人源性抗大疱性类天疱疮抗原BP180单链抗体的生物学功能研究
|
摘要
大疱性类天疱疮(bullous pemphigoid,BP)是一种好发于老年人,临床表现为紧张性水疱、大疱的慢性自身免疫性水疱性皮肤病,治疗目前主要依靠应用糖皮质激素和免疫抑制剂等方法。BP病情易反复发作,长期应用激素等治疗产生的副作用是临床实际工作中急需解决的问题[1]。
BP组织病理学表现为表皮下水疱形成,疱内可见到以嗜酸性粒细胞为主,包括淋巴细胞及中性粒细胞等多种炎细胞的浸润。患者血清中可以检测到特异性针对皮肤真表皮连接处的基底膜带(BMZ)的自身抗体,直接免疫荧光亦可以发现患者皮肤BMZ有抗体(IgG、IgE等)和补体沉积。目前研究认为BP的发病机制是由患者体内循环自身抗体介导补体激活、肥大细胞及中性粒细胞等炎细胞参与而最终导致皮肤真表皮交界处水疱发生的复杂过程[2]。
BP180属于胶原蛋白家族成员,位于皮肤基底膜带半桥粒上的一种Ⅱ型跨膜糖蛋白,又称ⅩⅦ型胶原(COL17)或大疱性类天疱疮抗原2 (BPAg2),在真表皮连接中发挥重要作用。目前认为BP180为BP致病性自身抗体所识别的主要靶抗原,其细胞外区域第16非胶原编码区(NC16A)是致病性自身抗体所识别的主要靶表位区[3]。BP致病性自身抗体与BP180等自身抗原特异性结合并激活补体,造成明显的免疫炎症反应,导致真表皮分离最终发生表皮下水疱。Nishie[4]等在BP180人源化动物模型上证实BP患者血清自身抗体在BP发病中发挥了关键作用,更深层次地揭示了BP的发病机理。在此过程中,自身抗体与BP180等自身抗原结合,进而激活补体是非常重要的环节。因此,我们通过制备人源性基因工程单链抗体,利用其能特异性识别靶抗原而又因不含有补体结合位点的Fc段,不能激活补体的特性,封闭与BP自身抗体结合的自身抗原表位,阻断自身抗体与自身抗原的结合,干预后续的免疫炎症反应,从而消除自身抗体的致病作用,探寻一种新的治疗BP的生物学方法。
我们前期构建了针对BP180-NC16A的单链抗体(single chain Fv,scFv),并实现了其可溶性表达及纯化[5, 6]。本研究对所获得的单链抗体进行功能学的鉴定,观察其对BP血清IgG自身抗体的竞争性抑制作用,构建BP体外冰冻切片模型,并在此模型中验证所获得的单链抗体可竞争性抑制由于BP自身抗体结合抗原引起后续免疫炎症反应,从而导致真表皮分离的发病过程。我们的研究为BP特异性的生物治疗提供实验依据,同时也为以自身抗体作为主要致病机制的自身免疫病治疗提供了一个新的研究方向。
目的:对人源性抗BP180单链抗体进行生物学功能的鉴定。
方法:蛋白亲和层析的方法纯化BP血清自身抗体,通过竞争性ELISA、竞争性免疫荧光和补体活化的竞争性抑制实验来观察所制备的单链抗体对自身抗体的竞争抑制作用。构建BP体外冰冻切片模型,利用此模型观察所制备的单链抗体对自身抗体所致真表皮分离的抑制作用。
结果:抗BP180-NC16A scFv对BP血清IgG抗体具有较好的抑制效果。竞争性ELISA各浓度组间A450值下降明显,在0-60μg范围内成剂量依赖关系,最大抑制率可达到69.50%(与对照组相比均有统计学意义,P<0.01);间接免疫荧光实验及补体活化的竞争抑制实验在抑制前可见线性荧光沉积条带,经scFv抑制后则无荧光条带。BP冰冻切片组织模型HE染色可见真表皮的分离,加入不同浓度的单链抗体后真表皮的分离受到明显的抑制。
结论:我们所制备的抗大疱性类天疱疮抗原BP180单链抗体对致病性自身抗体结合抗原具有一定的竞争抑制作用,为工程抗体封闭自身抗体抗原表位这一治疗策略提供了实验依据。
Bullous pemphigoid is a common autoimmune blistering disorder seen in the elderly,which is characterized clinically by tense subepidermal blister and bullae. Up to recently, systemic glucocorticosteroids have been the mainstay of therapy, frequently in combination with other immunosuppressants. Although the condition can be temporarily alleviated, the frequent recurrence of the disease and the severe side effects of glucocorticosteroids remain to be the most serious problems[1].
The major histologic feature of BP is subepidermal blisters with different inflammatory cells.The autoantibodies against the basement membrane zone (BMZ)in patients can be detected,and also show linear deposition of C3, IgG, and/or IgE along BMZ by direct immunofluorescence detection. Recent studies confirmed that the pathogenesis of BP contains an intact complement cascade,degranulation of resident mast cells and activation of infiltrating neutrophils which are required in the dermal-epidermal separation induced by BP autoantibodies[2].
BP180 is a member of the collagen protein family and is also referred to as type XVII collagen or BPAg2, which located at the hemidesmosome.It plays a important role in the pathomechanism of BP. The non-collagenous 16A domain (NC16A), located at the membrane-proximal region of BP180, is considered to be the major pathogenic epitope for BP autoantibodies[3].
The pathogenic autoantibodies of BP bind to the hemidesmosomal antigens such as BP180, which results in an intact complement cascade activation,degranulation of resident mast cells, activation of infiltrating inflammatory cells and proteinases , loss of cell-matrix adhesion structure and finally blister formation. Nishie[4]et al humanized the mouse BP autoantigen and developed BP-like skin lesions and a human disease phenotype. These findings clearly show that human autoantibodies against BP180 play a crucial role in blister formation in vivo. It was confirmed that the anti-BP180 autoantibodies trigger subepidermal vesiculation via the classical pathway of the complement system activation. It therefore makes sense to block the pathological action of autoantibodies by preventing them from binding to the corresponding pathogenic autoantigen using targeted genetically engineering scFv antibody.
