FasL

深度聚焦多肽、多肽偶联药物研发、质控、工艺开发，7月相约苏州，联系电话18116036798目前，治疗肿瘤的主要手段有手术切除、放射治疗和化学药物治疗。化疗药物不仅杀伤肿瘤细胞，同时也作用于广泛的正常细胞，产生各种各样的毒副作用。而化疗药物的耐药性也是其不能有效治疗肿瘤的原因。因此，研制高效、低毒副作用的抗肿瘤药物显得尤为重要。与传统化疗药物相比，多肽类药物具有分子量小、对肿瘤敏感、不易耐药、成本低等特点，这使得多肽类药物受到了国内外的积极研究与开发，此外，多肽也是肿瘤抗原中起作用的主要成分，多肽疫苗的研发也将为肿瘤患者带来希望。本文就多肽药物抗肿瘤的作用机制及多肽作为免疫检查点阻断剂的最新研究进展进行总结，一起来看看吧！一、多肽药物的抗肿瘤机制多肽是以氨基酸为基本单位组成的分子，分子量一般在10 KDa以下，介于化学小分子与生物制品之间，其主要特点是选择性高、作用浓度低。1、调节免疫功能 肿瘤免疫治疗是通过人体自身免疫系统对肿瘤做出反应，从而达到治疗癌症的目的。作为一种生物疗法，免疫治疗已成为除了手术治疗和放化疗之外的第4种重要的肿瘤治疗手段。免疫应答可以分为特异性免疫应答和非特异性免疫应答。非特异性免疫又称固有免疫，固有免疫细胞包括巨噬细胞、树突状细胞、NK细胞等，通过模式识别受体或有限多样性抗原识别受体对病原体等识别结合产生非特异性抗肿瘤等作用。Sun 等[18] 研究表明不同浓度的杏鲍菇菌丝体（PEMP）多肽能够刺激巨噬细胞释放NO、H2O2和TNF-α等抑制癌细胞的增殖，增强巨噬细胞的吞噬能力从而发挥抗肿瘤的作用。 特异性免疫应答可以分为T淋巴细胞介导的适应性免疫即细胞免疫和B淋巴细胞介导的体液免疫2种。T细胞可以分为CD4+T细胞和CD8+T细胞，在 特异性抗原部位发挥效应功能的主要是细胞毒性T 淋巴细胞（CTL）。CTL 是细胞免疫的主要效应细胞，特异性地杀伤胞内病原体感染细胞和肿瘤细胞等。Sun等研究开发人类磷脂酰乙醇胺结合蛋白4（hPEBP4）衍生的免疫原性肽来诱导针对乳腺癌的抗原特异性CTL，并鉴定了一种新的免疫原性肽 P40-48（TLFCQGLEV），其能够在 HLA-A2.1/Kb 转基因小鼠以及来自乳腺癌患者的外周血淋巴细胞中引发特异性CTL应答。用2种类似物接种诱导的CTL的继承性转移比天然肽更有效地抑制肿瘤生长。Xie等通过HLA结合预测算法搜索来自Eps8蛋白的新型人类白细胞抗原（HLA）-A*2402 限制性表位，来自 Eps8 的新的 HLA-A*2402 限制性表 的肽327（EFLDCFQKF）、534（KYAKSKYDF）和755（LFSLNKDEL）诱导肽特异性CTL分泌更高水平的IFN-γ并显示出增强的抗恶性癌细胞的细胞毒活性，通过抑制Eps8/EGFR相互作用，肽327和TAT327可以作为新的肽抑制剂，这可以提供治疗各种癌症的创新方法。B细胞作为体液免疫的主要细胞，其对肿瘤具有双重作用，即促进抗肿瘤反应和诱导免疫耐受。根据功能的不同，B细胞可分为效应性B细胞和调节性B细胞。其中调节性B细胞（Bregs）与肿瘤的关系十分密切，其肿瘤免疫中的作用机制主要有以下几个方面：一方面，Bregs可以通过分泌IL-10，减少Th1细胞和Th17等T细胞的分化，减少肿瘤抑制性细胞因子的表达从而促进肿瘤的生长；另一方面，Bregs可以通过分泌TGF-β、协同共刺激分子和IL-10一同诱导 Tregs的产生，从而通过Tregs抑制免疫效应细胞的功能以此保护肿瘤细胞。效应性B细胞分又1型和2型2种亚型。其中1型效应性B细胞（Be1）主要产生TNF-α、IFN-γ和IL-12等促炎细胞因子发挥抗肿瘤的作用；而2型效应性B细胞（Be2）主要产生IL-4、IL-13等细胞因子，Th2细胞反应有关。Huang等研究表明单独CpG足以抑制BRAF抑制剂诱导的抗肿瘤反应，表明在接受基于CpG的肽疫苗的小鼠中观察到的BRAF抑制剂的抗肿瘤活性受损主要取决于使用CpG。CpG增加了循环B细胞的数量，这产生了增加量的TNF-α，这有助于BRAF抑制剂对肿瘤的抗性增加。常旭、欧红利对美洲大蠊的抗肿瘤作用及对荷瘤小鼠免疫功能的影响等方面进行了研究，表明了美洲大蠊多肽是通过提高荷瘤小鼠免疫功能来实现抗 肿瘤作用的机制。图片来源：摄图网2、抑制肿瘤新生血管形成 新生血管是肿瘤获取营养保证自身发展的重要途径，肿瘤血管生成是肿瘤诱导体内多种因子共同作用的结果，其中主要是促进因子和抑制因子共同作用的结果，正常情况下两者处于平衡状态。在多种的促进因子中血管内皮生成因子（VEGF）是血管生成的重要调节物，其受体（VEGFR）主要有VEGFR-1、VEGFR-2、VEGFR-3 3种。目前研究的抗肿瘤肽类药物在抑制血管生成方面的机制是多数学者研究的热点。例如，Qi等研究发现金属蛋白酶-3（TIMP-3）的组织抑制剂通过其阻断VEGF与其受体VEGFR-2的结合的能力，是VEGF介导的血管生成和新血管形成的有效抑制剂。