlorobutanol at low concentrations. Phenol and m-cresol were not compatible with the protein. The I-optimal experimental design indicated that as an individual preservative, benzyl alcohol was promising. The model also indicated several effective combinations of preservatives that satisfied the antimicrobial efficacy and physical stability constraints. The preservative screening test and the experimental design approach were effective in identifying optimal concentrations of antimicrobial preservatives for a multidose protein formulation; (1) benzyl alcohol, and (2) the combination of methylparaben and chlorobutanol were screened as potential candidates to satisfy the regulatory requirements of various preservative efficacy tests.     

99mTc‐labeled rHuEpo for imaging of the erythropoietin receptor in tumors

To analyze erythropoietin receptor (EpoR) status in tumors, recombinant human erythropoietin (rHuEpo) was labeled with ^99mTc by ^99mTc‐centered 1‐pot synthesis, resulting in high radiochemical purity, stability, and biological activity. Both in vitro cell culture experiments and biodistribution studies of normal rats demonstrated successful EpoR targeting. The biodistribution of labeled rHuEpo in a NCI‐H1975 xenograft model showed tumor accumulation (tumor‐to‐muscle ratio, 4.27 ± 1.77), confirming the expression of active EpoR in tumors. Thus, as a novel single positron emission computerized tomography tracer for the imaging of EpoR expression in vivo, ^99mTc‐rHuEpo is effective for exploring the role of EpoR in cancer growth, metastasis and angiogenesis.     

Radionuclide probes for molecular imaging of pancreatic beta-cells

Islet transplantation is a promising treatment option for patients with type 1 diabetes (T1D); however, the fate of the graft over time remains difficult to follow, due to the lack of available tools capable of monitoring graft rejection and inflammation prior to islet graft loss. Due to the challenges imposed by the location of the pancreas and the sparsely dispersed beta-cell population within the pancreas, currently, the clinical verification of beta-cell abnormalities can only be obtained indirectly via metabolic studies, which typically is not possible until after a significant deterioration in islet function has already occurred. The development of non-invasive imaging methods for the assessment of the pancreatic beta-cells, however, offers the potential for the early detection of beta-cell dysfunction prior to the clinical onset of T1D and type 2 diabetes (T2D). Ideal islet imaging agents would have an acceptable residence time in the human body, be capable of providing high-resolution images with minimal uptake in surrounding tissues (e.g., the liver), would not be toxic to islets, and would not require pre-treatment of islets prior to transplantation. A variety of currently available imaging techniques, including magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging have been tested for the study of beta-cell diseases. In this article, we summarize the recent advances made in nuclear imaging techniques for non-invasive imaging of pancreatic beta-cells. The use of radioactive probes for islet imaging is also discussed.     

A kinetic enzyme immunoassay for the quantitation of antibodies to a humanized monoclonal antibody in human serum

A kinetic enzyme immunoassay was developed and validated to quantitate human antibodies to the humanized monoclonal antibody CAMPATH¹-1H (C1H) in human serum. The assay was configured using C1H-coated 96-well plates which were blocked with bovine serum albumin, and incubated with dilutions of human serum containing anti-C1H antibody. Antibody was detected using biotinylated C1H followed by streptavidin-conjugated alkaline phosphatase and p-nitrophenyl phosphate. Absorbance data were collected for 10 min, and mOD min⁻¹ data were exported to MultiCalc data analysis software. A 4-parameter logistic-log algorithm was shown to model the data through the range of the standard curve within 15% of nominal values. The overall assay performance coefficient of variation by ANOVA was 9.2%. The lower limit of detection was defined at 160 Units ml⁻¹. The anti-idiotype antibody standard stock solution is stable at 4°C and at −80°C for at least 11 months in buffer. The anti-idiotype antibody controls are stable for at least seven freeze–thaw cycles and at least 6 months in human serum stored at −20°C. A strategy was devised by which to establish the specific antibody potency for any given batch of anti-C1H antibody standard relative to the Reference Standard. This EIA has been used to quantify and characterize anti-C1H antibody in human serum in support of clinical safety and efficacy studies.     

一个新型α1-去甲肾上腺素受体的光亲和放射性碘探针的母体化合物的合成

A new and high-yielding procedure was described for the synthesis of 4-amino-6,7-dimethoxy-2[4-[4 (4-aminophenyl) butanoyl]-1-piperazinyl]-quinazoline Ⅶ, the key intermedi-ate for the newly designed potential α₁-AR affinity/photoaffinity probes Ⅷa/Ⅸa.     

Safety profile of tasimelteon,a melatonin MT1 and MT2 receptor agonist: pooled safety analyses from six clinical studies

Introduction: Tasimelteon, a novel circadian regulator, is the first product for the treatment of Non-24-hour Sleep-Wake Disorder (Non-24) approved by either the FDA or the European Medicines Agency (EMA). Tasimelteon is a potent and specific melatonin (MT₁ and MT₂) receptor agonist with 2 – 4 times greater affinity for the MT₂ receptor.