In our previous studies, the scFv antibody fragments were isolated and expressed, which specifically bind to BP180-NC16A, and purified by Ni affinity chromatography method[5, 6]. The purpose of the present study is to characterize the biological function of anti-BP180-NC16A scFv.
Objective: To characterize the function of human anti-BP180 single-chain Fv (scFv) antibody.
Methods: Anti-BP180 IgG autoantibodies were purified by Ni affinity chromatography from the sera of BP patients. The inhibitive effects of anti-BP180 scFv on the binding of BP-IgG autoantibodies to the recombinant NC16A fragment of human BP180 antigen or intact human tissue were observed by inhibition ELISA and inhibition immunofluorescence,respectively.BP frozen cryosections model was constructed to confirm the inhibitive effects of anti-BP180 scFv with variable concentration on the binding of BP-IgG autoantibodies with BP180.
Results: It was confirmed that the scFv had a dose-dependent inhibitive effect on the binding of BP-IgG autoantibodies against BP180 with the corresponding antigen at the given concentrations between 0 and 60μg/ml. The inhibitive rate was up to 69.50% (P<0.01). When the scFv concentration was higher than 40μg/ml, we found no linear fluorescence deposition along the dermal-epidermal junction (DEJ). We are successful to construct the BP frozen cryosections model,and dermal–epidermal separation can be seen using haematoxylin and eosin staining.In this model,we can observe that the scFv shows marked ability to inhibit the binding of BP autoantibodies and subsequent dermal-epidermal separation.
Conclusions: The anti-BP180 single-chain Fv antibody can markedly inhibit the binding of BP-IgG autoantibodies with BP180 and then affect subsequent complement activation in vitro.Our results provide experimental proof that the blocking of pathogenic epitopes using engineered scFv appears to be efficient as a method for BP therapy.
BP组织病理学表现为表皮下水疱形成,疱内可见到以嗜酸性粒细胞为主,包括淋巴细胞及中性粒细胞等多种炎细胞的浸润。患者血清中可以检测到特异性针对皮肤真表皮连接处的基底膜带(BMZ)的自身抗体,直接免疫荧光亦可以发现患者皮肤BMZ有抗体(IgG、IgE等)和补体沉积。目前研究认为BP的发病机制是由患者体内循环自身抗体介导补体激活、肥大细胞及中性粒细胞等炎细胞参与而最终导致皮肤真表皮交界处水疱发生的复杂过程[2]。
BP180属于胶原蛋白家族成员,位于皮肤基底膜带半桥粒上的一种Ⅱ型跨膜糖蛋白,又称ⅩⅦ型胶原(COL17)或大疱性类天疱疮抗原2 (BPAg2),在真表皮连接中发挥重要作用。目前认为BP180为BP致病性自身抗体所识别的主要靶抗原,其细胞外区域第16非胶原编码区(NC16A)是致病性自身抗体所识别的主要靶表位区[3]。BP致病性自身抗体与BP180等自身抗原特异性结合并激活补体,造成明显的免疫炎症反应,导致真表皮分离最终发生表皮下水疱。Nishie[4]等在BP180人源化动物模型上证实BP患者血清自身抗体在BP发病中发挥了关键作用,更深层次地揭示了BP的发病机理。在此过程中,自身抗体与BP180等自身抗原结合,进而激活补体是非常重要的环节。因此,我们通过制备人源性基因工程单链抗体,利用其能特异性识别靶抗原而又因不含有补体结合位点的Fc段,不能激活补体的特性,封闭与BP自身抗体结合的自身抗原表位,阻断自身抗体与自身抗原的结合,干预后续的免疫炎症反应,从而消除自身抗体的致病作用,探寻一种新的治疗BP的生物学方法。
我们前期构建了针对BP180-NC16A的单链抗体(single chain Fv,scFv),并实现了其可溶性表达及纯化[5, 6]。本研究对所获得的单链抗体进行功能学的鉴定,观察其对BP血清IgG自身抗体的竞争性抑制作用,构建BP体外冰冻切片模型,并在此模型中验证所获得的单链抗体可竞争性抑制由于BP自身抗体结合抗原引起后续免疫炎症反应,从而导致真表皮分离的发病过程。我们的研究为BP特异性的生物治疗提供实验依据,同时也为以自身抗体作为主要致病机制的自身免疫病治疗提供了一个新的研究方向。
目的:对人源性抗BP180单链抗体进行生物学功能的鉴定。
方法:蛋白亲和层析的方法纯化BP血清自身抗体,通过竞争性ELISA、竞争性免疫荧光和补体活化的竞争性抑制实验来观察所制备的单链抗体对自身抗体的竞争抑制作用。构建BP体外冰冻切片模型,利用此模型观察所制备的单链抗体对自身抗体所致真表皮分离的抑制作用。
结果:抗BP180-NC16A scFv对BP血清IgG抗体具有较好的抑制效果。竞争性ELISA各浓度组间A450值下降明显,在0-60μg范围内成剂量依赖关系,最大抑制率可达到69.50%(与对照组相比均有统计学意义,P<0.01);间接免疫荧光实验及补体活化的竞争抑制实验在抑制前可见线性荧光沉积条带,经scFv抑制后则无荧光条带。BP冰冻切片组织模型HE染色可见真表皮的分离,加入不同浓度的单链抗体后真表皮的分离受到明显的抑制。
结论:我们所制备的抗大疱性类天疱疮抗原BP180单链抗体对致病性自身抗体结合抗原具有一定的竞争抑制作用,为工程抗体封闭自身抗体抗原表位这一治疗策略提供了实验依据。
Bullous pemphigoid is a common autoimmune blistering disorder seen in the elderly,which is characterized clinically by tense subepidermal blister and bullae. Up to recently, systemic glucocorticosteroids have been the mainstay of therapy, frequently in combination with other immunosuppressants. Although the condition can be temporarily alleviated, the frequent recurrence of the disease and the severe side effects of glucocorticosteroids remain to be the most serious problems[1].
The major histologic feature of BP is subepidermal blisters with different inflammatory cells.The autoantibodies against the basement membrane zone (BMZ)in patients can be detected,and also show linear deposition of C3, IgG, and/or IgE along BMZ by direct immunofluorescence detection. Recent studies confirmed that the pathogenesis of BP contains an intact complement cascade,degranulation of resident mast cells and activation of infiltrating neutrophils which are required in the dermal-epidermal separation induced by BP autoantibodies[2].