Bracci 等研究的支链肽NT4结合HSPG并选择性靶向癌细胞和组织。NT4证明对内皮细胞增殖、迁移和管形成具有重要影响，特别是当由FGF2和凝血酶诱导时。此外，NT4对侵袭性肿瘤细胞迁移和侵袭具有重要作用。因此，抗肿瘤多肽类药物的抗血管生成机制为其成为肿瘤靶向药物的发展提供了线索。3、促进细胞凋亡 细胞凋亡又称细胞程序性死亡，是细胞正常死亡的主要方式，在清除细胞过程中起着重要的作用。当凋亡信号通路异常时就会出现多种疾病，其中就包括细胞癌变，细胞凋亡的抗性与肿瘤发生密切相关。在机体内，细胞凋亡有主要的2种途径：外源性通路，其主要是通过细胞表面的凋亡受体刺激激活的；内源性通路，除受凋亡信号激活外还有破坏线粒体膜完整性。与细胞凋亡密切相关的信号通路还有很多，比如Bcl-2家族和Fas/FasL途径。Bcl-2家族主要有2大类蛋白，一类为抗凋亡蛋白（Bcl-2），另一类为促凋亡蛋白（Bax），在肿瘤细胞中，抗凋亡蛋白（Bcl-2）过度表达导致细胞抗凋亡能力提高，肿瘤细胞的生长无法受到调控。多肽类药物诱导细胞凋亡可以通过影响Bcl-2家族蛋白。张丹等研究表明，美洲大蠊多肽可以通过上调Bax蛋白表达，下调Bcl-2蛋白表达，诱导人肝癌细胞SMMC-7721发生凋亡，从而抑制肿瘤细胞的增殖。因此，靶向调节Bcl-2家族成为治疗肿瘤的有效途径。另一个为 Fas/FasL 信号通路，是调节细胞凋亡的主要途径之一，Fas/FasL 信号通路的激活诱导细胞的凋亡。Kuo等研究表明从尼罗河罗非鱼中提取的低浓度海洋抗菌肽（AMP）MSP-4通过激活Fas/FasL信号通路诱导骨髓瘤MG63发生细胞凋亡从而发挥抗肿瘤作用。二、多肽作为免疫检查点抑制剂（一）单靶点多肽1、PD-1/PD-L1 多肽 高通量展示筛选技术在免疫检查点阻断肽的筛选中发挥着非常重要的作用。Tao等利用噬菌体展示技术筛选获得了靶向PD-1的多肽P-F4；Liu等利用同样的方法获得了靶向PD-L1的多肽CLP002；Li等首次利用细菌表面展示技术筛选获得了针对PD-L1的多肽TPP-1；Kamalinia等利用mRNA展示技术获得了PD-L1多肽SPAM。 然而，上述多肽均为L构型氨基酸组成的线性多肽，构象和抗酶解稳定性较差。为了获得构象更为稳定的多肽，环肽是一种较好的选择。2015 年，Maute 等利用酵母展示技术获得高亲和 PD-L1 的 PD-1 蛋白突变体，其亲和力是野生型 PD-1 的74000倍，该 PD-1 高亲和突变体已经被应用于靶向 PD-L1 多肽的设计。Jeong 等通过从上述 PD-1 蛋白突变体截取多肽片段，并利用树枝状大分子形成了多价的 PD-L1 多肽偶联物。无独有偶，Yin 等基于 PD-1 高亲和突变体，提出了 “多肽模拟设计”的概念，并设计了具有高亲和力和较 好抗肿瘤效果的 PD-L1 的大环肽 MOPD-1。PD-1/PD-L1 受配体相互作用是介导肿瘤免疫逃逸的关键因素之一，抑制其介导的负性信号通路能够打破机体已经建立的免疫耐受，并能使 PD-1 免疫细胞的功能耗竭得以改善。国内外已有多个课题组和公司也在研发 PD-1/PD-L1 的小分子药物，大多数小分子药物可以无障碍地进入细胞内部。与传统的放化疗相比，PD-1/PD-L1 的多肽阻断剂通常不会直接影响肿瘤细胞的增殖或迁移，而是通过调动免疫系统的功能而杀伤肿瘤细胞。此外，PD-1 主要在免疫耐受的维持阶段发挥作用，这意味着 PD-1/PD-L1 的阻断不会带来严重的不良反应。总之，PD-1/PDL1 的多肽阻断剂有望为肿瘤免疫治疗提供极具潜力的候选药物。2、其他免疫检查点多肽 以 PD-1/PD-L1 抗体为代表的基于免疫检查点阻断的肿瘤免疫治疗已经在临床治疗中取得了显著的效果。然而，在 PD-1 抗体治疗过程中，其他免疫检查点分子如 TIM-3 和 TIGIT 表达上调，会引起 PD-1 抗体治疗的适应性耐药。在黑色素瘤患者中，即便部分患者有较高的肿瘤突变负荷和炎性肿瘤微环境，TIGIT/PVR 通路仍会介导 PD-1 抗体治疗的耐药性。考虑到免疫反应的动态变化以及免疫检查点表达调控的复杂性，难以依靠任何单一的靶点实现最佳的治疗效果，针对其他免疫检查点通路的单抗药物研究也在进行中，而相关的多肽阻断剂研究也逐渐受到关注。 TIGIT/PVR 通路在肿瘤免疫抑制中发挥着重要的作用，阻断 TIGIT/PVR 能够增强 PD-1/PD-L1 阻断的治疗效果，并有望克服 PD-1/PD-L1 抗体治疗耐药。免疫检查点多肽阻断剂图片来源：参考文献[1]（二）双功能多肽 双特异性抗体是当前抗体药物研发的热点，已有多个双特异性抗体获得FDA批准用于临床研究。与双特异性抗体的作用相同，双功能多肽是将针对两个功能相关或互补的靶点的多肽分子进行偶联，偶联后的多肽不仅能够保留单靶点多肽的功能活性，还可以增加多肽的稳定性和半衰期。与单靶点多肽相比，双功能多肽可以同时针对生物体内不同的信号传导发挥功能，将不同靶点多肽进行合理设计还能够起到 1+1>2 的效果。