SAMHD1

近年来，CAR-T细胞治疗作为一种全新的肿瘤治疗手段，治疗效果优异，但是由于CAR-T治疗的生产成本、价格成本，使得CAR-T无法实现更大范围的供应，这也是CAR-T的销售增长缓慢的一大原因。延伸阅读：CAR-T 2022年大考成绩出炉！为了降低CAR-T生产成本，提高市场竞争力，研发人员进行了大量的工作，包括同种异体CAR-T、CAR-T自动化生产、CAR-T快速制备、体内CAR-T等等。延伸阅读：体内CAR-T细胞的技术路线CAR-T的快速制备，能够缩短生产时间，降低成本，包括生产空间和生产设备，以及人工费用等，能够大幅度缓解传统CAR-T生产所需要的生产资料，可以供给更多批次。CAR-T快速制备的机会当前全球范围内已有多款CAR-T产品获得批准上市，其大致生产制备流程基本相同：单采-分选-激活-转导-扩增-收获-包装和冻存-放行质检-回输。分选纯化：对单采产物进行T细胞分选纯化，获得目标的T细胞亚群，同时去除杂质，例如红细胞、抗凝剂等；激活/活化*：一般采用激活磁珠进行T细胞激活，然后将其放置于二氧化碳培养箱进行激活培养，T细胞激活能够提高CAR的转导率；转导CAR：完成激活的T细胞将进行CAR的转导，一般是通过病毒载体（慢病毒或逆转录病毒等）进行CAR的转导，使得T细胞转化为CAR-T细胞；扩增*：转导后，CAR-T细胞需要在生物反应器当中进行细胞扩增，直到细胞达到收获标准，包括活细胞数、体外效价、T细胞纯度；收获：收获工序包括清洗、去磁珠。包装和冻存：收获工序完成后，将在生物安全柜内完成分装，随后被保存于气相液氮罐中，放行质检合格后通知临床做回输准备。以上工序通常需要10-14天的工作时间，其中以细胞扩增所需的时间较长，为5天左右。延伸阅读：CAR-T生产工艺流程及优化考量在以上六个程序当中，CAR-T快速制备的机会在于激活与扩增这两个环节，通过删减和简化这两个环节，能够实现CAR-T在1-2天内的快速制备。挑战与一些解决方案但是激活与扩增两个环节在CAR-T细胞的生产中也具有重要意义：激活*：细胞激活能够促进CAR转导效率，静止T细胞的转导效率较低。如果要跳过或者优化这个环节，就必须解决静止T细胞的CAR转导效率过低的挑战。在现有的方案当中，对于这个环节有三种解决方式：a.使用具有更高转导效率的CAR基因载体；b.使用白介素等细胞因子进行促进转导效率；c.T细胞选用记忆T细胞，有部分研究显示，在不经活化的所有T细胞亚群中，记忆T细胞对CAR的转导率是最高的。扩增*：现在CAR-T剂量在106左右，通常从肿瘤患者体内采集到的T细胞数量制备成的CAR-T数量不足（主要也是CAR转导率低导致的），为了满足剂量要求达到最优疗效，需要在体外进行细胞扩增。因此快速制备平台还需要保证回输的CAR-T细胞剂量更低的同时，疗效不劣。不过由于快速制备不需要进行T细胞扩增，充分保留了T细胞干性，使得其在体内也可以进行一定的扩增，并且可能可以增强肿瘤细胞的清除效果。如何去考量和解决激活和扩增环节缺失，就成了CAR-T快速制备的关键，也是各家快速制备平台不同。举例：几个不同的快速制备平台（1）诺华 T-charge国际上，以诺华的CAR-T快速制备平台T-charge最为知名。T-charge平台的信息公开不多。根据诺华官网查询，T-charge平台删减了传统CAR-T细胞生产环节中的扩增部分，不需要在生物反应器当中进行CAR-T细胞扩增，而是将该环节置于患者体内。该平台能够保留T细胞干性，从而使得CAR-T产品具有更大的增殖潜力，以及更少的T细胞耗竭。这些特点可能会改善患者的预后，降低严重不良事件的风险。此前，诺华公布了其基于T-charge平台设计的一款靶向CD19的CAR-T细胞YTB323（rapcabtagene autoleucel）的数据，其door-to-door时间大约为10天左右；在DLBCL患者中CR（Month 3）可达到73%。延伸阅读：CAR-T“自卷”：生产周期降50%，剂量降25倍，CR73%（2）亘喜 FasTCAR在国内，亘喜生物的FasTCAR是进行比较早的CAR-T快速制备平台。根据公开资料，亘喜生物FasTCAR平台将传统生产中的激活、转导两个步骤简化和优化成同时进行，并且删减了扩增环节。FasTCAR平台利用专有的XLenti载体，同时激活和转导处于静息状态的T细胞。此外，亘喜生物表示，由于FasTCAR平台的产品具有较高的扩增和肿瘤细胞清除活性，因此可去除CAR-T细胞体外扩增的步骤，可以直接回输给患者。值得注意的是，亘喜生物基于FasTCAR平台开发的GC019F（CD19 CAR-T）和GC012F（BCMA/CD19 CAR-T）均已获得国家药品监督管理局的临床批件。（3）宾大团队 慢病毒转导静止T细胞2022年2月，宾大研究团队发表在期刊Nature Biomedical Engineering上的研究，就是一个经典的CAR-T快速制备方式。在该研究当中，研究人员通过慢病毒载体直接对静止T细胞进行转导，通过IL-7和IL-15等细胞因子的细胞培养基能够解决静止T细胞转导效率低下的问题，提高转导效率。此外，还可以通过工程病毒载体表达IL-7提高静止T细胞转导效率。同时，低浓度的核苷酸和其他限制性因子（如SAMHD1）均可限制静止T细胞逆转录率。小鼠模型中，通过这种方式设计的CD19 CAR-T可以显著地延长小鼠的生存期；与传统生产方式CAR-T相比，具有更优的抗白血病活性。（4）MASTER支架 CAR-T2022年3月，北卡罗来纳州立大学和北卡罗来那大学的研究人员开发了一种名为MASTER的多功能藻酸盐支架，MASTER技术集成了T细胞激活、重编程和体内扩增的步骤，将制造CAR-T的时间缩短到1天。图1 两种方法的路线图。研究人员从患者体内分离出T细胞，与CAR基因病毒载体混合，然后将混合物和藻酸盐支架（支架有激活T细胞的抗体以及促进增殖的白介素）混合后植入小鼠体内，CAR转导将发生在患者体内。在小鼠淋巴瘤异种移植模型中，这种基于MASTER支架在体内生成的CAR-T细胞，能够进入血液循环并控制远处肿瘤的生长。延伸阅读：120万的一针的CAR-T实现体内一天生产，期待成本下降 ！以上仅是举例，并不全面，如有缺漏，欢迎在留言区进行评论讨论。总结总结来说，CAR-T的快速制备平台关键在于激活，保证T细胞在静止状态下能够被转导成CAR-T，且具有高效率。不过目前来看，CAR-T快速制备还处于比较早期的状态，产品基本处于1期临床或以前。参考资料：1.https://ash.confex.com/ash/2022/webprogram/Paper162520.html2.https://www.novartis.com/research-development/technology-platforms/cell-therapy/charging-towards-next-generation-car-t3.亘喜生物官网：https://cn.gracellbio.com/4.Ghassemi, S., Durgin, J.S., Nunez-Cruz, S. et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 6, 118–128 (2022). https://doi-org.libproxy1.nus.edu.sg/10.1038/s41551-021-00842-65.Agarwalla, P., Ogunnaike, E.A., Ahn, S. et al. Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells. Nat Biotechnol 40, 1250–1258 (2022). https://doi-org.libproxy1.nus.edu.sg/10.1038/s41587-022-01245-x········

