双特异性抗体构建在肿瘤临床治疗中的应用

双特异性抗体(Bispecific antibody,BsAb)是具有两个不同抗原结合位点的抗体,可分为含Fc段和不含Fc段的BsAb,不同结构的BsAb具有不同的特点和应用领域。相比于传统的单克隆抗体,BsAb的灵敏度和特异性更高。更重要的是,BsAb具有募集免疫细胞、双重阻断信号通路等功能,在免疫诊断和治疗中扮演重要角色。随着全球环境的恶化以及人们生活习惯的不规律,肿瘤的发病率越来越高,成为仅次于心脑血管疾病的全球第二大致死疾病,全球每年有1200万新发癌症病例。肿瘤的治疗手段包括手术切除、放化疗和靶向治疗等。肿瘤免疫疗法是近几年新兴的治疗方法,其通过激发自身免疫系统的能力来清除肿瘤细胞。传统的单抗药物虽在肿瘤靶向治疗和免疫治疗中取得了一定的疗效,但肿瘤具有高度的异质性和可塑性,常常引发肿瘤耐药性的出现。双特异性抗体能够同时靶向多个靶点,目前已用于肿瘤的临床治疗,并取得了一定的治疗效果。文中就双特异性抗体在肿瘤临床治疗中的研究进展和应用作一综述。     

双特异性抗体的结构及应用研究进展

双特异性抗体(bi-specific antibody,BsAb)是具有双功能的抗体分子,可同时结合两种不同的抗原或表位,将T细胞和自然杀伤细胞重定向到肿瘤细胞,在功能分子(细胞)和靶细胞之间架起桥梁,产生导向性作用。目前,BsAb已广泛应用于癌症、炎症、病毒感染及其他疾病的治疗。现就BsAb的结构、应用等方面的研究进展作一综述。     

HIV双特异性抗体研究进展

双特异性抗体(bispecific antibody,BsAb)是一种人工制备的非自然抗体。该抗体含两种特异性结合位点,能在抗原抗体反应的同时结合多种功能分子与细胞,从而介入引导免疫反应的方向。上述特性使BsAb在对抗人免疫缺陷病毒(human immunodeficiency virus,HIV)时具有广谱效能。目前,BsAb已成为抗HIV研究领域中的一大热点。现就BsAb在抗HIV病毒中表位的设计选择与优化、作用机制等方面的研究进展作一概述。     

双特异性抗体及其在肿瘤治疗中的应用

双特异性抗体（BsAb）是改造抗体治疗效果的发展方向之一,现已成为抗体工程研究领域的热点。在过去20年的研究中．研究人员看到了常规BsAb的潜能以及它的不足。随着分子生物学技术的迅速发展,出现了利用基因工程手段构建的BsAb的多种模式,并且有多种BsAb制剂已经用于肿瘤的初期临床诊断和治疗。该文对BsAb最新的研究进展和肿瘤治疗中的应用进行了阐述。     

A型激酶锚定蛋白的结构和生物学功能

cAMP依赖性蛋白激酶A(PKA)通过A型激酶锚定蛋白(Akinaseanchorproteins,AKAPs)靶向亚细胞位点,PKA识别它的底物或效应蛋白,从而引导并放大cAMP信号的生物学效应。AKAPs是功能上相关的调节蛋白家族,具有结合PKA的保守区和引导AKAPPKA复合体到亚细胞位点的靶向区。AKAPs不仅与PKA相互作用,也与其它信号分子作用,主要是磷酸酶和激酶。AKAPPKA复合体可汇集和整合来自各种通路的信号,该复合体不仅可局部增强cAMP和其它信号,通过降低PKA的基础活性,还可发挥远程效应。     

抗α2δ1/CD3双特异性抗体的制备和功能的初步研究

目的:构建抗α2δ1和CD3的双特异性抗体,并在体外初步评价其杀伤肝癌细胞的功能。方法:通过基因工程技术,构建BiTE形式的anti-α2δ1/CD3双特异性抗体(BsAb),转染Expi 293F细胞96h后,使用镍离子亲和色谱纯化出双特异性抗体,使用流式细胞术检测anti-α2δ1/CD3BsAb对α2δ1和CD3的结合性质,使用Perkin Elmer Operetta高内涵成像仪测定anti-α2δ1/CD3BsAb介导细胞毒性T淋巴细胞(CTLs)对高表达α2δ1的人肝癌细胞Hep-12的杀伤效应,ELISA法检测杀伤过程中CTLs分泌hIL-2和hIFN-γ的变化。结果:anti-α2δ1/CD3 BsAb可以特异性结合α2δ1和CD3,anti-α2δ1/CD3 BsAb可以有效介导CTLs靶向杀伤高表达α2δ1的人肝癌细胞Hep-12,其介导杀伤Hep-12细胞的EC_(50)为8pmol/L,对于低表达α2δ1的人肝癌细胞Hep-11,anti-α2δ1/CD3 BsAb不能介导CTLs发挥杀伤作用,并且在杀伤过程中Hep-12细胞组CTLs释放的hIL-2和h IFN-γ比Hep11细胞组显著增多(P 0.05)。结论:anti-α2δ1/CD3 BsAb能有效介导CTLs体外杀伤高表达α2δ1的人肝癌细胞Hep-12,为双特异性抗体的肝癌免疫治疗奠定了一定的基础。     

