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评价的技术发展概述

药物的免疫原性是指药物刺激机体形成特异抗体或致敏淋巴细胞的性质。评价非期望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。免疫原性评价内容包括方法的开发、验证、药物诱导的抗体反应水平检测（滴度）、抗体的特性研究、抗体的中和活性测定、分析抗体产生对疗效、毒性和药动学的影响,并预测对人体潜在的免疫原性强弱。如何提高动物模型的预测性、优化检测技术、实现评价方法的规范化和标准化,是未来免疫原性评价亟需解决的问题。     

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。     

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。     

生物技术药物的发育和生殖毒性评价

由于生物技术药物与小分子药物本身存在差异,发育和生殖毒性(DART)评价应考虑受试物的种属特异性、免疫原性、生物活性和暴露情况,并采取灵活、个案处理和基于科学的方法进行研究.当传统种属(如啮齿类、兔)不是药理学相关物种时,DART评价首选非人灵长类动物模型.若生物技术药物只与人或黑猩猩产生交叉反应,DART评价可使用针对传统种属开发的同源蛋白(替代分子)作为受试物.此外,还可使用转基因动物进行DART评价.     

《生物技术药物安全性评价》书讯

药物安全性评价是新药研发中十分重要的研究内容之一。对于生物技术药物而言，由于其种类繁多，性质各异，机理复杂，并多具有明显的免疫原性等特点，使得其安全性评价研究变得更加复杂。随着我国生物技术药物的快速发展，对从事药物安全性评价研究的技术人员，既是机遇也是挑战。由中国药品生物制品检定所副所长王军志研究员主编（副主编：李波研究员、王秀文研究员）的《生物技术药物安全性评价》一书已于2008年3月首次出版。该专著内容凝聚了我国生物技术药物安全评价领域近年来最新研究成果，以便本领域科技工作者参考和借鉴。     

《生物技术药物安全性评价》出版

药物安全性评价永远是新药研发中十分重要的研究内容之一。对于生物技术药物而言，由于其种类繁多，性质各异，机理复杂，并多具有明显的免疫原性等特点，使得其安全性评价研究变的更加复杂。随着我国生物技术药物的快速发展，对从事药物安全性评价研究的技术人员，既是机遇也是挑战。由中国药品生物制品检定所副所长王军志研究员主编（副主编：     

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。     

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）。     

生物技术药品的药物经济学

随着生物技术药品的成熟和发展，需要用药物经济学原理和方法评价生物技术药品的特殊价值，本文介绍了药物经济学的原理，常用的评价方法和评价步骤，并结合生物技术药品的特点，以三种类型说明药物经济学的应用特征，药物经济学评价是评价药品评值的有力工具，对生物技术药品尤为重要。     

Strategies to estimate and improve drug tolerance in anti-drug antibody assays

Background: Drug tolerance of anti-drug antibody (ADA) assays is becoming increasingly important due to the high number of newly emerging long-acting drugs. Methods to estimate and improve drug tolerance in ADA assays are needed. Results: The relevance and precision of drug tolerance estimates in a bridging ELISA depended on characteristics and concentration of the surrogate control antibody together with assay cut point level. Stepwise and coincubation procedures were optimized for drug tolerance and sensitivity by adapting concentrations of reagents used for capture and detection of ADAs. In combination with acid treatment of samples, increase in drug tolerance of over 20-fold was achieved without losing sensitivity in two different assays. Acid treatment reduced drug interference observed in preclinical samples and prevented underestimation of ADA response. Conclusion: In a risk-based approach it may be possible to reliably predict in vivo drug tolerance using carefully chosen surrogate controls. General guidelines for development of drug-tolerant bridging ELISAs are presented.     

Comparison of assay formats for drug-tolerant immunogenicity testing

Background: Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development. Results: Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(?) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive. Conclusions: Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.     

Collaborative study using common samples to evaluate the performance of anti-drug antibody assays constructed by different companies

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.     

Evaluation of a generic immunoassay with drug tolerance to detect immune complexes in serum samples from cynomolgus monkeys after administration of human antibodies