In the tough war against glioblastoma, scientists are taking a cue from viruses on how to make the aggressive cancer more vulnerable to treatment. Their target is SAMHD1, a protein which can protect us from viral infections by destroying an essential building block of DNA that viruses and cancer need to replicate. In the tough war against glioblastoma, scientists are taking a cue from viruses on how to make the aggressive cancer more vulnerable to treatment. Their target is SAMHD1, a protein which can protect us from viral infections by destroying an essential building block of DNA that viruses and cancer need to replicate. But they've found SAMHD1 also has the seemingly contradictory skill of helping repair double-strand breaks in the DNA that if unrepaired can be lethal to any cell, including a cancer cell, and if mended incorrectly can result in genetic mutations that produce cancer. "When the DNA breaks, that is what actually interrupts the DNA replication and also the synthesis of proteins, so a double-strand break is lethal for cells," says Waaqo Daddacha, PhD, cancer biologist in the Department of Biochemistry and Molecular Biology at the Medical College of Georgia. Cancer cells, which are reproducing much more rapidly than most normal cells, are replicating even faster, so are impacted even more by these DNA breaks, which is why fundamental therapies like radiation and some chemotherapy drugs used to treat cancers make these lethal breaks. However, the aggressive brain cancer quickly becomes treatment-resistant and the average survival remains at about 15 months, Daddacha says. Now Daddacha and his colleagues report in the journal Cancers their surprising finding that in glioblastoma in humans both SAMHD1 and the essential DNA building block dNTP, which it can destroy, are highly expressed, indicating SAMHD1's likely importance to the brain tumor's aggressiveness and raising questions about what it's doing there. They were expecting high levels of dNTP because cancers need a ready supply of this building block to keep up their rapid pace of replicating and spreading, Daddacha says. Since dNTP levels were high, they were also expecting low levels of SAMHD1 would be present and that increasing its levels would help protect against glioblastoma. They would find the opposite to be true, indicating that like with so many innate properties cancer usurps, glioblastoma likely alters the function of SAMHD1. "Theoretically, since cancer cells divide rapidly and need more dNTP to do that, it seems logical that they would need less of this protein," Daddacha says. It also seems logical that SAMHD1 would be protective against cancer, like it is against viruses, Daddacha says. Based on what they found, the scientists instead decided to reduce SAMHD1 levels and that's where viruses' skill at eliminating the multitasking protein came in. Viruses deploy the protein viral protein X, or Vpx, to literally chop up SAMHD1 so they will have a ready supply of dNTP, a virus skill set first identified in HIV. So, the scientific team used a virus-like particle, called a vector, to deliver Vpx directly to the glioblastoma. These types of viral vectors already are used in people to deliver a variety of therapies, including some of the COVID-19 vaccines. They found by reducing SAMHD1, Vpx sensitized the brain tumor cells to the chemotherapy drug veliparib, which helps block DNA damage repair by cancer cells, and slowed cell growth of this fast-growing brain tumor. It also made the tumor cells more sensitive to temozolomide, or TMZ, another chemotherapy drug often used for glioblastoma, which disrupts the cell's DNA structure with the idea of killing the cell. By reducing SAMHD1, Vpx appeared to also reduce the innate skill called homologous recombination, which SAMHD1 promotes, and which enables a sound repair of double-strand breaks that also helps avoid cell mutations that could enable cancer. Reducing SAMHD1 levels enabled the combination therapies, including radiation, used