膜联蛋白A1及其与肿瘤关系的研究进展

膜联蛋白A1 (Annexin A1,ANXA1)是一种来源于脊柱(哺乳)动物的钙依赖性磷脂结合蛋白,是介导细胞内糖皮质激素抗炎作用的效应分子,在组织中广泛表达,参与细胞生长周期的各个阶段.其既可以可溶性形式存在,也可稳定或可逆结合于细胞骨架蛋白,调控细胞与细胞外基质的相互作用.大量的研究发现,AnnexinA1的表达在不同肿瘤组织中有差异,并且同一肿瘤不同类型中表达也不一样,其异常表达及细胞内定位改变可能跟多种恶性肿瘤的分化及转移相关.Annexin A1与肿瘤的密切关系,或许可使其发展为一个新的肿瘤标志物,为肿瘤的早期诊断、治疗及预后提供新的判断标准.因此,探讨Annexin A1与肿瘤的关系极具临床应用前景.     

CSF1与肿瘤微环境CSCD

肿瘤微环境是肿瘤细胞的复杂生态环境,细胞与环境共同进化,环境为细胞的恶性转化提供支持。在被招募到肿瘤发生位点的细胞中,巨噬细胞的数量最多,在肿瘤发展的各阶段都存在,人们将其称为肿瘤相关巨噬细胞(tumor associated macrophages,TAM)。CSF1(macrophage colony-stimulating factor 1)是目前公认的经典的促肿瘤细胞因子,它可招募巨噬细胞到肿瘤区域,并促进肿瘤细胞与巨噬细胞的相互作用,进而导致微环境中释放各种促肿瘤发展的生长因子,从而促进肿瘤的发生发展。本文主要阐述了CSF1在肿瘤微环境中发挥的功能作用,为针对CSF1/CSF1R信号而发展的肿瘤治疗策略提供更好的理论基础。     

CSF1与肿瘤微环境

肿瘤微环境是肿瘤细胞的复杂生态环境,细胞与环境共同进化,环境为细胞的恶性转化提供支持。在被招募到肿瘤发生位点的细胞中,巨噬细胞的数量最多,在肿瘤发展的各阶段都存在,人们将其称为肿瘤相关巨噬细胞(tumor associated macrophages,TAM)。CSF1(macrophage colony-stimulating factor 1)是目前公认的经典的促肿瘤细胞因子,它可招募巨噬细胞到肿瘤区域,并促进肿瘤细胞与巨噬细胞的相互作用,进而导致微环境中释放各种促肿瘤发展的生长因子,从而促进肿瘤的发生发展。本文主要阐述了CSF1在肿瘤微环境中发挥的功能作用,为针对CSF1/CSF1R信号而发展的肿瘤治疗策略提供更好的理论基础。     

可溶性耐药相关钙结合蛋白

可溶性耐药相关钙结合蛋白(sorcin)是一个21.6 kD的胞浆蛋白,具有典型的EF手臂(EF-hand)钙结合位点, 广泛存在于多种组织中,在心肌细胞中含量最丰富.Sorcin可与肌浆网钙离子通道RyR相互作用影响心肌细胞兴奋——收缩偶联.另一方面,sorcin在肿瘤耐药细胞中大量表达,它可以与细胞中钙离子结合,引起细胞内游离钙离子浓度下降,然后导致钙离子所介导的磷酸酶活性降低,使得具有排药功能的P-gp糖蛋白磷酸化水平下降,最终导致耐药.一旦明确引发和维持细胞耐药的作用机制,就可为克服肿瘤耐药提供新的靶点.同时随着RyR活性抑制作用研究的深入,有望通过sorcin转基因技术治疗心力衰竭.本文主要对sorcin的结构特点和生物学特性进行综述,并初步分析其耐药机制.     

