Development of antibody engineering-based cancer therapies

硫酸乙酰肝素蛋白聚糖（HSPG）调节许多细胞表面信号传导事件。它们是发育和疾病（如癌症）期间信号转导途径的细胞外调节剂。HSPG是细胞表面蛋白，主要由糖基磷脂酰肌醇（GPI）锚定的磷脂酰肌醇蛋白聚糖和跨膜多配体蛋白聚糖组成。在过去的15年里，Mitchell Ho博士在美国国家癌症研究所（NCI）的实验室已经开发了GPC 3和其他磷脂酰肌醇蛋白聚糖，如GPC 1和GPC 2，作为一个新的癌症治疗靶点家族，并产生了用于治疗实体瘤（包括肝癌，儿科癌症和其他实体瘤）的新型抗体和基于细胞的免疫技术。于2023财政年度，我们继续优化靶向GPC 3的CAR-T细胞，以治疗肝细胞癌（HCC）（肝癌的主要形式）。在我们之前的研究中（Li et al. Gastroenterology，2020），我们发现靶向YP 7抗体识别的近膜位点的CAR-T细胞比靶向HN 3识别的膜远端位点的CAR-T细胞更有效，表明目前的CAR形式不是针对靶向膜远端表位而定制的。在FY 2023中，使用hYP 7 Fv（膜近端）和HN 3 VH纳米抗体（膜远端）作为GPC 3靶向元件，我们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。我们产生了含有CD 8、CD 28、IgG 4和Fc结构域的CAR T细胞的多种排列。我们表明，工程化的HN 3 CAR T细胞可以通过铰链和跨膜结构域中的2个独立的协同变化来改善。表达含有IgG 4铰链区和CD 28跨膜结构域的HN 3 CAR（HN 3-IgG 4 H-CD 28 TM）的T细胞表现出高细胞毒性活性，并在免疫缺陷小鼠中引起完全的HCC肿瘤根除。HN 3-IgG 4 H-CD 28 TM CAR T细胞富集细胞毒性记忆性CD 8 + 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，与膜近端表位相比，改变靶向远端GPC 3表位的基于纳米抗体的CAR的铰链和跨膜结构域，导致强大的T细胞信号传导，并诱导HCC肿瘤的快速和持久根除。我们于2023财年在Hepatology Communications上发表了这项工作（Aarti Kolluri，Dan Li，Nan Li，Zhijian Duan，刘易斯R Roberts，Mitchell Ho。靶向磷脂酰肌醇蛋白聚糖3的人VH嵌合抗原受体T细胞在HCC临床前模型中消除肿瘤肝细胞通讯2023年1月18日;7（2）：e0022。doi：10.1097/HC9.0000000000022。eCollection 2023年2月1日）。除了GPC 3和GPC 2之外，在2023财年，Mitchell Ho博士的实验室开发了针对胰腺癌中表达的癌胚抗原GPC 1的新型CAR T细胞。我们报道了靶向GPC 1的基于单峰骆驼VHH纳米抗体（D4）的CAR T细胞的产生以及铰链（H）和跨膜结构域（TM）的优化以提高活性。我们发现，结构刚性的IgG 4 H和CD 28 TM结构域使两个D4片段接近，驱动CAR二聚化，并导致在雌性小鼠中具有低抗原密度的胰腺癌模型中增强的T细胞信号传导和肿瘤消退。此外，D4-IgG 4 H-CD 28 ™ CAR T细胞的基于单细胞的蛋白质组学和转录组学分析揭示了特异性基因（例如，HMGB 1）与高T细胞多功能性相关。我们的研究证明了基于VHH的CAR T用于胰腺癌治疗的潜力，并为开发靶向膜远端表位的有效CAR T细胞提供了工程策略。这项工作于2023年4月发表在Nature Communications上（Nan Li，Alex Quan，Dan Li，Jiajia Pan，Hua Ren，Gerard Hoeltzel，娜塔莉亚de瓦尔，Dana Ashworth，Weiming Ni，Jing Zhou，Sean Mackay，Stephen M休伊特，Raul Cachau，Mitchell Ho.具有CD 28跨膜结构域的IgG 4铰链改善了靶向胰腺癌中GPC 1的膜远端表位的基于VHH的CAR T细胞。Nat Commun. 2023年4月8日;14（1）：1986。doi：10.1038/s41467-023-37616-4。为了追求CAR-T细胞疗法的临床开发，我们与NCI CCR儿科肿瘤分支的Carol Thiele，Rosandra Kaplan和Rosa Nguyen合作，为NIH临床中心即将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，我们证明了我们的靶向GPC 2，CT 3的单链可变片段整合到具有CD 28铰链，CD 28跨膜和4-1BB共刺激结构域（CT3.28H）的CAR载体中，与其他测试的CAR构建体相比，引起了最好的临床前抗肿瘤活性。这种增强的活性与肿瘤微环境中CD 8+效应T细胞的富集和几种效应分子如GNLY、GZMB、ZNF 683和HMGN 2的上调有关。我们得出结论，鉴于CT3.28H的强大临床前活性，这些结果为神经母细胞瘤儿童的进一步临床试验奠定了有希望的基础。该研究发表在2023财年的Journal of Immunotherapy of Cancer上（Sun et al. 2023）。我们已经开发了GPC 3（hYP 7），GPC 2（CT 3）和间皮素（hYP 218）CAR T细胞，用于NIH治疗肝癌，间皮瘤和神经母细胞瘤的临床试验。Mitchell Ho博士从癌症登月计划和NCI CCR获得资金，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.