BP180 is a member of the collagen protein family and is also referred to as type XVII collagen or BPAg2, which located at the hemidesmosome.It plays a important role in the pathomechanism of BP. The non-collagenous 16A domain (NC16A), located at the membrane-proximal region of BP180, is considered to be the major pathogenic epitope for BP autoantibodies[3].
The pathogenic autoantibodies of BP bind to the hemidesmosomal antigens such as BP180, which results in an intact complement cascade activation,degranulation of resident mast cells, activation of infiltrating inflammatory cells and proteinases , loss of cell-matrix adhesion structure and finally blister formation. Nishie[4]et al humanized the mouse BP autoantigen and developed BP-like skin lesions and a human disease phenotype. These findings clearly show that human autoantibodies against BP180 play a crucial role in blister formation in vivo. It was confirmed that the anti-BP180 autoantibodies trigger subepidermal vesiculation via the classical pathway of the complement system activation. It therefore makes sense to block the pathological action of autoantibodies by preventing them from binding to the corresponding pathogenic autoantigen using targeted genetically engineering scFv antibody.
In our previous studies, the scFv antibody fragments were isolated and expressed, which specifically bind to BP180-NC16A, and purified by Ni affinity chromatography method[5, 6]. The purpose of the present study is to characterize the biological function of anti-BP180-NC16A scFv.
Objective: To characterize the function of human anti-BP180 single-chain Fv (scFv) antibody.
Methods: Anti-BP180 IgG autoantibodies were purified by Ni affinity chromatography from the sera of BP patients. The inhibitive effects of anti-BP180 scFv on the binding of BP-IgG autoantibodies to the recombinant NC16A fragment of human BP180 antigen or intact human tissue were observed by inhibition ELISA and inhibition immunofluorescence,respectively.BP frozen cryosections model was constructed to confirm the inhibitive effects of anti-BP180 scFv with variable concentration on the binding of BP-IgG autoantibodies with BP180.
Results: It was confirmed that the scFv had a dose-dependent inhibitive effect on the binding of BP-IgG autoantibodies against BP180 with the corresponding antigen at the given concentrations between 0 and 60μg/ml. The inhibitive rate was up to 69.50% (P<0.01). When the scFv concentration was higher than 40μg/ml, we found no linear fluorescence deposition along the dermal-epidermal junction (DEJ). We are successful to construct the BP frozen cryosections model,and dermal–epidermal separation can be seen using haematoxylin and eosin staining.In this model,we can observe that the scFv shows marked ability to inhibit the binding of BP autoantibodies and subsequent dermal-epidermal separation.
Conclusions: The anti-BP180 single-chain Fv antibody can markedly inhibit the binding of BP-IgG autoantibodies with BP180 and then affect subsequent complement activation in vitro.Our results provide experimental proof that the blocking of pathogenic epitopes using engineered scFv appears to be efficient as a method for BP therapy.
引文
[1] Mutasim D F. Autoimmune bullous dermatoses in the elderly: an update on pathophysiology, diagnosis and management[J]. Drugs Aging, 2010, 27(1): 1-19.
[2] Leighty L, Li N, Diaz L A, Liu Z. Experimental models for the autoimmune and inflammatory blistering disease, Bullous pemphigoid[J]. Arch Dermatol Res, 2007, 299(9): 417-422.
[3] Ujiie H, Shibaki A, Nishie W, Shimizu H. What's new in bullous pemphigoid[J]. J Dermatol, 2010, 37(3): 194-204.
[4] Nishie W, Sawamura D, Goto M, Ito K, Shibaki A, Mcmillan J R, Sakai K, Nakamura H, Olasz E, Yancey K B, Akiyama M, Shimizu H. Humanization of autoantigen[J]. Nat Med, 2007, 13(3): 378-383.
[5]王刚,刘玉峰.大疱性类天疱疮患者噬菌体抗体库的构建及抗BP180-NC16A抗体筛选[J].中国皮肤性病学杂志, 2008, 22(11): 655-657.
[6]孙娜娜,王欲晓,周丽君,王刚.人源性抗大疱性类天疱疮抗原BP180-NC16A单链抗体的构建及表达[J].中国皮肤性病学杂志, 2009, 23(2): 71-74.
[7] Di Zenzo G, Marazza G, Borradori L. Bullous pemphigoid: physiopathology, clinical features and management[J]. Adv Dermatol, 2007, 23: 257-288.
[8] Liu Z, Giudice G J, Swartz S J, Fairley J A, Till G O, Troy J L, Diaz L A. The role of complement in experimental bullous pemphigoid[J]. J Clin Invest, 1995, 95(4): 1539-1544.
[9] Wang G, Ujiie H, Shibaki A, Nishie W, Tateishi Y, Kikuchi K, Li Q, Mcmillan J R, Morioka H, Sawamura D, Nakamura H, Shimizu H. Blockade of autoantibody-initiated tissue damage by using recombinant fab antibodyfragments against pathogenic autoantigen[J]. Am J Pathol, 2010, 176(2): 914-925.
[10] Borradori L, Sonnenberg A. Hemidesmosomes: roles in adhesion, signaling and human diseases[J]. Curr Opin Cell Biol, 1996, 8(5): 647-656.
[11] Kiss M, Husz S, Janossy T, Marczinovits I, Molnar J, Korom I, Dobozy A. Experimental bullous pemphigoid generated in mice with an antigenic epitope of the human hemidesmosomal protein BP230[J]. J Autoimmun, 2005, 24(1): 1-10.
[12] Laffitte E, Burkhard P R, Fontao L, Jaunin F, Saurat J H, Chofflon M, Borradori L. Bullous pemphigoid antigen 1 isoforms: potential new target autoantigens in multiple sclerosis?[J]. Br J Dermatol, 2005, 152(3): 537-540.
[13] Zillikens D. BP180 as the common autoantigen in blistering diseases with different clinical phenotypes[J]. Keio J Med, 2002, 51(1): 21-28.
[14] Zillikens D, Rose P A, Balding S D, Liu Z, Olague-Marchan M, Diaz L A, Giudice G J. Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies[J]. J Invest Dermatol, 1997, 109(4): 573-579.
