cPLA2α

基于接受PD-1抑制剂治疗的患者的肿瘤组织和血液样本，Puig-Saus等发现，无论癌症患者对免疫治疗是否有反应，他们体内都存在能识别肿瘤新抗原的效应T细胞，而且都是只识别少数新抗原；不过，不同反应 PD-1抑制剂正式被用于临床的时间，眼看就要满10年了。 这近10年间，以免疫检查点抑制剂为代表的免疫治疗给癌症治疗带来了翻天覆地的变化，很多癌症的临床治疗指南因此改写，无数患者因此获得新生。 以至于我们很多时候都忘记了，能从免疫治疗中获益的其实只有一小部分癌症患者，大部分患者对免疫治疗没有反应，有些患者接受治疗后甚至出现了疾病进展。 因此，从免疫检查点抑制剂用于临床治疗开始，科学家就在寻找背后的原因。一些重要的发现，在一定程度上改善了免疫治疗的效果。然而，获益患者少的局面仍没有被打破。 今天，顶级期刊《自然》杂志发表了加州大学洛杉机分校Antoni Ribas和Cristina Puig-Saus团队的研究成果[1]。 基于接受PD-1抑制剂治疗的患者的肿瘤组织和血液样本，Puig-Saus等发现，无论癌症患者对免疫治疗是否有反应，他们体内都存在能识别肿瘤新抗原的效应T细胞，而且都是只识别少数新抗原；不过，不同反应状态患者体内的效应T细胞多样性存在巨大差异。 具体来说，能从免疫治疗中获益的患者，单个新抗原对应着多样性的效应T细胞克隆（一种新抗原能被多种T细胞受体识别）；而不能从免疫治疗中获益的患者，单个新抗原仅对应着少数效应T细胞克隆，甚至是单克隆（一种新抗原只能被一种T细胞受体识别）。 Puig-Saus团队的这项突破性研究，不仅让我们对患者的不同治疗反应有了新的理解，还为设计新型T细胞疗法指出了新方向。 论文首页截图 Puig-Saus团队开展的这个研究非常不易。 在他们开来，要想搞清楚免疫治疗后的新抗原特异性T细胞谱，至少有两个条件。 第一，需要一种极度灵敏的检测方法，能从少量血液样本的数百万个外周血单个核细胞（PBMCs）或少量活检肿瘤组织扩增的肿瘤浸润淋巴细胞（TILs）中，找到能识别数百种肿瘤新抗原的特异性效应T细胞。 第二，需要一种高效的新抗原特异性TCR（neoTCRs）的重建方法，在普通T细胞中逐一插入发现的neoTCRs，逐一验证功能，并系统性评估T细胞对匹配患者自体癌细胞系的识别和抗肿瘤活性。 在TCR-T细胞疗法公司PACT Pharma两项核心技术（ImPACT Isolation Technology®和PACT^NV™），分别帮助Puig-Saus团队解决了上述两个问题。 有了PACT Pharma的技术加持，Puig-Saus团队招募了11名接受过PD-1抑制剂治疗的黑色素瘤患者。其中患者1到患者7从免疫治疗中获得了持久的益处，生存时间在32个月到111个月之间，而且到本研究结束时所有患者依然存活；患者8到患者11，对免疫治疗没有反应，或者治疗后病情加重，在研究结束时这些患者都已去世。 研究人员还从所有患者身上采集肿瘤活检组织以及外周血，扩增了肿瘤浸润淋巴细胞，构建了癌细胞系，并给肿瘤组织和PBMCs做了全外显子组测序（WES）和RNA测序（RNA-seq）。工作量着实不小。 研究流程示意图 从WES数据来看，在所有患者体内发现的突变数量从31个（患者11）到3507个（患者1），中位数是380个。对免疫治疗有反应的7名患者中，非同义突变的中位数是506个；对免疫治疗没反应的4名患者这一数据为132个。虽然患者体内潜在的新抗原数量都不少，而且不同患者的变化非常大，但是被T细胞识别为新抗原的突变数量变化较小，从1到13不等。 在具体到TCR来看，对免疫治疗有反应的患者1-7体内，每个突变靶点对应的neoTCRs克隆型中位数是5种，每个患者体neoTCRs克隆型的中位数是41种。相比之下，四名对治疗没有临床反应的患者（8-11）体内，每个突变靶点对应的neoTCRs克隆型中位数是2种，每个患者体neoTCRs克隆型的中位数是6.5种。 不同患者的突变数量及被T细胞识别的突变数量 在后续的研究中，Puig-Saus逐一研究了每个患者体内T细胞的情况。 就拿患者1来说，他们从患者体内分离到571个新抗原特异性T细胞，包括针对11个突变的41种不同neoTCR克隆型。他们还从中筛选了针对9个突变的22种neoTCR克隆型，并通过CRISPR基因编辑技术将它们分别整合到健康供体的T细胞中，以研究这些neoTCR克隆型的功能。 在分析患者1不同时间点的样品时，他们还发现识别NUP188、UBE2J1和WDR1三个突变新抗原的neoTCR克隆型反复出现。这说明针对癌细胞新抗原的效应T细胞在患者1体内存在普遍的扩增。他们基于患者2和6的样本也开展了类似的研究，也发现了患者特异性的neoTCR克隆型。 患者1特异性T细胞的动态变化 在验证患者1体内存在的neoTCR克隆型的功能时，他们发现靶向其中5个基因突变（NRP1、TRAPPC10、UBE2J1、NUP188和WDR1）的neoTCR-T细胞对患者自体癌细胞系（M495）有中等以上的细胞毒性，靶向剩余4个基因突变（IFNLR1、PLA2G4A、SLC6A3和GSDMB）的neoTCR-T细胞对M495没有细胞毒性。所有靶向9个基因突变的neoTCR-T细胞，都对非匹配的黑色素瘤细胞系（M202）没有细胞毒性。 患者1的neoTCR克隆型功能验证 再来看不响应免疫治疗患者的数据。 在患者8的PBMC中只发现了8个neoTCR克隆型，它们分别针对NES、CTNNB1、POLR2G和SERPINH1四个基因突变。在患者9体内，发现了针对4个基因突变的5个neoTCR克隆型。在患者10体内，只发现了针对ACER3突变的两个neoTCR克隆型。在患者11体内，发现了针对5个基因突变的14个neoTCR克隆型。 Puig-Saus注意到，与对免疫治疗有反应的患者相比，这些对免疫治疗没反应的患者，不仅neoTCR克隆型少，而且在不同时间采集的血液和肿瘤样本也不经常出现。 此外，这四位患者neoTCR克隆型的功能分析发现，居然都对自体癌细胞系有毒性作用，而对非匹配的黑色素瘤细胞系没有毒性作用。 患者9的neoTCR克隆型在不同样本中随时间的变化 基于以上研究成果，Puig-Saus等认为，别看肿瘤中的突变那么多，实际上新抗原特异性的癌症免疫反应针对的只是数量有限突变（范围为3至13）。 不过，在对PD-1抑制剂有长期临床反应的患者中，新抗原特异性T细胞反应是多克隆的，即有多种新抗原特异性TCR克隆型针对同一个突变，而且在血液和肿瘤中都可以检测到这些新抗原特异性TCR克隆型。 因此，Puig-Saus等认为天然T细胞反应是通过不同的T细胞克隆型，靶向非常有限的突变来清除肿瘤的。这也为基于T细胞的免疫治疗，提供了新思路。 参考文献： [1].Puig-Saus, C., Sennino, B., Peng, S. et al. Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy. Nature, 2023. https://doi-org.libproxy1.nus.edu.sg/10.1038/s41586-023-05787-1