Current perspectives of bispecific antibody-based immunotherapy

The field of bispecific antibodies is an evolving field of research that has increasing clinical appeal.The fusion of two antibodies or antibody fragments introduced a new way to override natural specificity of T cell and induce effector responses against tumor targets in MHC-unrestricted manner. Initial experiences with bispecific antibodies demonstrate both the promise for and limitations of this anti-cancer strategy. Significant body of work has shown that bispecific antibodies have potential to induce T cell mediated anti-tumor responses in pre-clinical models. However, immunotherapy with bispecific antibodies in humans has yet to prove its value in clinical settings. In addition, the production of high-quality bispecific antibodies for clinical applications, the optimal size and avidity of bispecific antibodies, and in vivo T cell pre-activation remain critical issues. In this review, we summarize recent progress in bispecific antibody-based immunotherapy and address essential aspects of this anti-cancer strategy.     

Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcγRIII) on effector cells to an Fv specific for mouse or human CD32B (FcγRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.     

Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target cells.     

A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the “knob-into-hole” technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.     

Treatment of mice bearing BCL1 lymphoma with bispecific antibodies

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.     

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.     

Bispecific monoclonal antibody regulation of Fc gamma RIII-directed tumor cytotoxicity by large granular lymphocytes.

Bispecific monoclonal antibodies (BsMAbs) prepared by somatic cell fusion bind monovalently to their targets and yet are extremely potent enhancers of target cell lysis by relevant effector cells. The mechanisms underlying this efficiency are not known. To investigate this property, we studied the ability of selected antibodies to modulate potentiation of tumor lysis by a bispecific antibody (CL158) which targets Fc gamma RIII-expressing cells, via the 3G8 epitope, to malignant cells expressing CA19-9 antigen. Antibodies directed against the 3G8 and B73.1 epitopes of Fc gamma RIII efficiently inhibited BsMAb-mediated SW948 tumor cell lysis by interleukin-2 (IL-2)-activated lymphocytes (PBLs). Unexpectedly, Leu 19 antibody reversed antibody-dependent but not antibody-independent lysis of 51Cr-labeled SW948 cells by IL-2-activated PBLs in a concentration-dependent fashion. Leu 19 binds to CD56, a neural cell adhesion molecule (N-CAM) isoform expressed by large granular lymphocytes (LGLs). The effects of Leu 19 on bispecific antibody promotion of lysis were due to competition for binding to the 3G8 epitope of Fc gamma RIII and led to inhibition of binding between LGLs and SW948 cells. Leu 19 did not inhibit antibody-dependent lysis by the monospecific, bivalent IgG2a variant of CA19-9 antibody. These studies show that competition assays can be useful in dissecting the relevant mechanisms underlying BsMAb-promoted lysis. Steric constraints between effector cell trigger molecules (i.e., Fc gamma RIII) and CAM such as N-CAM may regulate the function of these molecules. Understanding the roles of diverse CAM in this phenomenon will facilitate efforts to expand and use defined effector cell populations with maximal lytic potential and to identify potentially responsive tumor phenotypes.     

Bispecific antibodies for cancer therapy

With 23 approvals in the US and other countries and 4 approvals outside US, antibodies are now widely recognized as therapeutic molecules. The therapeutic and commercial successes met by rituximab, trastuzumab, cetuximab and other mAbs have inspired antibody engineers to improve the efficacy of these molecules. Consequently, a new wave of antibodies with engineered Fc leading to much higher effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is being evaluated in the clinic, and several approvals are expected soon. In addition, research on a different class of antibody therapeutics, bispecific antibodies, has recently led to outstanding clinical results, and the first approval of the bispecific antibody catumaxomab, a T cell retargeting agent that was approved in the European Union in April 2009. This review describes the most recent advances and clinical study results in the field of bispecific antibodies, a new class of molecules that might outshine conventional mAbs as cancer immunotherapeutics in a near future.     

Biodistribution studies with tumor-targeting bispecific antibodies reveal selective accumulation at the tumor site

Bispecific antibodies are proteins that bind two different antigens and may retarget immune cells with a binding moiety specific for a leukocyte marker. A binding event in blood could in principle prevent antibody extravasation and accumulation at the site of disease. In this study, we produced and characterized two tetravalent bispecific antibodies that bind with high affinity to the alternatively-spliced EDB domain of fibronectin, a tumor-associated antigen. The bispecific antibodies simultaneously engaged the cognate antigens (murine T cell co-receptor CD3 and hen egg lysozyme) and selectively accumulated on murine tumors in vivo. The results, which were in agreement with predictions based on pharmacokinetic modeling and antibody binding characteristics, confirmed that bispecific antibodies can reach abluminal targets without being blocked by peripheral blood leukocytes.