Methods: Safety was assessed in two controlled and two open-label studies in blind individuals with Non-24 and in two controlled studies of primary insomnia. Periodic assessments included collection of adverse events (AEs), laboratory testing, electrocardiograms (ECGs), vital sign monitoring, physical examinations and assessment for the potential for suicide. One study included additional assessments for endocrine function.

Results: A total of 184 blind individuals with Non-24 received tasimelteon nightly with a median exposure > 1 year. In placebo-controlled studies, 387 patients with insomnia and 42 patients with Non-24 received tasimelteon nightly for 4 – 26 weeks. The total patient years exposure for the six studies assessed here is 258.64 patient years. Discontinuations due to AEs were similar across treatment groups. Overall in the clinical studies described here, AEs attributable to tasimelteon treatment were headache, diarrhea, dry mouth, alanine aminotransferase increased, somnolence, dizziness and nightmare/abnormal dreams. There were no clinically significant differences in treatment group with ECGs, vital signs, withdrawal, endocrine function and suicidality assessments.

Conclusion: Long-term tasimelteon administration was safe and well-tolerated. This is supported by placebo-controlled data in both Non-24 and insomnia patients.     

M1胆碱受体激动剂治疗阿尔茨海默病的研究进展

阿尔茨海默病(Alzheimer disease，AD)是一种以胆碱能神经元进行性退变、老年斑和神经元缠结为病理特征的神经退行性疾病。尽管AD发病机制尚未阐明，但β淀粉样肽沉积和tau蛋白磷酸化与胆碱能神经退变的恶性循环(vicious cycle)无疑是造成AD的重要病理机制之一。大量研究表明胆碱能神经突触后膜的M1受体的数目在整个病程中变化不大，M1受体选择性激动剂不但可以直接补偿胆碱能的功能，而且可以调节β淀粉样前体蛋白代谢和降低tau蛋白的过度磷酸化，有助于打破这一恶性循环，改善AD的学习记忆功能并延缓病情的发展。因此M1胆碱受体激动剂被认为是最有前途的治疗AD药物之一。目前Xanomeline、Sabcomeline等具有相对选择性M1受体激动剂业已进入新药临床试验阶段。     

Cognition and depression: the effects of fluvoxamine,a sigma‐1 receptor agonist,reconsidered

Cognitive impairment is a primary feature of patients with major depressive disorder (MDD) and is characterised by stress‐induced neural atrophy. Via alpha‐adrenergic, anti‐cholinergic and anti‐histaminic activities, several antidepressants can cause significant counter‐therapeutic cognitive impairment. Evidence is emerging of the involvement of sigma‐1 receptor agonism in the mechanism of action of some antidepressants, notably fluvoxamine. Sigma‐1 receptors are abundant in areas affected by depression/stress‐induced cerebral atrophy and their ligands have a unique pharmacological profile; they may promote neurogenesis and initiate adaptive neural plasticity as a protection/reaction to stress. Fluvoxamine, as a potent sigma‐1 receptor agonist, has shown ameliorating effects in animal models of psychosis, depression, stress, anxiety, obsessive‐compulsive disorder (OCD) and aggression and has been shown to improve cognitive impairments. In humans, fluvoxamine may repair central nervous system (CNS) atrophy and restore cognitive function. The current review explores the mechanisms through which sigma‐1 receptors can modulate cognitive function and examines how antidepressant therapy with fluvoxamine may help improve cognitive outcomes in patients with depression. Copyright © 2010 John Wiley & Sons, Ltd.     

Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours

Background and purpose:

The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications.

Experimental approach:

An anti–EGFR/EGFRvIII llama single-domain antibody (EG₂) and two higher valency format constructs, bivalent EG₂-hFc and pentavalent V2C-EG₂ sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies.

Key results:

Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG₂ to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG₂ and EG₂-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG₂, V2C-EG₂ and EG₂-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG₂-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG₂-hFc selective accumulation/retention in anatomically defined tumour regions.

Conclusions:

Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.     

Radioiodination,purification, and evaluation of antihuman tumor-derived immunoglobulin G light chain monoclonal antibody in tumor-bearing nude mice

We report the synthesis and biological evaluation of ¹³¹I-labeled antihuman tumor-derived immunoglobulin G (IgG) light chain monoclonal antibody (4E9) ([¹³¹I]I-4E9) as a promising probe for tumor imaging. [¹³¹I]I-4E9 was synthesized in radiochemical yield of 89.9 ± 4.7% with radiochemical purity of more than 99%. [¹³¹I]I-4E9 showed high stability in normal saline and human serum. In cell uptake studies, [¹³¹I]I-4E9 exhibited favorable binding affinity and high specificity in HeLa MR cells. In biodistribution studies, [¹³¹I]I-4E9 showed high tumor uptake, high tumor/non-tumor ratios, and specific binding in BALB/c nu/nu mice bearing human HeLa MR xenografts. Single-photon emission computerized tomography (SPECT) imaging of [¹³¹I]I-4E9 in the HeLa MR xenograft model demonstrated clear visualization of tumor after 48 h and confirmed specific binding in tumor. These findings suggest that [¹³¹I]I-4E9 possesses favorable biological characteristics and warrants further investigation as a prospective probe for imaging and treatment of cancers.     

