Development of new antibody-based cancer therapies

开发新的基于抗体的癌症疗法

• 批准号: 10702433
• 负责人: Mitchell Ho
• 金额: $ 162.98万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702433
• 关键词: Albumins; American; Animal Model; Antibodies; Antibody Therapy; Antigen Targeting; Antigens; Bacteriophages; Binding; Bispecific Antibodies; CAR T cell therapy; CCR; CD276 gene; Cancer Biology; Cancer Family; Canis familiaris; Cell Surface Proteins; Cell Therapy; Cell membrane; Cell physiology; Cell surface; Cells; Childhood Extracranial Solid Tumor; Chimeric Proteins; Clinical; Clinical Research; Clinical Trials; Communities; Data; Development; Disease; Down-Regulation; Epitopes; Event; Fusion Toxin; GPC3 gene; GPI Membrane Anchors; Glypican; Half-Life; Hematologic Neoplasms; Heparan Sulfate Proteoglycan; Human; Immunization; Immunosuppression; Immunotherapeutic agent; Immunotoxins; Infiltration; Journals; Laboratories; Libraries; Liver neoplasms; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant mesothelioma; Malignant neoplasm of liver; Malignant neoplasm of pancreas; Mediator of activation protein; Membrane; Mesothelioma; Methodology; Methods; Molecular; Molecular Biology; Monoclonal Antibodies; Muromonab-CD3; Mus; National Cancer Institute; Neoplasm Metastasis; Neuroblastoma; Oncolytic; Patients; Pediatric Neoplasm; Phage Display; Primary carcinoma of the liver cells; Production; Protein Biosynthesis; Protein Inhibition; Proteoglycan; Protocols documentation; Pseudomonas aeruginosa toxA protein; Publishing; Research; Role; Serum; Shark; Signal Transduction; Signal Transduction Pathway; Site; Solid Neoplasm; T-Lymphocyte; Technology; Therapeutic; Therapeutic antibodies; Tumor Antigens; United States National Institutes of Health; WNT Signaling Pathway; Work; Xenograft Model; antibody engineering; base; beta catenin; cancer cell; cancer therapy; chimeric antigen receptor; chimeric antigen receptor T cells; clinical development; cross reactivity; exhaustion; extracellular; immunogenic; improved; inducible gene expression; inhibiting antibody; irinotecan; malignant breast neoplasm; mesothelin; nanobodies; neoplastic cell; new technology; novel; novel strategies; overexpression; pancreatic neoplasm; preclinical development; preclinical study; programmed cell death ligand 1; programs; screening; small molecule; syndecan; targeted treatment; treatment strategy; tumor; tumor growth; tumor microenvironment; tumor-immune system interactions

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 14 years, Dr Mitchell Ho's laboratory at the National Cancer Institute (NCI) has studied GPC3 and other glypicans as a new family of cancer targets and developed novel antibody and cell-based immunotherapeutic technologies for treating solid tumors including liver cancer, pediatric cancers and other solid tumors. In FY2022, we created novel immunotoxins targeting GPC1 in pancreatic cancer and showed immunotoxins inhibited pancreatic cancer in mice and published our preclinical studies in Molecular Cancer Therapeutics [Pan et al. Molecular Cancer Therapeutics, 2022]. GPC1 is a cell surface proteoglycan that is upregulated in multiple types of human cancers including pancreatic cancer. We investigated whether GPC1 could be a target of antibody-toxin fusion proteins (i.e., immunotoxins) for treating pancreatic cancer. We constructed a panel of GPC1-targeted immunotoxins derived from a functional domain of Pseudomonas exotoxin A. An albumin-binding domain was also introduced into the anti-GPC1 immunotoxin to improve serum