生物技术药物免疫原性的评价及面临的挑战

随着大量生物技术药物的研发，免疫原性的评价成为阐明这些药物临床安全性和有效性的关键因素。评价非期望出现的免疫原性是生物技术药物临床前和临床评价的重要内容。完整的免疫原性评价应是方法学的验证，选择合理的免疫学和生物学方法检测抗体滴度的变化及分析抗体亚型和特性、测定抗体的中和活性、免疫复合物的形成及沉积，分析抗体产生对药物药动学、药效和毒性反应等影响。目前免疫原性评价面临的挑战是免疫学检测方法的优化以及提高动物和体外模型的预测性。     

生物技术药物免疫原性检测中改善药物耐受研究进展

随着大量生物技术药物的研发,生物技术药物的评价越来越受到重视.与传统小分子药物不同,生物技术药物在体内产生的抗药抗体(ADA)可能会中和药物的活性,造成在非临床和临床研究中观察到的效应并非药物真正的药理和毒性反应,因此需要对药物的免疫原性进行评价.通过检测ADA可评价药物的免疫原性.检测ADA的主要干扰物质为药物本身....     

生物技术药物临床前安全性评价中的免疫原性问题

由于生物技术药物具有结构和生物学性质的专一性和多样性,包括种属特异性、免疫原性和无法预料的多种组织亲和性等特性,常规的化学药和中药临床前安全性评价方法不一定适合生物药物。生物技术药物的高度种属特异性及其可能产生的免疫原性与体液和细胞免疫反应,外加在临床广泛应用中的多因性,对其临床前安全评价尚有较大的难度,研究很不充分。现有的临床前安全评价方法还很不完善,如未能选择相关动物种属和有针对性且灵敏的观察指标,给药方式与给药量的合理性也均有待探索,甚至还不能判定其在动物体内反应的临床意义,因此规范的评价方案有待在大量的实践中逐步完善。     

单抗类药物的免疫原性问题及其控制

单抗类药物(单克隆抗体药物和受体-Fc融合蛋白药物)近年来在癌症和自身免疫性疾病等领域取得了显著的疗效。根据诱发的抗药物抗体的类型不同,单抗类药物的免疫原性可能导致不同的临床后果,直至影响单抗类药物的有效性和安全性。单抗类药物免疫原性的发生机制尚没有明确的定论,可能与多种因素有关,其中蛋白质多聚体对免疫原性有显著影响。根据"质量源于设计"的原则,从单抗类药物的分子设计和工艺设计出发,采取人源化改造、开发人类抗体、检测和控制产品中多聚体含量等,有助于降低单抗类药物的免疫原性,实现单抗类药物的有效性、安全性和可生产性之间的平衡。     

单克隆抗体药物临床前安全性评价中的几个问题

单克隆抗体的本质是免疫球蛋白,与化学小分子药物和中药制剂相比,具有较慢的分布动力学、半衰期长、免疫原性强等特点.有些单克隆抗体的作用机制还不完全清楚,因此在单克隆抗体安全性评价实际操作时有一定难度.综述单克隆抗体临床前安全性评价中所遇到的问题,诸如动物种属/模型选择、药理/毒理学研究和免疫原性研究,为单克隆抗体的临床前安全性评价提供值得借鉴的方法和评价依据.     

生物技术药物临床前安全性评价中的免疫原性

生物技术药物的临床前安全性评价与化学药品和中药制剂相比较应注意其特殊性 ,常规的药物毒性试验方法不一定适合于生物药物 ,因为后者具有结构和生物学性质的专一性和多样性 ,包括种属特异性、免疫原性和无法预料的多种组织亲和性。近来该类药物临床前安全性评价中的相关动物种属特异性、免疫原性、体液与细胞免疫活性与联合用药反应等问题已引起广泛注意。生物技术药物由于高度种属特异性及其可能产生的免疫原性与免疫反应 ,外加在临床广泛应用中的多因性 ,对其临床前安全评价尚有较大的难度 ,现有生物技术药物的临床前安全评价方法还很不完善 ,如未能选择相关动物种属与有针对性而灵敏的观察指标 ,给药方式与给药量的合理性均有待探索 ,甚至还不能判定其在动物体内反应的临床意义 ,因此规范的评价方案有待在大量的实践中逐步完善。     

生物技术药物的免疫毒性和免疫原性研究

生物技术药物由于其结构、生产工艺、保存条件、可能存在的内源性对等物和作用靶点明确等固有的特点，因此在临床前与临床研究阶段有必要进行免疫毒性和免疫原性研究，也是我国申报生物技术药物临床试验常规需要进行评价的内容。免疫毒性是指受试品引起免疫抑制或增强、过敏反应或自身免疫反应，可能与药理活性相关（如抗排斥药物）或不相关（如部分抗肿瘤药物）。     