相较于 双特异性抗体，双功能多肽的设计和合成更为简便。 （三）自组装多肽 通过合理的设计，特定序列的多肽自身也能够自组装构建药物递送系统。Wang 等利用具有自组装功能的 G7 肽（GNNQQNY）将靶向 CD3 的多肽与靶向整合素 αvβ3 的RGD肽连接形成双功能多肽 anti-CD3-G7-RGD，该多肽在靶向 T 细胞的 CD3 受体的同时，利用 G7 肽原位自主装形成纳米寡聚物的特性，诱导 CD3 受体寡聚化进而激活 T 细胞。不仅如此， 双功能肽的 RGD 序列能够引导 T 细胞精准地识别并杀伤肿瘤细胞。Lv 等使 pep-20 在肿瘤组织中原位组装形成纳米纤维，打破肿瘤细胞高表达的 CD47 所介导的“不要吃我”信号，增强巨噬细胞对肿瘤细胞的吞噬效应，有效抑制了肿瘤生长。另外，也可以利用纳米载体将其他药物与免疫检查点阻断肽进行一体化设计，起到协同作用。Zhu 等将D PPA-1 肽用于与化疗药物阿霉素（DOX）通过基质金属蛋白酶 2（MMP2）的特异性底物序列进行偶联，自组装形成酶响应性的纳米颗 粒，通过胞吞作用促进药物在肿瘤的累积和渗透， D PPA-1 肽和 DOX 通过 MMP2 响应性的释放，不仅可以充分发挥化疗药物对肿瘤的杀伤作用，而且D PPA-1 肽能够增强 T 细胞功能。Cheng 等设计了由 3-二乙基氨基丙基异硫氰酸（DEAP）、MMP2 底物序列 和D PPA-1 组成的两亲性肽，该多肽与吲哚胺 2，3-双加氧酶 1（IDO1）的抑制剂 NLG919 通过疏水作用组装成纳米颗粒。该纳米颗粒在肿瘤微环境中具有双重刺激依次响应的优点：首先，通过 EPR 效应在肿瘤部位富集，DEAP 序列在弱酸环境下发生质子化使得纳米 结构膨胀；进而，高表达的 MMP2 识别底物序列并彻底崩解纳米颗粒，释放D PPA-1 肽和 NLG919。该设计充分结合多肽及小分子药物的性质和肿瘤微环境的特点，联合 PD-1/PD-L1 阻断和 IDO1 抑制剂，协同增强肿瘤组织 T 细胞的功能，诱导强大的抗肿瘤免疫反应。（四）核素标记多肽 阻断免疫检查点的肿瘤免疫治疗效果与免疫检查点的表达情况密切相关。通过检测免疫检查点的表达水平对患者进行筛选能够促进精准治疗。免疫组织 化学是目前检测免疫检查点表达的金标准。然而，该方法无法实时量化全身的表达水平。而在肿瘤发生、进展以及治疗过程中，检查点分子的表达是高度动态变化的。在特定的肿瘤类型或转移灶中，免疫检查点的表达也具有高度异质性。在患者选择和疗效监测方面，以高灵敏度和分辨率进行全身非侵入性免疫检查点表达检测非常重要。正电子发射断层扫描（PET）作为一种无创水平成像技术，可用于实时、重复和所有病灶的动态检测。极大方便了临床上筛选可能受益的患者、对免疫治疗进行疗效评价以及及时地进行治疗策略的调整。PET 成像已广泛用于多种免疫检查点的示踪，包括多种与放射性核素标记的 PD-1/PD-L1、TIGIT、LAG-3 和 TIM-3 抗体等。放射性核素标记的免疫检查点抗体组织渗透能力不足，而且由于抗体本身的半衰期较长，往往需要较长的时间才能实现较强的组织富集。低分子量的多肽因具有适当的半衰期和较强的实体瘤渗透能力成为 PET 成像探针的理想候选。靶向 PD-1 或 PD-L1 的高亲和力多肽，尤其是 TPP-1 和 WL12 已被用于各种放射性核素标记的 PET 成像。目前，放射性标记的免疫检查点抗体或多肽在 PET 成像和光学成像中的应用已经进行了广泛的临床前和初步临床研究，显示出其作为患者分层、疗效 的动态监测和预后工具的潜力。其中，低分子量多肽作为诊断探针具有实体瘤渗透能力强、不良反应小、半衰期适宜的良好特性，在 PET 成像中的应用已经崭露头角，也将为肿瘤患者的临床诊断和辅助治疗提供有力的工具。写在最后：近年来，全球多肽药物的市场销售额保持快速增长趋势，多肽药物成为中国“十四五”期间生物医药研究的重点方向之一。但多肽药物目前面临的困难依旧存在，仍需要致力于设计更为简便、有效的修饰策略来提高多肽的结构稳定性。另外，目前虽然在多肽药物口服方面取得了一些进展，但是在临床应用和递送技术方面仍未取得显著突破，这些问题亟需研究者们 设计更为巧妙的方法来解决，从而进一步推动多肽药物的临床应用。END参考文献：[1]牛潇爽, 胡争, 周秀曼, 高艳锋. 多肽药物在肿瘤免疫治疗中的研究进展[J]. 中国肿瘤临床, 2023, 50(6)：271-277. doi:10.12354/j.issn.1000-8179.2023.20221128[2]陈泽锋,白丽.抗肿瘤多肽类药物的来源及其机制的研究进展[J].免疫学杂志,2018,34(12):1099-1104.DOI:10.13431/j.cnki.immunol.j.20180171.[3]刘光照,张婕,殷润婷.抗肿瘤多肽的研究新进展[J].药学进展,2015,39(09):671-675.声明：本文出自医会宝编辑部，旨在为医疗专业人士传递更多医学信息。本文并不能取代医生的专业诊疗意见，如有罹患，需前往专业医院检查诊断。END深度聚焦多肽、多肽偶联药物研发、质控、工艺开发，7月相约苏州，联系电话18116036798戳“阅读原文”立即报名多肽药物论坛吧!

