提高抗体库容量和亲和力方法的新进展

抗体库为抗体工程带来了革命性的突破,其核心是将抗体的基因型与表型耦联,从抗体库中找到针对特异抗原的抗体基因。但是抗体工程发展所面临的两大难点是如何构建大库容量的抗体库和提高抗体的亲和力。抗体库方法和技术的出现使得改造工程抗体更方便、更有效。利用不同抗体库方法构建大库容量的突变抗体库,然后通过抗体库技术在体外模拟抗体亲和力成熟过程获得高亲和力的抗体。本文简要综述了抗体库方法和技术的最新进展。     

噬菌体展示技术在抗体亲和力成熟中的应用进展

抗体因其高度的特异性在诊断和靶向治疗上一直备受青睐,相关行业发展也是突飞猛进。噬菌体展示技术作为一种抗体库构建技术,不仅可应用于特异性抗体的筛选,也可应用于对已获得的低亲和力抗体进行亲和成熟的研究。该方法主要利用VH和VL的随机重组,能在一定程度上模拟体内抗体亲和力成熟的过程,使噬菌体展示技术在提升抗体亲和力方面拥有许多优势,以下对噬菌体展示技术在亲和力成熟方面的应用进行综述。     

抗CTLA-4嵌合抗体的制备及生物学活性鉴定

制备新型抗CTLA-4人鼠嵌合抗体并进行活性鉴定。通过杂交瘤技术获得高亲和力小鼠抗人CTLA-4单克隆抗体22G11和16C11;利用分子克隆技术将鼠源抗体可变区基因与人源抗体恒定区拼接后,最终通过CHO-K1工程株细胞表达高亲和力抗CTLA-4嵌合抗体。经SDS-PAGE电泳显示最终获得了纯度高于90%的CTLA-4嵌合抗体c22G11和c16C11,抗原结合活性结果表明两株嵌合抗体都能很好地与Jurkat细胞结合,竞争抑制实验表明它们都能与各自对应的鼠源抗体竞争。据此,本实验获得了两株抗人CTLA-4胞外区的高亲和力和特异性嵌合抗体。     

基因工程抗体亲和力成熟的策略

近年来，抗体因其高度的特异性在诊断和药物的靶向治疗上备受青睐，相关行业的发展也极为迅猛。在体内诊断和治疗上，基因工程抗体具有众多鼠源性单克隆抗体无可比拟的优势，但前者亲和力低是制约其实际应用的瓶颈问题。作者将就基因工程抗体亲和力成熟的策略及其应用进行综述。     

硫氰酸盐洗脱法测定噬菌体抗体的相对亲和力

目的　建立一种测定噬菌体抗体相对亲和力的方法。方法　参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法 ,在ELISA实验中以酶标抗M13为二抗检测 5个单克隆噬菌体抗体的相对亲和力 ,并与可溶性Fab段的相对亲和力进行比较。结果　噬菌体抗体可以耐受硫氰酸盐的洗脱 ,用硫氰酸盐洗脱法测定 5个噬菌体抗体所得亲和力排序与测定其相应可溶性抗体分子片段所得结果一致。结论　硫氰酸盐洗脱法可用于噬菌体抗体相对亲和力的测定。     

人源化鼠抗人纤维蛋白单链抗体的体外成熟

目的 提高人源化鼠抗人纤维蛋白单链抗体的亲和力和特异性，方法 以人源化鼠抗人纤维蛋白单链抗体为基础，通过合成部分随机化的突变引物和PCR等方法构建HCDR3和LCDR3的混合突库，然后利用噬菌体表面呈现技术对抗体库进行筛选，并利用本实验室建立的单链抗体－碱性磷酸酶系统进行单克隆的鉴定。结果 得到4株亲和力和特异性有明显改善的人源化抗体。结论 构建CDR3突变库是抗体体外成熟的一个有效方法，该研究为研制低免疫原性的导向溶栓制剂奠定了基础。     

用硫氰酸盐洗脱法筛选高亲和力噬菌体抗体

目的 :建立一种从抗体库中筛选高亲和力噬菌体抗体的方法。方法 :选择已知亲和力高低不同的噬菌体抗体以不同的比例组成模拟抗体库 ,在常规筛选程序的基础上 ,用不同浓度的硫氰酸盐进行洗脱 ,选择性去除低亲和力抗体 ,保留高亲和力体 ,对最后获得的噬菌体抗体制备质粒DNA进行内切酶谱分析 ,判断其中高亲和力抗体的比例。结果 :实验证明工作浓度硫氰酸盐的孵育不影响噬菌体颗粒的感染活性 ;抗体库筛选过程中用硫氰酸盐进行洗脱 ,发现随着硫氰酸盐浓度的增加 ,洗脱回收的噬菌体抗体中高亲和力克隆的比例逐渐升高。结论 :硫氰酸盐洗脱法用于噬菌体抗体的筛选有助于成功获得期望的高亲和力抗体。     