临床前生物技术药物诱导免疫复合物的形成机制及检测方法研究进展

生物技术药物的免疫原性在临床前药物安全性评价中表现为实验动物的超敏反应以及肾损伤,但其诱导免疫复合物形成的机制尚未完全明确。目前国内外主要通过检测动物体内循环态和沉积态的免疫复合物,来综合判断生物技术药物是否对动物造成免疫复合物性肾损伤。简述了临床前生物技术药物诱导免疫复合物的形成、清除、沉积、毒性反应以及检测方法的研究进展,以期为建立早期、简便、灵敏的生物技术药物免疫复合物性肾损伤的检测技术提供一定参考。     

抗体偶联药物临床前安全性评价考虑要点

抗体偶联药物（Antibody Drug Conjugate,ADC）,是通过连接子将高细胞毒性化合物连接到靶向肿瘤抗原的抗体上,利用抗体靶向识别将化合物递呈至肿瘤细胞表面,杀死肿瘤细胞。ADC药物同时具有抗体和化学药物属性,是将两者的优势结合,极大地提高了药物安全有效性。ADC药物结构复杂,在抗原识别表位、连接位点、连接子以及小分子药物各组分均存在特异性,所以在开展安全性评价研究时有一定的特殊性。本文将从动物种属选择、一般毒性研究、毒代动力学研究、组织交叉反应、免疫原性检测、安全药理研究、遗传毒性研究等方面,阐述ADC药物临床前安全性评价的考虑要点和研究策略。     

基因治疗产品的生物分析：方法开发的策略

基因治疗产品采用独特的递送和治疗机制,评估其安全性和有效性需要设计并开发非传统和全面的生物分析方法。在临床前和临床生物分析研究涉及药代动力学、药效学、免疫原性、生物标志物分析等方面。基因治疗产品的药代动力学和药效学研究需要确定载体、目的基因、以及目的基因表达产物蛋白的存在、作用部位和生物分布。而对于免疫原性的研究,则需要监测机体针对基因治疗载体所产生的抗体,以及可能需要在接受治疗前评估受试者的预存抗体。由于基因治疗产品表达的外源产物蛋白也可能引发机体的免疫反应,因此针对表达产物的抗体也是免疫原性的研究内容。本文从药代动力学、药效学与免疫原性分析研究方向,综述基因治疗产品的生物分析方法开发的关键点、策略和实践。     

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.     

Validation of a de-immunization strategy for monoclonal antibodies using cynomolgus macaque as a surrogate for human

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human–cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336–672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.     

抗体药物的国内外市场动态

生物制药是近年来医药行业中增长最迅速的行业之一,治疗性抗体作为生物药物中最耀眼的明珠已成为生物制药的最大产品类别,约占生物制药的35%。抗体药物在治疗以肿瘤为主的人类多种疾病方面起着重要作用。2011年,全球抗体药物的销售额达到480亿美元。我国当前正处在抗体药物快速发展阶段,目前SFDA共批准了17种单抗。预计到2015年,我国抗体销售额将达到325～650亿元。从产品来看,虽然我国已经有所突破,但数量和类型上,与国外的抗体药物还有很大差距。随着生物技术的不断发展,抗体药物的市场前景将会越来越广泛。     

Immunogenicity of rDNA-derived pharmaceuticals

Many biopharmaceuticals have been reported to induce the production of antibodies. Several factors influence immunogenicity, including the methods of production, formulation and storage as well as patient characteristics (e.g. type of disease and genetic background). Because immunogenicity is unpredictable, testing for antibodies should always be part of the evaluation of new biopharmaceuticals, although the biological consequences of these antibodies are rarely severe.     

Clinical concerns of immunogenicity produced at cellular levels by biopharmaceuticals following their parenteral administration into human body

Similar to the low molecular weight traditional drugs, biopharmaceuticals are capable of producing not only therapeutic effects but also side effects provided if the dose of these compounds exceeds certain concentration and/or if the exposure duration of these compounds at subtoxic doses is being lengthened. In addition, a major drawback of biopharmaceuticals is the risk of antibody formation. Following the administration of biopharmaceuticals into human body, the formation of antidrug–antibody (ADA) or neutralizing antibody and other general immune system effects (including allergy, anaphylaxis, or serum sickness) are of clinical concern regarding therapeutic efficacy and patient safety. For example, drug-induced neutralizing antibodies to erythropoietin (EPO) result in pure red cell aplasia, whereas drug-induced acquired anti-factor VIII antibodies worsen the pathology associated with hemophilia. Since most of the already developed or under development biopharmaceuticals are to some extent immunogenic, the regulatory agencies insist to conduct potential ADA formation during the drug development process itself. This review encompasses a short overview on the clinical concerns of immunogenicity produced at cellular levels by growth hormone, interferon-α, EPO, factor VIII, and factor IX following their parenteral administration into human body. Clinical concerns related to immunogenicity produced by the biosimilar versions of these drugs are also presented wherever possible.     