Publication of data detailing the development of our microRNA (miRNA)-based multiplex shRNA approach, which allows to down-regulate several genes simultaneously, in Molecular Therapy – Nucleic AcidsFive posters were presented at the 38th SITC Annual Meeting and two posters were presented at the 4th ICLE conference, with further developments in our multiplex shRNA platform and encouraging data from our NKG2D-based multi-specific CAR T-cell platformCelyad Oncology has received approximately EUR 9.8m in private placement commitments from historical shareholders MONT-SAINT-GUIBERT, Belgium, Nov. 09, 2023 (GLOBE NEWSWIRE) -- Celyad Oncology (Euronext: CYAD) (the “Company” or “Celyad Oncology”), a biotechnology company focused on innovative technologies for chimeric antigen receptor (CAR) T-cell therapies, today reports its financial results for the third quarter of 2023 and provides an update on recent business developments. “Celyad Oncology team is resolutely dedicated to harnessing our expertise, extensive know-how, and valuable intellectual property portfolio to address the existing challenges within CAR T-cell therapies. We are excited to report compelling data from our short hairpin RNA (shRNA)-based multiplexing platform, underscoring the remarkable versatility and adaptability of this technology. In addition, we are making significant progress with our multispecific CAR program, and we look forward to sharing an update at international conferences in the coming months. These achievements mark our commitment to advancing the frontiers of cellular immunotherapy and bringing innovative solutions to patients in need,” commented Georges Rawadi, Chief Executive Officer of the Company. Third quarter 2023 and recent corporate highlights: On August 24, 2023, the Company announced that it has obtained commitments from Fortress, Tolefi and other longstanding existing shareholders to subscribe to a capital increase of up to €9.8 million in 2 tranches: A first