[15] Hofmann S C, Tamm K, Hertl M, Borradori L. Diagnostic value of an enzyme-linked immunosorbent assay using BP180 recombinant proteins in elderly patients with pruritic skin disorders[J]. Br J Dermatol, 2003, 149(4): 910-912.
[16] Budinger L, Borradori L, Yee C, Eming R, Ferencik S, Grosse-Wilde H, Merk H F, Yancey K, Hertl M. Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls[J]. J Clin Invest, 1998, 102(12): 2082-2089.
[17] Black A P, Seneviratne S L, Jones L, King A S, Winsey S, Arsecularatne G,Wojnarowska F, Ogg G S. Rapid effector function of circulating NC16A-specific T cells in individuals with mucous membrane pemphigoid[J]. Br J Dermatol, 2004, 151(6): 1160-1164.
[18] Dopp R, Schmidt E, Chimanovitch I, Leverkus M, Brocker E B, Zillikens D. IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in Bullous pemphigoid: serum levels of these immunoglobulins reflect disease activity[J]. J Am Acad Dermatol, 2000, 42(4): 577-583.
[19] Sitaru C, Mihai S, Zillikens D. The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases[J]. Arch Dermatol Res, 2007, 299(1): 1-8.
[20] Liu Z. Are anti-BP180 IgG1 or IgG4 autoantibodies pathogenic?[J]. J Invest Dermatol, 2002, 119(5): 989-990.
[21] Tampoia M, Lattanzi V, Zucano A, Villalta D, Filotico R, Fontana A, Vena G A, Di Serio F. Evaluation of a new ELISA assay for detection of BP230 autoantibodies in bullous pemphigoid[J]. Ann N Y Acad Sci, 2009, 1173: 15-20.
[22] Suzuki M, Harada S, Yaoita H, Nakagawa H. Bullous pemphigoid IgG2 and IgG4 antibodies have a capability to inhibit a transient increase of intracellular Ca2+ induced by bullous pemphigoid IgG1 antibody[J]. Autoimmunity, 1999, 30(2): 85-91.
[23] Ujiie H, Shibaki A, Nishie W, Sawamura D, Wang G, Tateishi Y, Li Q, Moriuchi R, Qiao H, Nakamura H, Akiyama M, Shimizu H. A novel active mouse model for bullous pemphigoid targeting humanized pathogenic antigen[J]. J Immunol, 2010, 184(4): 2166-2174.
[24] Schmidt E, Wehr B, Tabengwa E M, Reimer S, Brocker E B, Zillikens D. Elevated expression and release of tissue-type, but not urokinase-type,plasminogen activator after binding of autoantibodies to bullous pemphigoid antigen 180 in cultured human keratinocytes[J]. Clin Exp Immunol, 2004, 135(3): 497-504.
[25] Liu Z, Li N, Diaz L A, Shipley M, Senior R M, Werb Z. Synergy between a plasminogen cascade and MMP-9 in autoimmune disease[J]. J Clin Invest, 2005, 115(4): 879-887.
[26] Zhao M, Trimbeger M E, Li N, Diaz L A, Shapiro S D, Liu Z. Role of FcRs in animal model of autoimmune bullous pemphigoid[J]. J Immunol, 2006, 177(5): 3398-3405.
[27] Leyendeckers H, Tasanen K, Bruckner-Tuderman L, Zillikens D, Sitaru C, Schmitz J, Hunzelmann N. Memory B cells specific for the NC16A domain of the 180 kDa bullous pemphigoid autoantigen can be detected in peripheral blood of bullous pemphigoid patients and induced in vitro to synthesize autoantibodies[J]. J Invest Dermatol, 2003, 120(3): 372-378.
[28] Lin M S, Fu C L, Giudice G J, Olague-Marchan M, Lazaro A M, Stastny P, Diaz L A. Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain[J]. J Invest Dermatol, 2000, 115(6): 955-961.
[29] Romagnani S. Human TH1 and TH2 subsets: regulation of differentiation and role in protection and immunopathology[J]. Int Arch Allergy Immunol, 1992, 98(4): 279-285.
[30] Giomi B, Caproni M, Calzolari A, Bianchi B, Fabbri P. Th1, Th2 and Th3 cytokines in the pathogenesis of bullous pemphigoid[J]. J Dermatol Sci, 2002, 30(2): 116-128.
[31] Nakashima H, Fujimoto M, Asashima N, Watanabe R, Kuwano Y, Yazawa N, Maruyama N, Okochi H, Kumanogoh A, Tamaki K. Serum chemokineprofile in patients with bullous pemphigoid[J]. Br J Dermatol, 2007, 156(3): 454-459.
[32] Cugno M, Tedeschi A, Asero R, Meroni P L, Marzano A V. Skin autoimmunity and blood coagulation[J]. Autoimmunity, 2010, 43(2): 189-194.
[33] Yasukawa S, Dainichi T, Kokuba H, Moroi Y, Urabe K, Hashimoto T, Furue M. Bullous pemphigoid followed by pustular psoriasis showing Th1, Th2, Treg and Th17 immunological changes[J]. Eur J Dermatol, 2009, 19(1): 69-71.
[34] Rensing-Ehl A, Gaus B, Bruckner-Tuderman L, Martin S F. Frequency, function and CLA expression of CD4+CD25+FOXP3+ regulatory T cells in bullous pemphigoid[J]. Exp Dermatol, 2007, 16(1): 13-21.
[35] Liu Z, Shapiro S D, Zhou X, Twining S S, Senior R M, Giudice G J, Fairley J A, Diaz L A. A critical role for neutrophil elastase in experimental bullous pemphigoid[J]. J Clin Invest, 2000, 105(1): 113-123.
[36] Yu X, Holdorf K, Kasper B, Zillikens D, Ludwig R J, Petersen F. FcgammaRIIA and FcgammaRIIIB are required for autoantibody-induced tissue damage in experimental human models of bullous pemphigoid[J]. J Invest Dermatol, 2010, 130(12): 2841-2844.
[37] Nelson K C, Zhao M, Schroeder P R, Li N, Wetsel R A, Diaz L A, Liu Z. Role of different pathways of the complement cascade in experimental bullous pemphigoid[J]. J Clin Invest, 2006, 116(11): 2892-2900.
