Development of new antibody-based cancer therapies

硫酸乙酰肝素蛋白多糖(HSPGs)调节多种细胞表面信号事件。它们是发育和癌症等疾病过程中信号转导通路的细胞外调节器。HSPGs是一种细胞表面蛋白，主要由糖基磷脂酰肌醇(GPI)锚定的葡聚糖和跨膜联糖组成。在过去的十多年里，Mitchell Ho博士在国家癌症研究所(NCI)的实验室将GPC3和其他Glypicans作为一个新的癌症靶标家族进行了研究。在过去的一年里，我们评估了GPC1-6在癌症中的表达[Li等人。J组织化学细胞化学2020年；PMID：32623934]。我们和其他人已经证实Glypcan-3(GPC3)是治疗肝细胞癌(HCC)的新靶点，肝细胞癌是最常见的原发性肝癌。我们通过杂交瘤和噬菌体展示技术制备了几种针对GPC3的抗体(如YP7、HN3)。在2020财年，我们使用人源化YP7(HYP7)和HN3抗体创建了嵌合抗原受体(CARS)，并展示了单一治疗hYP7 CAR T细胞诱导肝癌小鼠模型的肿瘤消退[Li等人。胃肠病学，2020年；PMID：32060001]。我们从健康供者和肝癌患者的外周血中采集单个核细胞，以人源化的YP7(HYP7)和HN3抗体为基础建立CAR T细胞，这两种抗体分别与GPC3的C叶和N叶具有高亲和力。我们还开发了水滴数字聚合酶链式反应和基因组测序方法来分析小鼠的持久性CAR T细胞。我们发现，注射CAR(HYP7)T细胞在第3周消除了66%的小鼠肿瘤，而CAR(HN3)T细胞并没有减少肿瘤负担。给予CAR(HYP7)T细胞的小鼠在用额外的Hep3B细胞再次攻击后仍然没有肿瘤。CAR T细胞诱导穿孔素和颗粒酶介导的肝癌细胞凋亡，并降低活化的β-连环素水平。注射CAR(HYP7)T细胞的小鼠通过抗原诱导的选择，T细胞和多功能CAR T细胞亚群持续扩增。注射CAR T细胞后，肿瘤微环境和脾组织中可观察到这些T细胞，持续时间长达7周。注射前CAR(HN3)和CAR(HYP7)T细胞的整合部位是随机分布的，而CAR(HYP7)T细胞在注射后5周整合到NUPL1的比例为3.9%，而CAR(HYP7)T细胞在注射后7周的整合部位为18.1%，而CAR(HN3)和CD19 CAR(HYP7)T细胞没有共同的整合部位。总之，在异种移植或直肠癌的小鼠中，CAR(HYP7)T细胞可能通过诱导穿孔素和颗粒酶介导的细胞凋亡或减少肿瘤细胞中的Wnt信号来消除GPC3阳性的肝癌细胞。针对GPC3的CAR T细胞可能被开发用于治疗肝细胞癌患者。在2020财年，我们在小鼠身上产生了半衰期更长、抗肿瘤效果更好的基于HN3的免疫毒素，并将我们的发现发表在了《肝病》杂志上。肝病，PMID：31520528,2020年]。HN3是一种人源单结构域抗体，它识别GPC3核心蛋白中的一个新的功能位点，并通过阻断WNT和YAP癌症信号来抑制肝癌细胞的增殖[冯等，PNAS，PMID：23471984,2013年；Gao等，自然通讯，PMID：25758784,2015年]。使用我们针对人纳米体(HN3)的GPC3免疫毒素治疗肝细胞癌，通过阻断蛋白质合成和下调Wnt信号通路，导致有效的肿瘤消退。然而，免疫原性和较短的血清半衰期可能会限制免疫毒素过渡到临床的能力。为了解决这些问题，我们设计了基于HN3的免疫毒素，使其含有各种灭活的假单胞菌外毒素(PE)结构域。这包括HN3-T20，它被修改以去除T细胞表位，并包含PE结构域II截断。我们将它们与我们之前报道的B细胞去免疫免疫毒素(HN3-mPE24)和我们最初的带有野生型PE结构域的HN3免疫毒素(HN3-PE38)进行了比较。所有免疫毒素均与人GPC3有较高的亲和力，HN3-T20的Kd值为7.4 nM。在二磷酸腺苷-核糖化实验中，HN3-T20与野生型免疫毒素相比保持了73%的酶活性。有趣的是，实时细胞生长抑制实验表明，在10天的实验中，62.5 ng/mL(1.6 nM)的单剂量HN3-T20能够抑制几乎所有细胞的增殖。为了提高HN3-T20‘S的血清保留率，我们测试了添加链球菌白蛋白结合域(ABD)和针对小鼠和人血清白蛋白的骆驼单域抗体片段的效果。为了检测小鼠血清中的免疫毒素，我们建立了一种高灵敏度的酶联免疫吸附试验，发现HN3-Abd-T20的血清半衰期是HN3-T20的45倍(326分钟对7.3分钟)；因此，添加ABD导致HN3-Abd-T20在1 mg/kg时介导的肿瘤消退。总之，我们的数据表明，含有ABD的去免疫HN3-T20免疫毒素是准备在临床试验中评估的高效疗法，用于肝癌的治疗。

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 ten 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. In the past year, we have evaluated expression of glypicans (GPC1-6) in cancer [Li et al. J Histochem Cytochem 2020; PMID: 32623934]. We and others have validated glypican-3 (GPC3) as a new therapeutic target in hepatocellular carcinoma (HCC), the most common form of primary liver cancers. We produced several antibodies (e.g., YP7, HN3) targeting GPC3 either by hybridoma and phage display technologies. In FY2020, we created chimeric antigen receptors (CARs) using humanized YP7 (hYP7) and HN3 antibodies and showed