重新编程T细胞以攻击实体瘤

StephenW

Reprograming T cells to attack solid tumors

by Agency for Science, Technology and Research (A*STAR), Singapore
A colourful battle: Live human liver cancer cells (green) are attacked by engineered T cells (blue), resulting in a dead liver cancer cell (red). White blood cells called monocytes (orange) can prevent T cells reaching and killing the cancer cells.

An attractive approach to liver cancer treatment is to bolster the body's own immune defenses. Now an international team involving A*STAR researchers has genetically engineered human T cells to endow them with both specificity against liver cancer cells in patients with Hepatitis B, and the potential for long-term antitumor efficacy.

Hepatocellular carcinoma (HCC) is a common form of liver cancer, and a leading cause of cancer deaths worldwide. It often arises in people with chronic liver damage associated with hepatitis B virus (HBV) infection.

"An overarching research aim is to develop next-generation immunotherapies for HBV-related HCC and other solid tumors, including secondary metastases," says project co-leader Andrea Pavesi, at A*STAR's Institute of Molecular and Cell Biology (IMCB).

Solid tumors are particularly challenging—compared to blood cancers, for example—because they are difficult for immune cells to infiltrate. Nevertheless, two types of immunotherapy show excellent promise. Checkpoint inhibitor therapy typically uses antibodies to block inhibitory molecules that stop T cells attacking tumors. While, in adoptive cell therapy, patient-derived T cells are modified outside the body to increase their potency before being injected back into the patient.

Both checkpoint inhibitor and adoptive cell therapy have revolutionized cancer treatment for some patients. However, T cells with desired specificities are rare and difficult to isolate and manipulate.

"We sought to combine advantages of the two approaches while overcoming limitations," says Itziar Otano, former IMCB visiting researcher and first author of the paper1.

The researchers first engineered T cells to express a cell surface protein called a T cell receptor (TCR) tailored to recognize HBV-infected liver cancer cells.

"The TCR 'hook' links with a corresponding HBV-derived hook on the cancer cell," says Pavesi. "By using genetic engineering, we effectively increase the pool of T cells potentially able to 'see' and attack solid tumors."

A second problem to overcome is the gradual loss of efficacy that occurs when T cells are repetitively stimulated, a phenomenon known as T cell exhaustion. The receptor protein PD-1 is a central mediator of T cell exhaustion and a target of checkpoint inhibitor therapy.

"The restoration of immune control of cancer through PD-1 blockade was first shown in animal studies," explains Pavesi. "Subsequently, ground-breaking results have been achieved for melanoma patients. In parallel, T cell adoptive therapy is showing great results for both leukemia and melanoma."

Despite successes, the use of PD-1-blocking antibodies has disadvantages: it requires multiple dosing, and these antibodies also affect non-target cells expressing PD-1, triggering autoimmune disease in some patients.

"We reasoned that an elegant solution would be to genetically knockdown PD-1 just on T cells of the desired specificity," says Otano.

To achieve this, the researchers used gene editing to knockdown PD-1 expression on T cells that had been engineered to recognize HBV-infected liver tumor cells.

To assess the functional consequences of the dual manipulation for anti-tumor immunity, they used an innovative 3-D microfluidic device. T cells injected into fluid-filled microchannels interacted with gel-immobilized cancer cells in the central region of the device, recapitulating the tumor microenvironment.

"The beauty of our microfluidic device is that it allows us to study cancer cell killing by T cells with minimal experimental noise," explains Pavesi. "More generally, the use of microfluidic devices to test possible treatments under conditions mimicking those of target tissues could reduce clinical trial failures."

Using their device, the researchers found that PD-1 knockdown increased immediate T cell killing of HBV-infected liver cancer cells. However, repetitive stimulation eventually induced other inhibitory pathways that compensated for PD-1 knockdown and resulted in premature T cell senescence and death.

"Future work must address the emergence of alternative inhibitory pathways," cautions Pavesi, "Nevertheless, our study provides proof of principle that a dual approach to genetically reprogram human T cells can invigorate their ability to attack solid tumors."