tranche of 2.0 million was disbursed in the context of authorized capital as of September 4, 2023; andA second tranche to be subscribed by Fortress is subject to the approval by the extraordinary shareholders’ meeting. Following this private placement, the Company believes that its existing cash and cash equivalents should be sufficient, based on the current scope of activities, to fund operating expenses and capital expenditure requirements into the second quarter of 2025. Celyad Oncology has relocated from September 25, 2023 into a new research facility which fits better its current needs after the strategic shift. The Company remains headquartered at the Axis Parc, Mont-Saint-Guibert, Belgium but with its new business location at Dumont 9. Operational highlights shRNA multiplexing platform: Data validating our microRNA (miRNA)-based multiplex shRNA approach, which allows to down-regulate several genes simultaneously, were presented at the 4th International Conference on Lymphocyte Engineering (ICLE) in Munich (September 12-14) and at the 38th Annual Meeting of the Society for Immunotherapy of Cancer (SITC) in San Diego (November 1-5) and posters are available on the company’s website, at https://celyad.com/our-science-technology/publications/;With our approach we proved the feasibility of the simultaneous knock-down of 4 co-inhibitory receptors (PD-1, LAG-3, TIM-3 and CD95) to decrease the expression of exhaustion markers at the surface of CAR T-cells and the feasibility of this approach to improve allogeneic CAR T-cell viability by allowing evasion from graft-versus-host disease (GvHD), host-versus-graft (HvG) reaction and CD95L-induced autophagy.The results detailing the technical aspects of the development of this platform and showcasing the easiness, efficiency, and tunability of this technology to knock-down up to four target genes simultaneously have been published in Molecular Therapy – Nucleic Acids as of September 20. NKG2D-based CAR T-cells: We have developed different CD19/NKG2DL, BCMA/NKG2DL and PSMA/NKG2DL multi-specific CAR T-cells, utilizing both tandem