single treatment of hYP7 CAR T cells induced tumor regression in liver cancer mouse models [Li et al. Gastroenterology, 2020; PMID: 32060001]. We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. We also developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. We found that injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active beta-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. In conclusion, in mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC. In FY2020, we generated HN3-based immunotoxin with a longer half-life and better anti-tumor efficacy in mice and published our finding in Hepatology [Fleming et al. Hepatology, PMID: 31520528, 2020]. HN3 is a human single-domain antibody that recognizes a novel functional site in the core protein of GPC3 and inhibits proliferation of HCC cells via blocking Wnt and Yap cancer signaling [Feng et al., PNAS, PMID: 23471984, 2013; Gao et al., Nature Communications, PMID: 25758784, 2015]. Treatment of hepatocellular carcinomas using our glypican-3 (GPC3)-targeting human nanobody (HN3) immunotoxins causes potent tumor regression by blocking protein synthesis and down-regulating the Wnt signaling pathway. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. To address these concerns, we engineered HN3-based immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3-T20, which was modified to remove T-cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B-cell deimmunized immunotoxin (HN3-mPE24) and our original HN3-immunotoxin with a wild-type PE domain (HN3-PE38). All of our immunotoxins displayed high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. HN3-T20 retained 73% enzymatic activity when compared with the wild-type immunotoxin in an adenosine diphosphate-ribosylation assay. Interestingly, a real-time cell growth inhibition assay demonstrated that a single dose of HN3-T20 at 62.5 ng/mL (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20's serum retention, we tested the effect of adding a streptococcal albumin-binding domain (ABD) and a llama single-domain antibody fragment specific for mouse and human serum albumin. For the detection of immunotoxin in mouse serum, we developed a highly sensitive enzyme-linked immunosorbent assay and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20 (326 minutes vs. 7.3 minutes); consequently, addition of an ABD resulted in HN3-ABD-T20-mediated tumor regression at 1 mg/kg. In conclusion, our data indicate that ABD-containing deimmunized HN3-T20 immunotoxins are high-potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.