Development of a Cross-Reactive Monoclonal Antibody for Detecting the Tumor Stroma
Development of a Cross-Reactive Monoclonal Antibody for Detecting the Tumor Stroma
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DOI：
10.1021/acs.bioconjchem.9b00206
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发表时间：
2019-05-01
影响因子：
4.7
通讯作者：
LeBeau, Aaron M.
中科院分区：
文献类型：
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作者：
Hintz, Hallie M.;Cowan, Aidan E.;LeBeau, Aaron M.
在这里，我们发现了一种能选择性地与人和小鼠成纤维细胞激活蛋白α(FAP)结合的单抗，FAP是一种在癌症相关成纤维细胞(CAF)上过度表达的丝氨酸蛋白酶，使其成为辅助和教唆肿瘤微环境的有吸引力的治疗靶点。从初步筛选的抗B12轻链的鼠源单链抗体噬菌体展示文库中筛选出抗B12的先导抗体B12，并将其克隆到全长人重组人FAP磁珠上。重组人免疫球蛋白1(Ig G)载体，表达为嵌合单抗(B12 Ig G)。我们设计了一种耐药的前列腺癌细胞系CWR-R1-EnzR，用于表达人FAP以用于抗体鉴定和验证(R1-EnzR(FAP))。B12免疫球蛋白通过流式细胞仪选择性地结合到R1EnzR(FAP)细胞上，并在体外通过共聚焦显微镜被内化。在R1-EnzR(FAP)和亲代异种移植模型中，进一步评价了B12免疫球蛋白作为近红外(NIR)光学成像探针的作用。在R1EnzR(FAP)移植瘤中观察到高摄取和滞留的NIR探针，并在亲代移植瘤中检测到内源性小鼠基质来源FAP的表达。通过免疫组织化学对这些模型的体外评估记录了B12免疫球蛋白对人和小鼠FAP表达细胞的定位。
Here, we document the discovery of a monoclonal antibody that selectively binds to both human and murine fibroblast activation protein alpha (FAP), a serine protease that is overexpressed on cancer-associated fibroblasts (CAFs), making it an attractive therapeutic target for the aiding and abetting tumor microenvironment. The lead antibody, B12, was identified from a naive murine single-chain variable fragment antibody phage display library screened against light chains of B12 were cloned into full-length human recombinant human FAP on magnetic beads. The heavy and immunoglobulin 1 (IgG) vectors and expressed as a chimeric monoclonal antibody (B12 IgG). We engineered a drug-resistant prostate cancer cell line, CWR-R1-EnzR, to express human FAP for antibody characterization and validation (R1-EnzR(FAP)). B12 IgG selectively bound to the R1-EnzR(FAP) cells by flow cytometry and was internalized in vitro by confocal microscopy. B12 IgG was further evaluated as a near-infrared (NIR) optical imaging probe in R1-EnzR(FAP) and parental xenograft models. High tumor uptake and retention of the NIR probe was observed in the R1-EnzR(FAP) xenografts, and endogenous expression of murine stromal origin FAP was detected in the parental xenografts. Ex vivo evaluation of these models by immunohistochemistry documented B12 IgG localization to both human and murine FAP-expressing cells.