More information: Itziar Otano et al. Molecular Recalibration of PD-1+ Antigen-Specific T Cells from Blood and Liver, Molecular Therapy (2018). DOI: 10.1016/j.ymthe.2018.08.013

StephenW

重新编程T细胞以攻击实体瘤

新加坡科学，技术和研究机构（A * STAR）
多彩的战斗：活的人类肝癌细胞（绿色）受到工程化T细胞（蓝色）的攻击，导致死亡的肝癌细胞（红色）。称为单核细胞（橙色）的白细胞可以阻止T细胞到达并杀死癌细胞。

治疗肝癌的一种有吸引力的方法是加强身体自身的免疫防御能力。现在，一个由A * STAR研究人员组成的国际团队已经对人类T细胞进行基因工程改造，赋予他们对乙型肝炎患者肝癌细胞的特异性，以及长期抗肿瘤疗效的潜力。

肝细胞癌（HCC）是肝癌的常见形式，并且是全世界癌症死亡的主要原因。它通常出现在与乙型肝炎病毒（HBV）感染相关的慢性肝损伤患者中。

“总体研究目标是为HBV相关的HCC和其他实体肿瘤（包括继发性转移瘤）开发下一代免疫疗法，”A * STAR分子与细胞生物学研究所（IMCB）的项目联合负责人Andrea Pavesi说。

例如，与血癌相比，实体瘤特别具有挑战性 - 因为它们难以渗透免疫细胞。然而，两种类型的免疫疗法显示出极好的前景。检查点抑制剂治疗通常使用抗体来阻断抑制T细胞攻击肿瘤的抑制性分子。然而，在过继细胞疗法中，患者来源的T细胞在体外被修饰以在注射回患者之前增加其效力。

检查点抑制剂和过继细胞疗法都为一些患者的癌症治疗带来了革命性的变化。然而，具有所需特异性的T细胞是罕见的并且难以分离和操作。

“我们试图结合这两种方法的优势，同时克服限制，”前IMCB访问研究员，论文第一作者Itziar Otano说。

研究人员首先设计了T细胞来表达称为T细胞受体（TCR）的细胞表面蛋白，以识别HBV感染的肝癌细胞。

“TCR'钩'与癌细胞上相应的HBV衍生钩相连，”Pavesi说。 “通过使用基因工程，我们有效地增加了可能'看到'并攻击实体肿瘤的T细胞库。”

要克服的第二个问题是当T细胞被重复刺激时发生的功效逐渐丧失，这种现象称为T细胞衰竭。受体蛋白PD-1是T细胞衰竭的中枢介质和检查点抑制剂治疗的靶标。

“通过PD-1阻断恢复癌症的免疫控制首先在动物研究中得到证实，”Pavesi解释道。 “随后，黑色素瘤患者取得了突破性的成果。同时，T细胞过继治疗对白血病和黑色素瘤都有很好的效果。”

尽管成功，PD-1阻断抗体的使用具有缺点：它需要多次给药，并且这些抗体也影响表达PD-1的非靶细胞，在一些患者中引发自身免疫疾病。

“我们推断，优雅的解决方案是在特定的特异性T细胞上基因敲除PD-1，”Otano说。

为了实现这一目标，研究人员利用基因编辑技术来抑制T细胞上PD-1的表达，该细胞经过工程改造可识别HBV感染的肝肿瘤细胞。

为了评估双重操作对抗肿瘤免疫的功能性后果，他们使用了创新的3-D微流体装置。注入充满液体的微通道中的T细胞与装置中心区域中凝胶固定的癌细胞相互作用，重现肿瘤微环境。

“我们的微流体装置的美妙之处在于它使我们能够以最小的实验噪音研究T细胞杀死癌细胞，”Pavesi解释道。 “更一般地说，使用微流体装置在模仿目标组织的条件下测试可能的治疗方法可以减少临床试验失败。”

使用他们的设备，研究人员发现PD-1敲低增加了立即T细胞杀死HBV感染的肝癌细胞。然而，重复刺激最终诱导其他抑制性途径，其补偿PD-1敲低并导致过早的T细胞衰老和死亡。

“未来的工作必须解决替代抑制途径的出现，”Pavesi警告说，“尽管如此，我们的研究提供了原理的证据，即对人类T细胞进行遗传重编程的双重方法可以激发他们攻击实体肿瘤的能力。”

更多信息：Itziar Otano等。来自血液和肝脏的PD-1 +抗原特异性T细胞的分子重新校准，分子疗法（2018）。 DOI：10.1016 / j.ymthe.2018.08.013