Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.
PLoS One. 2021 Apr 23;16(4):e0250175. doi: 10.1371/journal.pone.0250175. eCollection 2021.
CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated in Escherichia coli support the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21-220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.
CD44 是一种跨膜糖蛋白，与透明质酸结合，在许多细胞过程中发挥作用，并在多种细胞类型中表达。它在干细胞和癌症中上调。抗 CD44 单克隆抗体影响细胞运动和聚集，并抑制肿瘤发生和转移。在这里，我们描述了四种新的抗 CD44 单克隆抗体，它们源自注射表达 CD44 同种型 12 的质粒的小鼠的 B 细胞。这四种单克隆抗体结合到 CD44 同种型的末端、细胞外、保守结构域。根据癌细胞裂解物的 western blot 模式的差异，这四种抗 CD44 mAb 分为三个不同的类别，包括 P4G9、P3D2 和 P3A7，以及 P3G4。用大肠埃希菌产生的肽进行斑点分析支持这样的结论，即单克隆抗体识别 N 端保守区 21-220 位氨基酸之间未糖基化的序列，用人类胚胎肾 293 细胞产生的肽进行分析表明，这些单克隆抗体仅在糖基化后才与这些肽结合。用三种癌细胞系的裂解物进行的 western blot 分析表明，几种 CD44 同种型在抗 CD44 靶区未糖基化。通过将乳腺癌衍生的致瘤细胞系 MDA-MB-231 的细胞与抗 CD44 单克隆抗体 P3D2 共注射到小鼠的乳腺脂肪垫中，测试了单克隆抗体在阻断肿瘤发生中的潜在用途。用 MDA-MB-231 细胞加抗 IgG 注射的五只对照小鼠均形成可触及的肿瘤，而用 MDA-MB-231 细胞加 P3D2 注射的六只试验小鼠中只有一只形成微小肿瘤，其余五只无肿瘤，表明这四种抗 CD44 mAb 可能具有治疗作用。