A double antigen bridging immunogenicity ELISA for the detection of antibodies to polyethylene glycol polymers

Introduction

Polyethylene glycol (PEG) polymers attached to biotherapeutic molecules enhance in vivo delivery and stability of these large molecular weight drugs. However, these polymers may by themselves be immunogenic and elicit antibodies that can reduce the efficacy of the drug and contribute to potential patient morbidity. A double antigen bridging ELISA immunogenicity assay for the detection of anti-drug antibodies (ADAs) specific to PEG polymers of various sizes has been developed.

Methods

Hapten-labeled conjugate of 40 kDa PEG polymer was synthesized and used in a double antigen bridging ELISA. The hapten-labeled PEG is incubated with the patient sample, then this mixture is added to a 96-well microplate precoated with 40 kDa PEG, allowing PEG-specific ADA to form a bridge complex with the PEG conjugate and the PEG coated on the microplate. After incubation, the reaction mixture is removed and replaced by horseradish peroxidase (HRP)-labeled anti-hapten antibody. After sufficient incubation, the plate is washed and substrate reagent is added. Enzyme color development, directly proportional to ADA, is stopped after 20 min with 2N sulfuric acid and the absorbance in each well is measured at 450/630 nm. Dose response, drug tolerance, matrix effects, reproducibility, specificity/free drug depletion experiments and screening cut-point determination of 350 naïve normal human sera were performed.

Results

Using an anti-PEG mouse monoclonal IgM as a positive control, a reproducible dose response curve was demonstrated for the PEG Immunogenicity ELISA. Pre-existing PEG-specific antibodies which were proven to be highly specific to the PEG polymer structure were found in 15 human serum samples in a total population of 350 naïve donors. The assay exhibited no significant matrix effects and was shown to be highly reproducible.

Discussion

A double antigen bridging immunogenicity assay for the detection of antibodies to PEG in the typical polymer size ranges used in biotherapeutics has been successfully developed in ELISA format. The antibodies detected in positive samples displayed a diverse spectrum of specificities for different PEG polymer lengths and linking functional groups. The discovery of 15 confirmed positive samples among 350 naïve patient samples calls into focus the need for testing PEG-specific immunogenicity of PEGylated biotherapeutics.     

Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA

The evaluation of the potential immunogenicity of therapeutic proteins in nonclinical safety studies has become complicated by the development of biopharmaceuticals that are dosed at high concentrations and/or have long half lives. These products remain in the circulation of the test system for extended periods of time, resulting in samples containing high concentrations of drug that interfere with standard immunogenicity assays. This protocol describes a novel solid-phase extraction with acid dissociation (SPEAD) sample treatment that removes the interfering therapeutic protein "drug" from the sample prior to performance of a direct immunoassay for detection of anti-drug antibodies (ADA). A biotin-avidin capture technique is used to physically separate ADA and ADA:Drug complexes from the drug and the sample matrix. The acid dissociation step removes the ADA from the biotin-avidin complex, and detection is performed by simple direct enzyme-linked immunoassay (ELISA). The SPEAD treatment allows for detection of ADA in an ELISA format and results in a >10-100-fold increase in residual drug tolerance as compared to an immunoassay without the sample treatment. The method can be used for serum samples from all species, but is presented here as an assay for detection of anti-drug antibodies in cynomolgus monkey serum.     

Submicron Size Particles of a Murine Monoclonal Antibody Are More Immunogenic Than Soluble Oligomers or Micron Size Particles Upon Subcutaneous Administration in Mice

Protein aggregates are one of the several risk factors for undesired immunogenicity of biopharmaceuticals. However, it remains unclear which features determine whether aggregates will trigger an unwanted immune response. The aim of this study was to determine the effect of aggregates' size on their relative immunogenicity. A monoclonal murine IgG1 was stressed by exposure to low pH and elevated temperature followed by stirring to obtain aggregates widely differing in size. Aggregate fractions enriched in soluble oligomers, submicron size particles and micron size particles were isolated via centrifugation or size-exclusion chromatography and characterized physicochemically. The secondary and tertiary structures of aggregates were altered in a similar way for all the fractions, while no substantial chemical degradation was observed. Development of anti-drug antibodies was measured after subcutaneous administration of each enriched fraction to BALB/c mice. Among all tested fractions, the most immunogenic was the one highly enriched in submicron size particles (～100-1000 nm). Fractions composed of micron size (>1-100 μm) particles or soluble oligomers (<100 nm) were not immunogenic under the dosing regimen studied in this work. These results show that aggregate size is an important factor for protein immunogenicity.