Pharmacokinetics,pharmacodynamics and clinical efficacy of omalizumab for the treatment of asthma

Introduction: Omalizumab is a subcutaneously administrated monoclonal anti-IgE antibody indicated in adults, adolescents and children 6 years of age and older with moderate to severe allergic asthma uncontrolled by conventional pharmacological treatments and sensitization to at least one perennial allergen.

Area covered: This drug evaluation summarizes published data on pharmacokinetic and pharmacodynamic properties of omalizumab, on clinical efficacy and safety, including real-world evidence, and provides a medico-economic evaluation of the drug.

Expert opinion: Omalizumab represents an efficient therapeutic option for the management of patients with uncontrolled moderate/severe allergic asthma. It provides a significant reduction in the asthma exacerbation rate with a steroid-sparing effect, an improvement in quality of life in adults and adolescents, despite a lack of evidence about its efficacy specifically in severe allergic asthma. Clinical trials have demonstrated its efficacy in the pediatric population but further real-life evidence is expected to better characterize long-term effects in this population. There is still some debate about the optimal treatment duration but, to date, it is recommended not to stop the treatment as cessation has resulted in symptom recurrence. Omalizumab is an expensive treatment, but a key therapeutic option when used for uncontrolled severe allergic asthma.     

Interactions of GR127935, a 5-HT1B/D receptor ligand, with functional 5-HT receptors

GR127935 (N-[methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl) [1, 1-biphenyl]-4-carboxamide hydrochloride) has been recently introduced as an experimental tool to antagonize 5-HT_1B/D receptor-mediated functional responses. The compound indeed exhibits a very high affinity and selectivity for 5-HT_1B/D binding sites and it antagonizes a number of 5-HT_1B/D receptor-mediated responses. The present experiments were performed to investigate the selectivity of GR127935 against functional responses mediated by 5-HT₁-like, ‘orphan’ 5-HT₁-like (5-ht₇?), 5-HT₂, 5-HT₃ or 5-HT₄ receptors in several invivo preparations. Intravenous (i.v.) treatment with GR127935 (300μg?kg^-1) potently antagonized decreases in total carotid blood flow as well as hypotensive responses induced by the 5-HT₁-like receptor agonist sumatriptan in rabbits. I.v. bolus injections of GR127935 (up to 500 and/or 1500μg?kg^-1) did not significantly modify 5-HT-induced: (i) tachycardia in the pig (5-HT₄ receptor-mediated) and cat (‘orphan’ 5-HT₁-like or, perhaps, 5-ht₇ receptor-mediated); (ii) depressor effects in the rat and cat (‘orphan’ 5-HT₁-like or 5-ht₇ receptor-mediated); (iii) vonBezold-Jarisch reflex in the rat or the early phase of the urinary bladder contraction in the cat (both 5-HT₃ receptor-mediated). In contrast, high doses (500-1500μg?kg^-1) of GR127935 suppressed 5-HT-induced pressor responses in the rat and cat and urinary bladder contractions (secondary phase) in the cat as well as the DOI ((±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride)-induced pressor responses in the rat, which are all mediated by 5-HT_2A receptors. In conclusion, the present study demonstrates that GR127935 is a selective 5-HT_1B/D receptor antagonist devoid of interactions at ‘orphan’ 5-HT₁-like (5-ht₇?), 5-HT₃ and 5-HT₄ receptors. However, GR127935 possesses a moderate 5-HT_2A receptor blocking property, which is consistent with its binding profile (pK_i: 7.4). Lastly, in view of the potent antagonist action of GR127935, the sumatriptan-induced hypotension in rabbits seems to be mediated by 5-HT_1B/D receptors.     

Biochemical and behavioral studies of the 5-HT1B receptor agonist,CP-94,253

CP-94,253, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine, a new serotonergic ligand, was found to exhibit significantly greater binding affinity at 5-HT_1B receptors than at 5-HT_1A or 5-HT_1C receptors. Saturation studies showed CP-94,253 to be a competitive inhibitor of [¹²⁵l]iodocyanopindolol binding to 5-HT_1B sites. Its competition curve with this radioligand was shifted to the right (decreased affinity) in the presence of Gpp(NH)p, indicating an agonist function for CP-94,253. Oral administration of CP-94,253 to rats caused inhibition of food intake, decrease in body weight gain, and hyperlocomotion, effects apparently elicited via activation of 5-HT_1B receptors. © 1992 Wiley-Liss, Inc.