[38] Heimbach L, Li Z, Berkowitz P, Zhao M, Li N, Rubenstein D, Diaz L A, Liu Z. C5A receptor on mast cells is critical for the autoimmune skin blistering disease bullous pemphigoid[J]. J Biol Chem, 2011.
[39] Chen R, Fairley J A, Zhao M L, Giudice G J, Zillikens D, Diaz L A, Liu Z.Macrophages, but not T and B lymphocytes, are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation[J]. J Immunol, 2002, 169(7): 3987-3992.
[40] Stahle-Backdahl M, Inoue M, Guidice G J, Parks W C. 92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen[J]. J Clin Invest, 1994, 93(5): 2022-2030.
[41] Borrego L, Maynard B, Peterson E A, George T, Iglesias L, Peters M S, Newman W, Gleich G J, Leiferman K M. Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins[J]. Am J Pathol, 1996, 148(3): 897-909.
[42] Gounni A S, Wellemans V, Agouli M, Guenounou M, Hamid Q, Beck L A, Lamkhioued B. Increased expression of Th2-associated chemokines in bullous pemphigoid disease. Role of eosinophils in the production and release of these chemokines[J]. Clin Immunol, 2006, 120(2): 220-231.
[43] Hertl M, Eming R, Veldman C. T cell control in autoimmune bullous skin disorders[J]. J Clin Invest, 2006, 116(5): 1159-1166.
[44] Laszlo S, Neumann S, Hertl M, Hunzelmann N. Generation of increased numbers of HLA-DR(high) IgG+ plasma cells in the peripheral blood of patients with bullous pemphigoid: NC16a-specific cells belong to the short-lived plasma blast population[J]. J Invest Dermatol, 2010, 130(12): 2838-2841.
[45] Liu Z, Diaz L A, Troy J L, Taylor A F, Emery D J, Fairley J A, Giudice G J. A passive transfer model of the organ-specific autoimmune disease, bullouspemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180[J]. J Clin Invest, 1993, 92(5): 2480-2488.
[46] Liu Z, Zhao M, Li N, Diaz L A, Mayadas T N. Differential roles for beta2 integrins in experimental autoimmune bullous pemphigoid[J]. Blood, 2006, 107(3): 1063-1069.
[47] Liu Z, Sui W, Zhao M, Li Z, Li N, Thresher R, Giudice G J, Fairley J A, Sitaru C, Zillikens D, Ning G, Marinkovich M P, Diaz L A. Subepidermal blistering induced by human autoantibodies to BP180 requires innate immune players in a humanized bullous pemphigoid mouse model[J]. J Autoimmun, 2008, 31(4): 331-338.
[48] Nishie W, Sawamura D, Natsuga K, Shinkuma S, Goto M, Shibaki A, Ujiie H, Olasz E, Yancey K B, Shimizu H. A novel humanized neonatal autoimmune blistering skin disease model induced by maternally transferred antibodies[J]. J Immunol, 2009, 183(6): 4088-4093.
[49] Zillikens D, Schmidt E, Reimer S, Chimanovitch I, Hardt-Weinelt K, Rose C, Brocker E B, Kock M, Boehncke W H. Antibodies to desmogleins 1 and 3, but not to BP180, induce blisters in human skin grafted onto SCID mice[J]. J Pathol, 2001, 193(1): 117-124.
[50] Zone J J, Taylor T, Hull C, Schmidt L, Meyer L. IgE basement membrane zone antibodies induce eosinophil infiltration and histological blisters in engrafted human skin on SCID mice[J]. J Invest Dermatol, 2007, 127(5): 1167-1174.
[51] Fairley J A, Burnett C T, Fu C L, Larson D L, Fleming M G, Giudice G J. A pathogenic role for IgE in autoimmunity: bullous pemphigoid IgE reproduces the early phase of lesion development in human skin grafted to nu/nu mice[J]. J Invest Dermatol, 2007, 127(11): 2605-2611.
[52] Olasz E B, Roh J, Yee C L, Arita K, Akiyama M, Shimizu H, Vogel J C, Yancey K B. Human bullous pemphigoid antigen 2 transgenic skin elicits specific IgG in wild-type mice[J]. J Invest Dermatol, 2007, 127(12): 2807-2817.
[53] Sitaru C, Schmidt E, Petermann S, Munteanu L S, Brocker E B, Zillikens D. Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin[J]. J Invest Dermatol, 2002, 118(4): 664-671.
[54] Mihai S, Chiriac M T, Herrero-Gonzalez J E, Goodall M, Jefferis R, Savage C O, Zillikens D, Sitaru C. IgG4 autoantibodies induce dermal-epidermal separation[J]. J Cell Mol Med, 2007, 11(5): 1117-1128.
[55] Loo W J, Burrows N P. Management of autoimmune skin disorders in the elderly[J]. Drugs Aging, 2004, 21(12): 767-777.
[56] Khumalo N, Kirtschig G, Middleton P, Hollis S, Wojnarowska F, Murrell D. Interventions for bullous pemphigoid[J]. Cochrane Database Syst Rev, 2005(3): D2292.
[57] Joly P, Fontaine J, Roujeau J C. The role of topical corticosteroids in bullous pemphigoid in the elderly[J]. Drugs Aging, 2005, 22(7): 571-576.
[58] Herrero-Gonzalez J E, Sitaru C, Klinker E, Brocker E B, Zillikens D. Successful adjuvant treatment of severe bullous pemphigoid by tryptophan immunoadsorption[J]. Clin Exp Dermatol, 2005, 30(5): 519-522.
[59] Peterson J D, Chan L S. Effectiveness and side effects of anti-CD20 therapy for autoantibody-mediated blistering skin diseases: A comprehensive survey of 71 consecutive patients from the Initial use to 2007[J]. Ther Clin Risk Manag, 2009, 5(1): 1-7.
[60] Saouli Z, Papadopoulos A, Kaiafa G, Girtovitis F, Kontoninas Z. A newapproach on bullous pemphigoid therapy[J]. Ann Oncol, 2008, 19(4): 825-826.
