Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors.
Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors.
复制标题
DOI：
10.1186/s12885-015-1608-z
复制
发表时间：
2015-09-03
期刊：
影响因子：
3.8
通讯作者：
Claffey KP
中科院分区：
文献类型：
--
作者：
Devarakonda CV;Kita D;Phoenix KN;Claffey KP
单克隆抗体已被用于有效治疗各种肿瘤。我们以前建立了一个独特的策略，以确定肿瘤特异性抗体捕获B细胞反应对乳腺肿瘤抗原从患者来源的前哨淋巴结。这种方法的最初应用导致了肿瘤特异性单域抗体的鉴定。在本文中，我们优化了我们以前的策略，通过产生重链抗体（HCAbs），以克服单域抗体的不足。在这里，我们确定并表征了重链抗体（HCAb 2），其靶向乳腺肿瘤细胞而不是正常细胞上的细胞表面HSP 90抗原。使用体外表达系统产生来自4名乳腺癌患者的8个HCAb。使用流式细胞术和免疫荧光针对正常乳腺细胞（MCF 10A，HMEC）和肿瘤细胞系（MCF 7，MDA-MB-231）筛选HCAb，以鉴定细胞表面靶向和肿瘤特异性抗体。通过使用免疫荧光筛选一组原代人乳腺正常和肿瘤组织来验证用细胞系观察到的结果。分别为两个HCAbs（HCAb 1和HCAb 2）的抗原进行了鉴定，使用免疫沉淀，然后质谱。最后，我们在NOD scid gamma小鼠中产生了MDA-MB-231异种移植肿瘤，并进行了HCAb 1和HCAb 2的体内肿瘤靶向分析。流式细胞术筛选显示，与MCF 10A和MCF 7细胞相比，HCAb 2选择性地结合于MDA-MB-231细胞的表面。HCAb 2在MDA-MB-231细胞上显示点状膜染色，并且与正常乳腺组织相比优先结合人乳腺肿瘤组织。在原发性乳腺癌组织中，HCAb 2与E-cadherin阳性和阴性肿瘤细胞均呈阳性结合。我们鉴定并证实HCAb 2的靶抗原为热休克蛋白90（HSP 90）。HCAb 2在体内也选择性靶向MDA-MB-231异种移植肿瘤细胞，几乎不靶向小鼠正常组织。最后，HCAb 2特异性靶向钙连接蛋白阴性异种移植肿瘤细胞。从我们的筛选方法，我们确定HCAb 2作为乳腺肿瘤特异性重链抗体靶向细胞表面HSP 90。HCAb 2在体内也能靶向MDA-MB-231肿瘤细胞，表明HCAb 2可能是一种理想的肿瘤靶向抗体。本文的在线版本（doi：10.1186/s12885-015-1608-z）包含补充材料，可供授权用户使用。
Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells. Eight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2. Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells. From our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody. The online version of this article (doi:10.1186/s12885-015-1608-z) contains supplementary material, which is available to authorized users.