for glioblastoma to work more synergistically as intended, and reconfirmed SAMHD1's role in enabling glioblastoma's usual treatment resistance, the scientists write. In a mouse model with the human brain tumor cells, they found that reducing SAMHD1 slowed tumor growth and genetically knocking out SAMHD1 improved survival, the scientific team reports. As another piece of the emerging puzzle, when the scientists removed SAMHD1, dNTP levels did increase some but not dramatically, like they have seen in some other cell types, more evidence that while SAMHD1 still has some impact on degrading this DNA building block, in this scenario, it clearly has yet another function, Daddacha says. He suspects that the real benefit of high levels of SAMHD1 to glioblastoma is "self-protection" relying most on the protein's innate ability to help repair double-strand DNA breaks. There is overwhelming evidence that glioblastoma's skill at repairing double-strand DNA breaks is key to its treatment resistance, which makes targeting the repair process logical, Daddacha says. One of the ways cancer takes over a protein for its own purposes is by modifying its function so, for example, it could shift SAMHD1 into primarily DNA repair mode and reduce its natural ability to degrade dNTP, and Daddacha suspects glioblastoma is changing SAMHD1's function. "Clearly it's using it to survive," Daddacha says, which is likely at least a part of how glioblastoma is so tenacious, and a piece of the big puzzle needed to one day better treat the deadly cancer. Their findings indicate that SAMHD1 can be targeted and eliminated using the viral protein Vpx in glioblastoma, Daddacha says, but notes that much work remains before the findings and the tool can be used to improve glioblastoma treatment. Next steps include learning more about what SAMHD1 is doing in glioblastoma, and how high levels of it can coexist with high levels of dNTP, which it should destroy. "We will try to discover if SAMHD1 is actually degrading dNTP in cancer cells," says Daddacha. It may be that the high levels of SAMHD1 may also help keep high levels of dNTP in check, he says, because everything needs balance. They also are refining the safe, specific technique of delivering Vpx with the idea that one day it could also be used in people, and to determine if the technique impairs glioblastoma's growth in other ways as well. Glioblastoma is a lethal cancer that is always considered as an aggressive, stage 4 tumor at diagnosis with patients living an average of 15 months after diagnosis, Daddacha says. By the time symptoms like headaches and seizures emerge, the tumor already has progressed. Standard treatment includes surgery to remove as much of the tumor as possible, radiation and chemotherapy. SAMHD1, or sterile alpha motif and HD domain-containing protein 1, is omnipresent in cells, including in our brain cells. It is believed to keep viruses like HIV-1 from replicating by cutting and destroying dNTPs, or deoxynucleoside triphosphates, which normal cells, cancer cells and viruses all need to reproduce. "We make our DNA with a chain of dNTPs," Daddacha says. SAMHD1 also likely normally works to help regulate the amount of dNTPs the body needs, he says. While working on his PhD at the University of Rochester, Daddacha was part of the team that discovered that SAMHD1 degrades the DNA building block dNTP. While doing his postdoctoral studies at Winship Cancer Institute at Emory University in Atlanta, he further explored SAMHD1's role with DNA and found it actually has a key role in the DNA repair pathway. Daddacha joined the MCG faculty in 2019. The research was funded by the National Cancer Institute.