Halo Therapeutics LTD is preparing for clinical trials into pivotal, cost-effective antiviral treatments for Covid-19, after discovering a molecule which changes the shape of the virus’s spike protein and in so doing inhibits the virus’ ability to enter cells Studies show the treatments are potentially ‘pan-corona antivirals’ in that they will work against all coronavirus strains - including the highly contagious ‘UK’, ‘South African’ and Brazilian’ variants The company is preparing for clinical trials. If approved, the antivirals could be used by patients globally at the first sign of Covid-19 symptoms – stopping the virus in its tracks Bristol, UK – 13th April 2021: A team of top scientists from the University of Bristol have announced the formation of a new biotech company that is developing ground-breaking and newly patented potential treatments for Coronavirus. Bristol-based Halo Therapeutics is founded by the team of leading scientists who made a recent breakthrough discovery, which was published in Science Magazine. They found that exposing the SARS-CoV-2 (Coronavirus) virus to a free fatty acid called linoleic acid locks the virus’s spike protein into a closed, non-infective form stopping it in its tracks. The company is now preparing to make an application to start clinical trials with infected patients*. If proven to be effective, the antivirals could be used by people of all ages world wide at the first sign of Covid-19 symptoms, or if they have been in contact with someone with the virus, preventing the virus from taking hold and stopping further transmission. Lab studies indicate the antiviral will work against all pathogenic coronavirus strains including the highly contagious ‘UK’, ‘South African’ and ‘Brazilian’ variants by preventing the virus from penetrating cells in the nose, throat and lungs. The treatments under development by Halo Therapeutics include a nasal spray and an asthma-type inhaler, and offer the possibility of a game-changing pan-coronavirus antiviral to treat patients at all stages of the disease and to reduce the transmission of the virus. The Halo Therapeutics team is currently engaging investors to help finance multiple parallel clinical trials. If approved, the antiviral treatments could potentially start rolling out to patients globally. Professor Imre Berger, Director of the Max Planck-Bristol Centre for Minimal Biology at Bristol and one of the team leading the drug’s development, explains: “The aim of our treatment is to significantly reduce the amount of virus that enters the body and to stop it from multiplying. Then, even if people are infected with the virus or exposed to it, they will not become ill because the antiviral prevents the virus from spreading to the lungs and beyond. Importantly, because the viral load will be so low it will likely also stop transmission.” Professor Christiane Berger-Schaffitzel from Bristol’s School of Biochemistry, added: “Our vision is that at the first sign of the disease, whether you come into contact with someone who has Covid-19 or you have early symptoms, you would self-medicate at home to stop the virus in its tracks and prevent you from getting ill.” Professor Adam Finn from Bristol Medical School and Children Vaccine Centre says: “As the virus mutates there is a real risk that presently available vaccines diminish in their protective effect and people could develop the disease again. We need an array of readily-available, cost-effective antiviral treatments that work across all virus strains and complement vaccination efforts.” The Science: The scientific results, published in the academic journal Science Magazine revealed that exposing the SARS-CoV-2 (Coronavirus) virus to a free fatty acid called linoleic acid locks the virus’s spike protein into a closed, non-infective form