[61] Szabolcs P, Reese M, Yancey K B, Hall R P, Kurtzberg J. Combination treatment of bullous pemphigoid with anti-CD20 and anti-CD25 antibodies in a patient with chronic graft-versus-host disease[J]. Bone Marrow Transplant, 2002, 30(5): 327-329.
[62] Lanschuetzer C M, Olasz E B, Lazarova Z, Yancey K B. Transient anti-CD40L co-stimulation blockade prevents immune responses against human bullous pemphigoid antigen 2: implications for gene therapy[J]. J Invest Dermatol, 2009, 129(5): 1203-1207.
[63]朱振洪余勤万海同.基因工程小分子抗体的研究进展[J].生物技术通讯, 2008(Z1): 59-62.
[64] Cheng W W, Allen T M. The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment[J]. Expert Opin Drug Deliv, 2010, 7(4): 461-478.
[65] Smith R, Tarner I H, Hollenhorst M, Lin C, Levicnik A U, Fathman C G, Nolan G P. Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis[J]. Gene Ther, 2003, 10(15): 1248-1257.
[66] Meng F, Stassen M H, Schillberg S, Fischer R, De Baets M H. Construction and characterization of a single-chain antibody fragment derived from thymus of a patient with myasthenia gravis[J]. Autoimmunity, 2002, 35(2): 125-133.
[67] Li Q, Ujiie H, Shibaki A, Wang G, Moriuchi R, Qiao H J, Morioka H, Shinkuma S, Natsuga K, Long H A, Nishie W, Shimizu H. Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16Adomain induces blisters via complement activation in experimental bullous pemphigoid model[J]. J Immunol, 2010, 185(12): 7746-7755.
[2] Leighty L, Li N, Diaz L A, Liu Z. Experimental models for the autoimmune and inflammatory blistering disease, Bullous pemphigoid[J]. Arch Dermatol Res, 2007, 299(9): 417-422.
[3] Ujiie H, Shibaki A, Nishie W, Shimizu H. What's new in bullous pemphigoid[J]. J Dermatol, 2010, 37(3): 194-204.
[4] Nishie W, Sawamura D, Goto M, Ito K, Shibaki A, Mcmillan J R, Sakai K, Nakamura H, Olasz E, Yancey K B, Akiyama M, Shimizu H. Humanization of autoantigen[J]. Nat Med, 2007, 13(3): 378-383.
[5]王刚,刘玉峰.大疱性类天疱疮患者噬菌体抗体库的构建及抗BP180-NC16A抗体筛选[J].中国皮肤性病学杂志, 2008, 22(11): 655-657.
[6]孙娜娜,王欲晓,周丽君,王刚.人源性抗大疱性类天疱疮抗原BP180-NC16A单链抗体的构建及表达[J].中国皮肤性病学杂志, 2009, 23(2): 71-74.
[7] Di Zenzo G, Marazza G, Borradori L. Bullous pemphigoid: physiopathology, clinical features and management[J]. Adv Dermatol, 2007, 23: 257-288.
[8] Liu Z, Giudice G J, Swartz S J, Fairley J A, Till G O, Troy J L, Diaz L A. The role of complement in experimental bullous pemphigoid[J]. J Clin Invest, 1995, 95(4): 1539-1544.
[9] Wang G, Ujiie H, Shibaki A, Nishie W, Tateishi Y, Kikuchi K, Li Q, Mcmillan J R, Morioka H, Sawamura D, Nakamura H, Shimizu H. Blockade of autoantibody-initiated tissue damage by using recombinant fab antibodyfragments against pathogenic autoantigen[J]. Am J Pathol, 2010, 176(2): 914-925.
[10] Borradori L, Sonnenberg A. Hemidesmosomes: roles in adhesion, signaling and human diseases[J]. Curr Opin Cell Biol, 1996, 8(5): 647-656.
[11] Kiss M, Husz S, Janossy T, Marczinovits I, Molnar J, Korom I, Dobozy A. Experimental bullous pemphigoid generated in mice with an antigenic epitope of the human hemidesmosomal protein BP230[J]. J Autoimmun, 2005, 24(1): 1-10.
[12] Laffitte E, Burkhard P R, Fontao L, Jaunin F, Saurat J H, Chofflon M, Borradori L. Bullous pemphigoid antigen 1 isoforms: potential new target autoantigens in multiple sclerosis?[J]. Br J Dermatol, 2005, 152(3): 537-540.
[13] Zillikens D. BP180 as the common autoantigen in blistering diseases with different clinical phenotypes[J]. Keio J Med, 2002, 51(1): 21-28.
[14] Zillikens D, Rose P A, Balding S D, Liu Z, Olague-Marchan M, Diaz L A, Giudice G J. Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies[J]. J Invest Dermatol, 1997, 109(4): 573-579.
[15] Hofmann S C, Tamm K, Hertl M, Borradori L. Diagnostic value of an enzyme-linked immunosorbent assay using BP180 recombinant proteins in elderly patients with pruritic skin disorders[J]. Br J Dermatol, 2003, 149(4): 910-912.
[16] Budinger L, Borradori L, Yee C, Eming R, Ferencik S, Grosse-Wilde H, Merk H F, Yancey K, Hertl M. Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls[J]. J Clin Invest, 1998, 102(12): 2082-2089.
[17] Black A P, Seneviratne S L, Jones L, King A S, Winsey S, Arsecularatne G,Wojnarowska F, Ogg G S. Rapid effector function of circulating NC16A-specific T cells in individuals with mucous membrane pemphigoid[J]. Br J Dermatol, 2004, 151(6): 1160-1164.
[18] Dopp R, Schmidt E, Chimanovitch I, Leverkus M, Brocker E B, Zillikens D. IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in Bullous pemphigoid: serum levels of these immunoglobulins reflect disease activity[J]. J Am Acad Dermatol, 2000, 42(4): 577-583.
[19] Sitaru C, Mihai S, Zillikens D. The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases[J]. Arch Dermatol Res, 2007, 299(1): 1-8.
[20] Liu Z. Are anti-BP180 IgG1 or IgG4 autoantibodies pathogenic?[J]. J Invest Dermatol, 2002, 119(5): 989-990.
