Development of antibody engineering-based cancer therapies

硫酸肝素蛋白聚糖（HSPGs）调节许多细胞表面信号转导事件。它们是发育和疾病（如癌症）过程中信号转导通路的细胞外调节剂。HSPGs是细胞表面蛋白，主要由糖基磷脂酰肌醇（GPI）锚定的glypicans和跨膜syndecans组成。在过去的15年里，Mitchell Ho博士在国家癌症研究所（NCI）的实验室开发了GPC3和其他glypicans，如GPC1和GPC2，作为一个新的癌症治疗靶点家族，并产生了新的抗体和基于细胞的免疫治疗技术，用于治疗实体肿瘤，包括肝癌、儿童癌症和其他实体肿瘤。在2023财年，我们继续优化靶向GPC3的CAR-T细胞治疗肝细胞癌（HCC），这是肝癌的一种主要形式。在我们之前的研究中(Li et al.；胃肠病学，2020)，我们发现靶向YP7抗体识别的膜近端部位的CAR- t细胞比靶向HN3识别的膜远端部位的CAR- t细胞更有效，这表明目前的CAR格式不是针对膜远端表位量身定制的。在2023财年，我们使用hYP7 Fv（膜近端）和HN3 VH纳米体（膜远端）作为GPC3靶向元件，试图确定不同结构和大小的铰链和跨膜部分如何影响CAR - t细胞功能。我们生成了含有CD8、CD28、IgG4和Fc结构域的CAR - T细胞的多种排列。我们表明，工程HN3 CAR - T细胞可以通过铰链和跨膜结构域的两个独立的协同变化来改善。表达含有IgG4铰链区和CD28跨膜结构域的HN3 CAR （HN3- igg4h - cd28tm）的T细胞表现出高的细胞毒活性，并在免疫缺陷小鼠中完全根除HCC肿瘤。HN3-IgG4H-CD28TM CAR - T细胞富集细胞毒性记忆CD8+ T细胞和NFAT信号，并降低HCC细胞中的β -连环蛋白水平。我们的研究结果表明，改变基于纳米体的CAR靶向远端GPC3表位的铰链和跨膜结构域，而不是膜-近端表位，可以导致强大的t细胞信号传导，并诱导快速持久地根除HCC肿瘤。我们于2023财年在《肝病通讯》（Aarti Kolluri, Dan Li, Nan Li, Zhijian Duan, Lewis R Roberts, Mitchell Ho）上发表了这项工作。靶向glypican 3的人基于vh的嵌合抗原受体T细胞在HCC临床前模型中消除肿瘤。国际肝病杂志，2018;7(2):e0022。doi: 10.1097 / HC9.0000000000000022。藏品2023年2月1日)。除了GPC3和GPC2，在2023财年，Mitchell Ho博士的实验室开发了针对GPC1的新型CAR - T细胞，GPC1是一种在胰腺癌中表达的癌胎儿抗原。我们报道了基于单峰骆驼VHH纳米体（D4）的靶向GPC1的CAR - T细胞的产生，并优化了铰链(H)和跨膜结构域（TM）以提高活性。我们发现，在低抗原密度的雌性小鼠胰腺癌模型中，结构刚性的IgG4H和CD28TM结构域使两个D4片段接近，驱动CAR二聚化，并导致t细胞信号传导增强和肿瘤退化。此外，基于单细胞的D4-IgG4H-CD28TM CAR - T细胞蛋白质组学和转录组学分析揭示了与高T细胞多功能性相关的特定基因（例如HMGB1）。我们的研究证明了基于vhh的CAR - T治疗胰腺癌的潜力，并为开发靶向膜远端表位的强效CAR - T细胞提供了一种工程策略。这项研究发表在2023年4月的《自然通讯》杂志上（作者：李楠、Alex Quan、Dan Li、潘家佳、任华、Gerard Hoeltzel、Natalia de Val、Dana Ashworth、倪伟明、周静、Sean Mackay、Stephen M Hewitt、Raul Cachau、Mitchell Ho）。IgG4与CD28跨膜结构域的铰链改善了胰腺癌中靶向GPC1膜远端表位的VHH-based CAR - T细胞。自然科学学报。2023年4月8日；14(1):1986。doi: 10.1038 / s41467 - 023 - 37616 - 4。为了追求CAR-T细胞疗法的临床发展，我们与NCI CCR儿科肿瘤科的Carol Thiele， Rosandra Kaplan和Rosa Nguyen合作，为即将在NIH临床中心进行的治疗神经母细胞瘤的临床试验生产临床就绪的CAR-T细胞。通过我们的合作，我们证明了将靶向GPC2、CT3的单链可变片段整合到具有CD28铰链、CD28跨膜和4-1BB共刺激结构域（CT3.28H）的CAR载体中，与其他已测试的CAR构建物相比，具有最佳的临床前抗肿瘤活性。这种增强的活性与肿瘤微环境中CD8+效应T细胞的富集和几种效应分子（如GNLY、GZMB、ZNF683和HMGN2）的上调有关。我们的结论是，考虑到CT3.28H强大的临床前活性，这些结果为进一步在儿童神经母细胞瘤中进行临床试验奠定了有希望的基础。该研究发表于2023财年的《Journal of Immunotherapy of Cancer》（Sun et al. 2023）。我们已经开发出GPC3 （hYP7）、GPC2 （CT3）和间皮素（hYP218） CAR - T细胞用于NIH的临床试验，用于治疗肝癌、间皮瘤和神经母细胞瘤。Mitchell Ho博士获得了癌症登月计划和NCI CCR的资金，以支持NIH治疗癌症患者的临床试验。

Heparan sulfate proteoglycans (HSPGs) regulate numerous cell surface signaling events. They are extracellular modulators of signal transduction pathways during development and diseases such as cancer. HSPGs are cell-surface proteins that mainly consist of glycosylphosphatidylinositol (GPI)-anchored glypicans and transmembrane syndecans. In the last over 15 years, Dr Mitchell Ho's laboratory at the National Cancer Institute (NCI) has developed GPC3 and other glypicans such as GPC1 and GPC2 as a new family of cancer therapeutic targets and generated novel antibody and cell-based immunotherapeutic technologies for treating solid tumors including liver cancer, pediatric cancers, and other solid tumors. In FY2023, we