stopping it in its tracks. While it is known that linoleic acid is indispensable for many cellular functions including inflammation control and maintaining cell membrane elasticity, its central role in Covid-19 has only recently been discovered. In Covid-19 patients, the body’s limited store of linoleic acid is depleted. This depletion is sensed by an enzyme called cPLA2 which tries to re-supply fatty acid levels in the cell by releasing free fatty acid storage pools from inside the cell. The virus utilizes the released free fatty acids to build specially purposed micro-compartments in which it replicates. In addition, dysregulated cPLA2 activity causes the membranes of infected cells to stiffen and lose elasticity. In the lungs, this triggers oxygen permeation breakdown that is known to cause breathing difficulties. Ultimately, uncontrolled free fatty acid release contributes proinflammatory precursors which trigger the cytokine storm observed in more severe forms of COVID-19. Linoleic acid can be purified from plant oils, e.g. sunflower oil, and is scalable to meet global demand. There is a strong potential to create a very cost effective pan-coronavirus treatment. The study leading to the discovery included experts from the Max Planck-Bristol Centre, the School of Biochemistry, the Bristol UNCOVER Group and was supported by funds from the Elizabeth Blackwell Institute with additional support from Oracle high-performance cloud computing and Genscript. ENDS NOTES TO EDITORS: For further information or to arrange an interview with the Halo Therapeutics and Bristol research team please contact Victoria Holden at Transatlantic Entertainment at victoria@transatlanticent.com / 07803 328 465 *Phase 2 clinical trial: A phase II clinical trial tells doctors more about how safe the treatment is and how well it works. Doctors also test whether a new treatment works. What is linoleic acid? Linoleic acid is a free fatty acid, which is indispensable for many cellular functions. The human body cannot produce linoleic acid: instead, the body absorbs this essential molecule through diet. Intriguingly, linoleic acid plays a vital role in inflammation and immune modulation, which are both key elements of Covid-19 disease progression. Linoleic acid is also needed to maintain cell membranes in the lungs so that we can breathe properly. Linoleic acid is also a central controlling switch for enzymes that release stored free fatty acid from cell membranes. About Halo Therapeutics Ltd Bristol-based biotech start-up Halo Therapeutics halo-therapeutics.com develops pan-coronavirus antivirals and anti-inflammatory therapies for coronavirus infection and is backed by private investors. About Professor Christiane Berger-Schaffitzel Christiane Schaffitzel is also a Wellcome Trust Investigator and Academic Lead of the Wellcome Trust/BBSRC-funded GW4 Cryo-EM Facility at the University of Bristol, and co-founder and CTO of Halo Therapeutics. About Professor Imre Berger Imre Berger is also Director of BrisSynBio, a BBSRC/EPSRC Research Centre for Synthetic Biology in Bristol and a partner in the Wellcome Trust COVID-19 Protein Portal Consortium CPPC. BrisSynBio is part of the Bristol BioDesign Institute. He is co-founder and CSO of Halo Therapeutics. Support our COVID-19 research Bristol’s researchers are part of a global network of scientists responding urgently to the challenge of the coronavirus pandemic.