人源性抗乳腺癌单链抗体库的筛选与初步鉴定

目的:本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定。方法:以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况。结果:成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白。结论:本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库。研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础。     

四环素对侵袭性牙周炎血清抗牙龈卟啉菌IgG抗体亲和力影响的研究

目的：评价结合四环素的牙周非手术治疗对侵袭性牙周炎（AgP）患者牙周附着水平（AL）和血清抗牙龈卟啉菌（Pg）抗体亲和力水平的影响。 方法:研究对象由25名侵袭性牙周炎患者、20名牙周健康者（HS）组成。AgP患者在接受机械性牙周治疗后给服四环素(0.5 g/d)，3个月疗程结束后对病人进行评估。牙周治疗前及治疗3个月、6个月和12个月后常规临床检查、记录牙周附着水平，通过硫氰酸铵分离试验测定血清抗牙龈卟啉菌脂多糖（LPS）的抗体亲和力。 结果:牙周治疗后，牙周附着水平有显著改善(P<0.01);AgP患者血清抗Pg抗体亲和力明显下降(P<0.01)。 结论:结合四环素的机械性牙周治疗能显著降低AgP患者血清抗Pg抗体的亲和力对侵袭性牙周炎可获得满意的疗效。     

联合应用错配PCR和交替延伸PCR法提高抗TNFα抗体亲和力

目的：通过突变方法提高抗TNFα单链抗体pscTNF的亲和力。方法：以pscTNF质粒为模板，应用错配PCR构建突变抗体库，筛选亲和力得到改善的变种，再以所获变种为模板，用交替延伸PCR技术，再次构建突变库，筛选活性得到改良的克隆。以斑点ELISA方法及硫氰酸盐洗脱ELISA法评估其亲和力的改善。结果：从错配PCR抗体库中筛选得到7个活性有所增强的变种，再通过交替延伸PCR使这7个变种的突变位点交换重组，获得了亲和力进一步提高的克隆。结论：联合应用错配PCR和交替延伸PCR法可提高单链抗体亲和力。     

Single-domain antibodies for biomedical applications

Abstract

Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.     

基因工程双价抗体在肿瘤中的研究进展

基因工程抗体技术的发展加速了单链抗体的应用,但其稳定性差,亲和力低,功能单一,体内清除过快等特点影响了它的广泛应用。双价抗体作为一种新型小分子抗体,具有双价的结合位点,能够使抗原分子上的两个表位交联或使两个分子连接,可以模拟完整的单克隆抗体的抗原抗体反应,其构建方法有亮氨酸拉链法、利用部分抗体恒定区法、连接肽法、利用双聚化结构法、knobs into holes技术等,在乳腺癌、 直肠癌、淋巴瘤等的诊治方面均有很好的应用价值。     

8th FIMSA/IIS Advanced Course on Immunology: Focus on Clinical Immunology

Anticalins^® are engineered ligand-binding proteins based on the human lipocalin scaffold. Their architecture is characterized by a rigid β-barrel that supports four structurally hypervariable loops. Similar to antibodies, these loops form the natural ligand-binding site, usually for vitamins, hormones or secondary metabolites. Anticalins with novel specificities can be engineered by reshaping this loop region, using targeted random mutagenesis in combination with functional display and guided selection. Several drug candidates with specificities for exogenous low-molecular-weight substances, peptides and even protein targets (e.g., several disease-related cell surface receptors) have been obtained in this way. Owing to their exquisite specificity and high affinity, Anticalins are particularly attractive as antagonists for the manipulation of immune mechanisms, leading to either inhibitory or stimulatory effects. Compared with antibodies, Anticalins offer several practical advantages as they are much smaller, consist of a single polypeptide chain and can be produced easily in microbial expression systems.     

Development of Novel Monoclonal Antibodies for the,Analysis of Functional Sites in FGF-2

Abstract

Fibroblast growth factor 2 (FGF-2) can function as a potent mitogen, as well as a survival factor for a variety of mammalian cell types. The biological effects of FGF-2 are mediated by its interaction with two types of cellular binding sites: (1) high affinity tyrosine kinase receptors; and (2) low affinity heparan sulfate proteoglycans (HSPGs) on the cell surface. Although numerous FGF-2 antibodies have been used previously to analyze its biological actions, few studies have utilized antibodies to analyze domains within FGF-2 involved in its interactions with the two binding sites. In this report, we describe the generation and use of two monoclonal antibodies against human recombinant FGF-2 (254F1 and 2S6A12) that inhibit FGF-2 function. However, these antibodies appear to target preferentially different domains within the FGF-2 molecule, and therefore differentially influence the interactions of FGF-2 with its low and high affinity receptors. 254F1 is a more effective inhibitor of the high affinity, receptor tyrosine kinase binding site, whereas 256A12 appears to be a better inhibitor of the low affinity, HSPG interactions. We also demonstrate that the two antibodies are potent inhibitors of FGF-2 stimulated vascular cell proliferation, and as such have potential use in the treatment of vascular hyperproliferative diseases.     