continued to optimize the CAR-T cells targeting GPC3 for treating hepatocellular carcinoma (HCC), a major form of liver cancer. In our previous study (Li et al. Gastroenterology, 2020), we found CAR-T cells targeting a membrane-proximal site recognized by YP7 antibody were more potent than the CAR-T cells targeting a membrane distal site recognized by HN3, indicating current CAR formats are not tailored toward targeting membrane distal epitopes. In FY2023, using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced beta-catenin levels in HCC cells. Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane-proximal epitope, leads to robust T-cell signaling and induces swift and durable eradication of HCC tumors. We published this work in FY2023 in Hepatology Communications (Aarti Kolluri, Dan Li, Nan Li, Zhijian Duan, Lewis R Roberts, Mitchell Ho. Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC. Hepatol Commun. 2023 Jan 18;7(2):e0022. doi: 10.1097/HC9.0000000000000022. eCollection 2023 Feb 1.). In addition to GPC3 and GPC2, in FY2023 Dr. Mitchell Ho's lab developed novel CAR T cells targeting GPC1, an oncofetal antigen expressed in pancreatic cancer. We reported the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We found that a structurally rigid IgG4H and CD28TM domain brought the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells revealed specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. Our study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes. This work was published in Nature Communications in April 2023 (Nan Li, Alex Quan, Dan Li, Jiajia Pan, Hua Ren, Gerard Hoeltzel, Natalia de Val, Dana Ashworth, Weiming Ni, Jing Zhou, Sean Mackay, Stephen M Hewitt, Raul Cachau, Mitchell Ho. The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer. Nat Commun. 2023 Apr 8;14(1):1986. doi: 10.1038/s41467-023-37616-4. To pursue the clinical development of CAR-T cell therapy, we have collaborated with Carol Thiele, Rosandra Kaplan, and Rosa Nguyen in the Pediatric Oncology Branch at the CCR, NCI to produce clinical-ready CAR-T cells for an upcoming clinical trial treating neuroblastoma in the NIH Clinical Center. In a major effort through our collaboration, we demonstrated that our single-chain variable fragment targeting GPC2, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H) elicited the best preclinical anti-tumor activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. We concluded that given the robust preclinical activity of CT3.28H, these results form a promising basis for further clinical testing in children with neuroblastoma. The work was published in the Journal of Immunotherapy of Cancer in FY2023 (Sun et al. 2023). We have developed GPC3 (hYP7), GPC2 (CT3) and mesothelin (hYP218) CAR T cells for clinical trials at the NIH for treating liver cancer, mesothelioma, and neuroblastoma. Dr. Mitchell Ho has obtained funds from the Cancer Moonshot program and the NCI CCR to support these clinical trials for treating cancer patients at the NIH.