[21] Tampoia M, Lattanzi V, Zucano A, Villalta D, Filotico R, Fontana A, Vena G A, Di Serio F. Evaluation of a new ELISA assay for detection of BP230 autoantibodies in bullous pemphigoid[J]. Ann N Y Acad Sci, 2009, 1173: 15-20.
[22] Suzuki M, Harada S, Yaoita H, Nakagawa H. Bullous pemphigoid IgG2 and IgG4 antibodies have a capability to inhibit a transient increase of intracellular Ca2+ induced by bullous pemphigoid IgG1 antibody[J]. Autoimmunity, 1999, 30(2): 85-91.
[23] Ujiie H, Shibaki A, Nishie W, Sawamura D, Wang G, Tateishi Y, Li Q, Moriuchi R, Qiao H, Nakamura H, Akiyama M, Shimizu H. A novel active mouse model for bullous pemphigoid targeting humanized pathogenic antigen[J]. J Immunol, 2010, 184(4): 2166-2174.
[24] Schmidt E, Wehr B, Tabengwa E M, Reimer S, Brocker E B, Zillikens D. Elevated expression and release of tissue-type, but not urokinase-type,plasminogen activator after binding of autoantibodies to bullous pemphigoid antigen 180 in cultured human keratinocytes[J]. Clin Exp Immunol, 2004, 135(3): 497-504.
[25] Liu Z, Li N, Diaz L A, Shipley M, Senior R M, Werb Z. Synergy between a plasminogen cascade and MMP-9 in autoimmune disease[J]. J Clin Invest, 2005, 115(4): 879-887.
[26] Zhao M, Trimbeger M E, Li N, Diaz L A, Shapiro S D, Liu Z. Role of FcRs in animal model of autoimmune bullous pemphigoid[J]. J Immunol, 2006, 177(5): 3398-3405.
[27] Leyendeckers H, Tasanen K, Bruckner-Tuderman L, Zillikens D, Sitaru C, Schmitz J, Hunzelmann N. Memory B cells specific for the NC16A domain of the 180 kDa bullous pemphigoid autoantigen can be detected in peripheral blood of bullous pemphigoid patients and induced in vitro to synthesize autoantibodies[J]. J Invest Dermatol, 2003, 120(3): 372-378.
[28] Lin M S, Fu C L, Giudice G J, Olague-Marchan M, Lazaro A M, Stastny P, Diaz L A. Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain[J]. J Invest Dermatol, 2000, 115(6): 955-961.
[29] Romagnani S. Human TH1 and TH2 subsets: regulation of differentiation and role in protection and immunopathology[J]. Int Arch Allergy Immunol, 1992, 98(4): 279-285.
[30] Giomi B, Caproni M, Calzolari A, Bianchi B, Fabbri P. Th1, Th2 and Th3 cytokines in the pathogenesis of bullous pemphigoid[J]. J Dermatol Sci, 2002, 30(2): 116-128.
[31] Nakashima H, Fujimoto M, Asashima N, Watanabe R, Kuwano Y, Yazawa N, Maruyama N, Okochi H, Kumanogoh A, Tamaki K. Serum chemokineprofile in patients with bullous pemphigoid[J]. Br J Dermatol, 2007, 156(3): 454-459.
[32] Cugno M, Tedeschi A, Asero R, Meroni P L, Marzano A V. Skin autoimmunity and blood coagulation[J]. Autoimmunity, 2010, 43(2): 189-194.
[33] Yasukawa S, Dainichi T, Kokuba H, Moroi Y, Urabe K, Hashimoto T, Furue M. Bullous pemphigoid followed by pustular psoriasis showing Th1, Th2, Treg and Th17 immunological changes[J]. Eur J Dermatol, 2009, 19(1): 69-71.
[34] Rensing-Ehl A, Gaus B, Bruckner-Tuderman L, Martin S F. Frequency, function and CLA expression of CD4+CD25+FOXP3+ regulatory T cells in bullous pemphigoid[J]. Exp Dermatol, 2007, 16(1): 13-21.
[35] Liu Z, Shapiro S D, Zhou X, Twining S S, Senior R M, Giudice G J, Fairley J A, Diaz L A. A critical role for neutrophil elastase in experimental bullous pemphigoid[J]. J Clin Invest, 2000, 105(1): 113-123.
[36] Yu X, Holdorf K, Kasper B, Zillikens D, Ludwig R J, Petersen F. FcgammaRIIA and FcgammaRIIIB are required for autoantibody-induced tissue damage in experimental human models of bullous pemphigoid[J]. J Invest Dermatol, 2010, 130(12): 2841-2844.
[37] Nelson K C, Zhao M, Schroeder P R, Li N, Wetsel R A, Diaz L A, Liu Z. Role of different pathways of the complement cascade in experimental bullous pemphigoid[J]. J Clin Invest, 2006, 116(11): 2892-2900.
[38] Heimbach L, Li Z, Berkowitz P, Zhao M, Li N, Rubenstein D, Diaz L A, Liu Z. C5A receptor on mast cells is critical for the autoimmune skin blistering disease bullous pemphigoid[J]. J Biol Chem, 2011.
[39] Chen R, Fairley J A, Zhao M L, Giudice G J, Zillikens D, Diaz L A, Liu Z.Macrophages, but not T and B lymphocytes, are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation[J]. J Immunol, 2002, 169(7): 3987-3992.
[40] Stahle-Backdahl M, Inoue M, Guidice G J, Parks W C. 92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen[J]. J Clin Invest, 1994, 93(5): 2022-2030.
[41] Borrego L, Maynard B, Peterson E A, George T, Iglesias L, Peters M S, Newman W, Gleich G J, Leiferman K M. Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins[J]. Am J Pathol, 1996, 148(3): 897-909.
[42] Gounni A S, Wellemans V, Agouli M, Guenounou M, Hamid Q, Beck L A, Lamkhioued B. Increased expression of Th2-associated chemokines in bullous pemphigoid disease. Role of eosinophils in the production and release of these chemokines[J]. Clin Immunol, 2006, 120(2): 220-231.
[43] Hertl M, Eming R, Veldman C. T cell control in autoimmune bullous skin disorders[J]. J Clin Invest, 2006, 116(5): 1159-1166.