Fc Receptors as Targets for Immunotherapy

Human membrane and soluble Fee receptors (FcεRI, FcεRII/CD23) and Fcγ receptors (FcγRI/CD64, FCγRII/CD32, FcγRIII/CD 16) have been implicated in a number of diseases. Their functional roles such as capture and clearance of immune complexes, antibody-dependent cell cytotoxicity, or cytokine or inflammatory mediator release, make them potential targets for immuno-intervention. In the present review, we will describe how membrane and soluble human FcεR and FcγR have been already used as targets/tools for immuno-interventions by using monoclonal and bispecific engineered antibodies. Some therapeutic uses of these molecules both in cancer, infectious, and auto-immune diseases are presented.     

Activating and inhibitory Fcγ receptors in immunotherapy: being the actor or being the target

Membrane Fcγ receptors (FcγRs) can act either as potent activators of effector cell functions or as inhibitors of receptor-mediated cell activation following engagement by IgG antibodies bound to their target molecules. The remarkable ability of activating FcγRs to trigger antibody-dependent cellular cytotoxicity, cytokine release and phagocytosis/endocytosis followed by antigen presentation has stimulated the development of a number of therapeutic monoclonal antibodies whose Fc regions have been engineered to optimize their effector functions, mostly their killing activities. Conversely, the demonstration that inhibitory FcγRs can block or downmodulate effector functions has led to the concept that targeting these receptors is of interest in a number of pathologies. The use of bispecific antibodies leading to the crosslinking of FcγRIIB with activating receptors could induce immunomodulation in autoimmune or allergic diseases. Alternatively, the use of cytotoxic/antagonist anti-FcγRIIB antibodies could kill FcγRIIB-positive tumor cells or prevent the downmodulation of activating receptors. Thus, antibodies engineered to preferentially target activating or inhibitory FcγRs are currently being designed for therapeutic use.     

Humanized antibodies as potential drugs for therapeutic use

Antibodies have been used therapeutically to treat a variety of clinical conditions. The introduction of monoclonal antibodies (mAb) and, recently, engineered antibodies has greatly refined and expanded the therapeutic potential of this modality of treatment. Expanded use will depend on improvement in their efficacy (affinity and specificity), demonstration of their safety, and reduction of their immunogenicity depending on the size, suboptimal biodistribution and pharmacokinetics. To surmount these problems the molecules have to be redesigned and the basic issues of how monoclonal antibodies kill cells reinvestigated. The review will survey the literature for humanized antibodies in clinical trials and the perspective of the use of mAbs or engineered antibodies in clinical practice.     

Theoretical considerations of the ability of monoclonal antibodies to detect antigenic differences between closely related variants, with particular reference to heterospecific reactions

In theory monoclonal antibodies can be used to analyse antigenic determinants in great detail by correlating differences in antibody affinity for variant antigens with their amino acid differences. In particular, heteroclitic antibodies should be detected, which would normally be masked in a polyclonal antiserum. Recognition of such antibodies may be important for our understanding of the scope of antibody repertoires particularly when the immunogen is closely related to a component of the immunised animal. In practice the immunoassays commonly used to measure affinity differences between different antigens fall short of these capabilities. Mathematical studies were carried out to identify factors controlling the sensitivity of 4 types of assay to differences in affinities for different antigens. The most important factors controlling assay sensitivity were found to be the ratio of antibody affinity (K) to epitope density in direct binding assays, the ratio of K to antibody concentration in liquid phase competition assays, and the ratio of solid phase to liquid phase values of K for solid-phase competition assays. It is predicted that a combination of solid-phase competition assay with high epitope density and direct binding assay with low epitope density would result in optimal detection of heteroclitic antibodies and small differences in antibody affinity for cross-reactive antigens.     

A potassium ion channel is involved in cytokine production by activated human macrophages

Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0.5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.     

Development trends for generation of single-chain antibody fragments

Recombinant antibodies are increasingly being employed as therapeutic agents especially in combination with anti-cancer drugs. The single-chain antibody fragments are small antigen-binding proteins which provide the most commonly used antibody formats for diagnostic and therapeutic purposes. These antibody fragments have more rapid tumor penetration and clearance from the serum relative to full-length monoclonal antibodies. There are in vitro antibody-display technologies such as phage display, cell surface display, ribosome display and mRNA display that can be used to isolate high specificity and affinity single-chain antibodies against a wide variety of targets. We review these strategies for generation of stable and active antibody fragments in the present article.