half-life. Small-molecule screening was performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. We showed that GPC1 was internalized upon antibody binding. Anti-GPC1 immunotoxins alone inhibited tumor growth in a pancreatic cancer xenograft model. The immunotoxin treatment reduced active beta-catenin expression in tumor cells. Furthermore, immunotoxins containing an albumin-binding domain in combination with irinotecan caused pancreatic tumor regression. GPC1 expression was reduced by the immunotoxin treatment due to the degradation of the internalized GPC1 and its short cellular turnover rate. Our data indicate that the GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment strategy for patients with pancreatic tumors. We also summarized our work in studying GPC1 as an immunotherapeutic target in pancreatic cancer in a review article published in the American Journal of Physiology Cell Physiology [Pan and Ho, Am J Physiol Cell Physiol. 2021]. In FY2022, we also developed novel PD-L1 targeted shark VNAR single-domain-based CAR-T cell strategy for treating breast cancer and liver cancer [Li et al. Molecular Therapy Oncolytics 2022]. Chimeric antigen receptor (CAR)-T cell therapy shows excellent potency against hematological malignancies, but it remains challenging to treat solid tumors, mainly because of a lack of appropriate antigenic targets and an immunosuppressive tumor microenvironment (TME). The checkpoint molecule programmed death-ligand 1 (PD-L1) is widely overexpressed in multiple tumor types, and the programmed death-ligand 1 (PD-1)/PD-L1 interaction is a crucial mediator of immunosuppression in the TME. In this new study, we constructed a semi-synthetic shark VNAR phage library and isolated anti-PD-L1 single-domain antibodies. Among these VNARs, B2 showed cross-reactivity to human, mouse, and canine PD-L1, and it partially blocked the interaction of human PD-1 with PD-L1. CAR (B2) T cells specifically lysed human breast cancer and liver cancer cells by targeting constitutive and inducible expression of PD-L1 and hindered tumor metastasis. Combination of PD-L1 CAR (B2) T cells with CAR T cells targeted by GPC3 (a liver cancer-specific antigen) regresses liver tumors in mice. We concluded that PD-L1-targeted shark VNAR single-domain-based CAR-T therapy is a novel strategy to treat breast and liver cancer. This study provides a rationale for potential use of PD-L1 CAR-T cells as a monotherapy or in combination with a tumor-specific therapy in clinical studies. Besides cell-based therapy, we also developed bispecific antibody hYP7-OKT3-hFc targeting GPC3 and showed its potent activity in mice [Chen et al. Mol Cancer Ther 2022]. We also published three protocols regarding the construction and production of CAR-T cells and immunotoxins for cancer therapy in STAR Protocols (Cell Press) and Methods in Molecular Biology [Li et al. STAR Protocols 2021; Li and Ho, Methods in Molecular Biology 2022; Fleming and Ho, Methods in Molecular Biology 2022]. We shared our protocols and methodologies in the scientific community to help advance the CAR-T field quickly. To develop CAR-T for treating other solid tumors, we collaborated with Dr. Ira Pastan's lab and Dr. Raffit Hassan's lab to develop novel CAR-T cell therapies targeting mesothelin [Tomar et al., Mol Cancer Ther 2022; Liu et al., PNAS, 2022] and with Javed Khan's lab and Brad St Croix's lab to develop novel bicistronic CAR against GPC2 or CD276 in neuroblastoma [Tian et al., J Clin Invest. 