Current state of the art bridging ELISA technologies for detection of anti-drug antibodies (ADAs) against therapeutic antibodies bear the risk of false-negative results due to interference by circulating drug. Methods to remove the drug in the sample or sample pre-treatment techniques such as acid dissociation of the immune complexes are limited, laborious and may destroy ADAs resulting again in false-negative results. The immune complex ELISA described in this publication provides a simple solution. It is designed to analyze samples from cynomolgus monkeys dosed with human antibodies; it can be used for all human antibodies since it is independent of the specific antibody and its target. The generic applicability of the ADA assay is enabled by the use of (1) a murine anti-human Fc monoclonal antibody (MAb) as capture reagent; (2) a murine anti-cynomolgus monkey IgG MAb as detection reagent; and (3) an ADA positive control conjugate consisting of cynomolgus IgG complexed with human IgG. In its basic version, the generic ADA ELISA specifically detects only immune complexes formed in vivo. Validation of the ADA assay revealed a lower limit of quantitation of 15.6 ng/mL in serum samples. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 8%. Matrix effects were low as evidenced by a mean recovery of 95%. In vitro pre-incubation of the serum samples with drug makes also the free ADA in the sample amenable to measurement by the immune complex ELISA as demonstrated by analysis of ADAs from two cynomolgus monkey studies with two different antibodies. The generic and versatile nature of this ADA assay favors its use in pilot pharmacokinetic and safety studies in cynomolgus monkeys during candidate selection of antibodies. The assay can help to explain unexpected drug clearance profiles, loss of efficacy or safety events caused by immune complexes and guide further development.     

Advancements in Understanding Immunogenicity of Biotherapeutics in the Intraocular Space

Therapeutic breakthroughs in a number of retinal degenerative diseases have come about through the development of biotherapeutics administered directly into the eye. As a consequence of their use, we have gained more insight into the immune privileged status of the eye and the various considerations that development, manufacturing, and use of these drugs require. It has been observed that therapeutic proteins injected into the vitreous can elicit an immune response resulting in the production of anti-drug antibodies (ADAs) which can have clinical consequences. This review includes discussion of the anatomy, physiology, and specific area of the eye that are targeted for drug administration. The various immunologic mechanisms involved in the immune responses to intraocularly administered protein are discussed. This review entails discussion on chemistry, manufacturing, and control (CMC) and formulation-related issues that may influence the risk of immunogenicity. Based on the available immunogenicity profile of the marketed intraocular drugs and their reported adverse events, the animal models and the translational gap from animals to human are discussed. Thus, the objective of this review article is to assess the factors that influence immunogenicity in relation to intraocular administration and the steps taken for mitigating immunogenicity risks.     

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.     

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.     

Immunogenicity of therapeutics: a matter of efficacy and safety

Importance of the field: The unwanted immunogenicity of therapeutic proteins is a major concern regarding patient safety. Furthermore, pharmacokinetic, pharmacodynamic and clinical efficacy can be seriously affected by the immunogenicity of therapeutic proteins. Authorities have fully recognized this issue and demand appropriate and well-characterized assays to detect anti-drug antibodies (ADAs).

Areas covered in this review: We provide an overview of the immunogenicity topic in general, the regulatory background and insight into underlying immunological mechanisms and the limited ability to predict clinical immunogenicity a priori. Furthermore, we comment on the analytical testing approach and the status-quo of appropriate method validation.

What the reader will gain: The review provides insight regarding the analytical approach that is expected by regulatory authorities overseeing immunogenicity testing requirements. Additionally, the factors influencing immunogenicity are summarized and key references regarding immunogenicity testing approaches and method validation are discussed.

Take home message: The unwanted immunogenicity of protein therapeutics is of major concern because of its potential to affect patient safety and drug efficacy. Analytical testing is sophisticated and requires more than one assay. Because immunogenicity in humans is hardly predictable, assay development has to start in a timely fashion and for clinical studies immunogenicity assay validation is mandatory prior to analyzing patient serum samples. Regarding ADAs, the question remains as to when such antibodies are regarded of clinical relevance and what levels are, if at all, acceptable. In summary, the detection of ADAs should raise the awareness of the physician concerning patient safety and of the sponsor/manufacture concerning the immunogenic potential of the drug product.     

Pharmacological strategies for mitigating anti-TNF biologic immunogenicity in rheumatoid arthritis patients

Tumor necrosis factor alpha (TNFα) inhibitors are a mainstay of treatment for rheumatoid arthritis (RA) patients after failed responses to conventional disease-modifying antirheumatic drugs (DMARDs). Despite the clinical efficacy of TNFα inhibitors (TNFi), many RA patients experience TNFi treatment failure due to the development of anti-drug antibodies (ADAs) that can neutralize drug levels and lead to RA disease relapse. Methotrexate (MTX) therapy with concomitant TNFα inhibitors decreases the risk of TNFi immunogenicity, but additional and/or alternative strategies are needed to reduce MTX-associated toxicities and to further increase its potency for preventing TNFα inhibitor immunogenicity. In this review, we highlight the limitations of MTX for mitigating TNFα inhibitor immunogenicity, and we discuss potential alternative pharmacological targets for decreasing the risk of immunogenicity during TNFα inhibitor therapy based on the key kinases, second messengers, and shared signaling mechanisms of lymphocyte receptor signaling.