[44] Laszlo S, Neumann S, Hertl M, Hunzelmann N. Generation of increased numbers of HLA-DR(high) IgG+ plasma cells in the peripheral blood of patients with bullous pemphigoid: NC16a-specific cells belong to the short-lived plasma blast population[J]. J Invest Dermatol, 2010, 130(12): 2838-2841.
[45] Liu Z, Diaz L A, Troy J L, Taylor A F, Emery D J, Fairley J A, Giudice G J. A passive transfer model of the organ-specific autoimmune disease, bullouspemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180[J]. J Clin Invest, 1993, 92(5): 2480-2488.
[46] Liu Z, Zhao M, Li N, Diaz L A, Mayadas T N. Differential roles for beta2 integrins in experimental autoimmune bullous pemphigoid[J]. Blood, 2006, 107(3): 1063-1069.
[47] Liu Z, Sui W, Zhao M, Li Z, Li N, Thresher R, Giudice G J, Fairley J A, Sitaru C, Zillikens D, Ning G, Marinkovich M P, Diaz L A. Subepidermal blistering induced by human autoantibodies to BP180 requires innate immune players in a humanized bullous pemphigoid mouse model[J]. J Autoimmun, 2008, 31(4): 331-338.
[48] Nishie W, Sawamura D, Natsuga K, Shinkuma S, Goto M, Shibaki A, Ujiie H, Olasz E, Yancey K B, Shimizu H. A novel humanized neonatal autoimmune blistering skin disease model induced by maternally transferred antibodies[J]. J Immunol, 2009, 183(6): 4088-4093.
[49] Zillikens D, Schmidt E, Reimer S, Chimanovitch I, Hardt-Weinelt K, Rose C, Brocker E B, Kock M, Boehncke W H. Antibodies to desmogleins 1 and 3, but not to BP180, induce blisters in human skin grafted onto SCID mice[J]. J Pathol, 2001, 193(1): 117-124.
[50] Zone J J, Taylor T, Hull C, Schmidt L, Meyer L. IgE basement membrane zone antibodies induce eosinophil infiltration and histological blisters in engrafted human skin on SCID mice[J]. J Invest Dermatol, 2007, 127(5): 1167-1174.
[51] Fairley J A, Burnett C T, Fu C L, Larson D L, Fleming M G, Giudice G J. A pathogenic role for IgE in autoimmunity: bullous pemphigoid IgE reproduces the early phase of lesion development in human skin grafted to nu/nu mice[J]. J Invest Dermatol, 2007, 127(11): 2605-2611.
[52] Olasz E B, Roh J, Yee C L, Arita K, Akiyama M, Shimizu H, Vogel J C, Yancey K B. Human bullous pemphigoid antigen 2 transgenic skin elicits specific IgG in wild-type mice[J]. J Invest Dermatol, 2007, 127(12): 2807-2817.
[53] Sitaru C, Schmidt E, Petermann S, Munteanu L S, Brocker E B, Zillikens D. Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin[J]. J Invest Dermatol, 2002, 118(4): 664-671.
[54] Mihai S, Chiriac M T, Herrero-Gonzalez J E, Goodall M, Jefferis R, Savage C O, Zillikens D, Sitaru C. IgG4 autoantibodies induce dermal-epidermal separation[J]. J Cell Mol Med, 2007, 11(5): 1117-1128.
[55] Loo W J, Burrows N P. Management of autoimmune skin disorders in the elderly[J]. Drugs Aging, 2004, 21(12): 767-777.
[56] Khumalo N, Kirtschig G, Middleton P, Hollis S, Wojnarowska F, Murrell D. Interventions for bullous pemphigoid[J]. Cochrane Database Syst Rev, 2005(3): D2292.
[57] Joly P, Fontaine J, Roujeau J C. The role of topical corticosteroids in bullous pemphigoid in the elderly[J]. Drugs Aging, 2005, 22(7): 571-576.
[58] Herrero-Gonzalez J E, Sitaru C, Klinker E, Brocker E B, Zillikens D. Successful adjuvant treatment of severe bullous pemphigoid by tryptophan immunoadsorption[J]. Clin Exp Dermatol, 2005, 30(5): 519-522.
[59] Peterson J D, Chan L S. Effectiveness and side effects of anti-CD20 therapy for autoantibody-mediated blistering skin diseases: A comprehensive survey of 71 consecutive patients from the Initial use to 2007[J]. Ther Clin Risk Manag, 2009, 5(1): 1-7.
[60] Saouli Z, Papadopoulos A, Kaiafa G, Girtovitis F, Kontoninas Z. A newapproach on bullous pemphigoid therapy[J]. Ann Oncol, 2008, 19(4): 825-826.
[61] Szabolcs P, Reese M, Yancey K B, Hall R P, Kurtzberg J. Combination treatment of bullous pemphigoid with anti-CD20 and anti-CD25 antibodies in a patient with chronic graft-versus-host disease[J]. Bone Marrow Transplant, 2002, 30(5): 327-329.
[62] Lanschuetzer C M, Olasz E B, Lazarova Z, Yancey K B. Transient anti-CD40L co-stimulation blockade prevents immune responses against human bullous pemphigoid antigen 2: implications for gene therapy[J]. J Invest Dermatol, 2009, 129(5): 1203-1207.
[63]朱振洪余勤万海同.基因工程小分子抗体的研究进展[J].生物技术通讯, 2008(Z1): 59-62.
[64] Cheng W W, Allen T M. The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment[J]. Expert Opin Drug Deliv, 2010, 7(4): 461-478.
[65] Smith R, Tarner I H, Hollenhorst M, Lin C, Levicnik A U, Fathman C G, Nolan G P. Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis[J]. Gene Ther, 2003, 10(15): 1248-1257.
[66] Meng F, Stassen M H, Schillberg S, Fischer R, De Baets M H. Construction and characterization of a single-chain antibody fragment derived from thymus of a patient with myasthenia gravis[J]. Autoimmunity, 2002, 35(2): 125-133.
[67] Li Q, Ujiie H, Shibaki A, Wang G, Moriuchi R, Qiao H J, Morioka H, Shinkuma S, Natsuga K, Long H A, Nishie W, Shimizu H. Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16Adomain induces blisters via complement activation in experimental bullous pemphigoid model[J]. J Immunol, 2010, 185(12): 7746-7755.