2022]. We developed hYP218 (against membrane-proximal epitope) CAR T cells [Tomar et al. Mol Cancer Ther 2022]. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers. In another study led by Dr. Pastan's lab and our lab, we have identified shed mesothelin as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed mesothelin and merits further preclinical development [Liu et al., PNAS, 2022]. CAR-T cell therapies targeting single antigens perform poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T-cell persistence and exhaustion. A project led by Dr. Khan's lab, Dr. St Croix's lab and our lab aimed to identify optimal CARs against GPC2 or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. We made a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T-cells effectively eliminated tumor cells expressing GPC2 or CD276. For clinical development, we have developed GPC3 (hYP7), GPC2 (CT3) and mesothelin (hYP218) CAR T cells for the clinical trials at the NIH for treating liver cancer, mesothelioma, and neuroblastoma as supported by the Cancer Moonshot program and the NCI CCR.

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14年里，Mitchell Ho博士在美国国家癌症研究所（NCI）的实验室研究了GPC3和其他glypicans作为一个新的癌症靶点家族，并开发了新的抗体和基于细胞的免疫治疗技术，用于治疗包括肝癌、儿童癌症和其他实体肿瘤在内的实体肿瘤。在FY2022，我们在胰腺癌中创造了新的靶向GPC1的免疫毒素，并在小鼠中显示了免疫毒素对胰腺癌的抑制作用，并在Molecular cancer Therapeutics上发表了我们的临床前研究[Pan等]。分子肿瘤治疗[j]。GPC1是一种细胞表面蛋白多糖，在包括胰腺癌在内的多种人类癌症中表达上调。我们研究了GPC1是否可以作为抗体-毒素融合蛋白（即免疫毒素）治疗胰腺癌的靶点。我们构建了从假单胞菌外毒素a的功能域衍生的gpc1靶向免疫毒素，并将白蛋白结合域引入抗gpc1免疫毒素中以提高血清半衰期。进行小分子筛选以确定伊立替康与免疫毒素具有协同作用。我们发现GPC1在抗体结合后被内化。抗gpc1免疫毒素单独抑制胰腺癌异种移植模型的肿瘤生长。免疫毒素治疗降低了肿瘤细胞中活性β -连环蛋白的表达。此外，含有白蛋白结合结构域的免疫毒素与伊立替康联合可引起胰腺肿瘤消退。由于内化GPC1降解，细胞周转率短，免疫毒素处理降低了GPC1的表达。我们的数据表明，GPC1靶向免疫毒素通过降解内化的GPC1、下调Wnt信号和抑制蛋白质合成来抑制胰腺肿瘤的生长。因此，抗gpc1免疫毒素联合伊立替康为胰腺肿瘤患者提供了一种潜在的新治疗策略。我们还在《美国生理学细胞生理学杂志》上发表的一篇综述文章中总结了我们在研究GPC1作为胰腺癌免疫治疗靶点方面的工作[Pan and Ho， Am J Physiol Cell Physiol. 2021]。在FY2022，我们还开发了新的PD-L1靶向鲨鱼VNAR单域CAR-T细胞策略，用于治疗乳腺癌和肝癌[Li等]。Molecular Therapy oncology [j]。嵌合抗原受体(CAR)-T细胞疗法对血液系统恶性肿瘤具有良好的疗效，但治疗实体肿瘤仍然具有挑战性，主要是因为缺乏适当的抗原靶点和免疫抑制肿瘤微环境（TME）。检查点分子程序性死亡配体1 （PD-L1）在多种肿瘤类型中广泛过表达，程序性死亡配体1 (PD-1)/PD-L1相互作用是TME中免疫抑制的重要介质。在这项新研究中，我们构建了半合成鲨鱼VNAR噬菌体文库，并分离了抗pd - l1单域抗体。在这些VNARs中，B2对人、小鼠和犬的PD-L1具有交叉反应性，部分阻断了人PD-1与PD-L1的相互作用。CAR (B2) T细胞通过靶向PD-L1的组成性和诱导性表达，特异性裂解人乳腺癌和肝癌细胞，抑制肿瘤转移。PD-L1 CAR (B2) T细胞与GPC3（肝癌特异性抗原）靶向的CAR - T细胞联合治疗小鼠肝脏肿瘤。我们得出结论，pd - l1靶向鲨鱼VNAR单域CAR-T疗法是一种治疗乳腺癌和肝癌的新策略。这项研究为PD-L1 CAR-T细胞在临床研究中作为单一疗法或与肿瘤特异性疗法联合使用的潜在应用提供了理论依据。除了基于细胞的治疗，我们还开发了针对GPC3的双特异性抗体hYP7-OKT3-hFc，并在小鼠中显示了其强大的活性[Chen等]。[j]。我们还在STAR protocols （Cell Press）和Methods in Molecular Biology上发表了三个关于CAR-T细胞和免疫毒素用于癌症治疗的构建和生产的方案[Li et al]。STAR协议2021；Li and Ho, Methods in Molecular Biology 2022；Fleming和Ho，方法在分子生物学2022]。我们在科学界分享了我们的协议和方法，以帮助快速推进CAR-T领域。为了开发CAR-T治疗其他实体肿瘤，我们与Ira Pastan博士的实验室和Raffit Hassan博士的实验室合作开发了针对间皮素的新型CAR-T细胞疗法[Tomar等人，Mol Cancer Ther 2022；Liu et al.， PNAS， 2022]并与Javed Khan的实验室和Brad St Croix的实验室合作开发新的双路CAR靶向神经母细胞瘤中的GPC2或CD276 [Tian et ., J clinin Invest. 2022]。我们开发了hYP218（针对膜近端表位）CAR - T细胞[Tomar等]。[j]。我们的研究结果表明，靶向靠近细胞膜的间皮素表位的hYP218 CAR - T细胞对间皮素阳性肿瘤非常有效，并且与持久性和肿瘤浸润增加有关。这些结果支持其用于治疗表达间皮素的癌症患者的临床开发。在Pastan博士的实验室和我们的实验室领导的另一项研究中，我们已经确定脱落间皮素是成功的抗体治疗的主要障碍，并制备了一种抑制脱落的单克隆抗体，并使活性不被脱落间皮素阻断的非常活跃的CAR - T细胞，值得进一步的临床前开发[Liu等人，PNAS, 2022]。针对单一抗原的CAR-T细胞疗法在实体瘤的临床试验中表现不佳，这是由于肿瘤相关抗原（TAAs）的异质性表达、有限的t细胞持久性和耗竭。由Khan博士的实验室、St Croix博士的实验室和我们的实验室领导的一个项目旨在确定抗GPC2或CD276 （B7-H3）的最佳car - car，它们在神经母细胞瘤（NB）中高度但异质性表达，这是一种致命的儿童颅外实体瘤。我们做了一个双路“或”车（BiCisCAR）。BiCisCAR t细胞能有效消除表达GPC2或CD276的肿瘤细胞。在临床开发方面，我们已经开发出GPC3 （hYP7）、GPC2 （CT3）和间皮素（hYP218） 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，得到了癌症登月计划和NCI CCR的支持。