constructs – that encompass the extracellular domain of the natural NKG2D receptor fused to a scFv targeting CD19, BCMA or PSMA, or dual constructs – that co-express the NKG2D-based CAR with an anti-CD19, anti-BCMA or anti-PSMA CAR, respectively;Our data provides the proof-of-concept that NKG2DL are valuable targets in a multispecific CAR approach;Specifically, we showed that CD19/NKG2DL multispecific CAR T-cells, and in particular dual receptors, are highly effective in vitro against CD19+ and CD19- cell lines and against CD19+ primary B-cell acute lymphoblastic leukemia (B-ALL) cells. In vivo, CD19/NKG2DL tandem CAR T-cells outperforms dual CAR T-cells in controlling tumor growth in an aggressive B-ALL relapse model.In vitro data generated with BCMA/NKG2DL and PSMA/NKG2DL multispecific CAR T-cells further validate this approach and its application in other hematological and solid indications.Preliminary data were presented at the 4th ICLE conference in Munich (September 12-14) and at the 38th SITC meeting in San Diego (November 1-5) and posters are available on the company’s website, at https://celyad.com/our-science-technology/publications/. Financial highlights – Third quarter 2023 financial review As of September 30, 2023, the Company had cash and cash equivalents of €2.3 million. Net cash burn during the third quarter of 2023 amounted to €2.7 million, in line with expectations. The Company projects that its existing cash and cash equivalents should be sufficient to fund operating expenses and capital expenditure requirements into the first quarter of 2024. After due consideration of detailed budgets and estimated cash flow forecasts for the years 2023 and 2024, the Company continues to project that its existing cash and cash equivalents will not be sufficient to fund its estimated operating and capital expenditures over at least the next 12 months from the date that this release is issued. On August 24, 2023, the Company announced that it has obtained commitments from Fortress, Tolefi and other longstanding existing shareholders to subscribe to a capital increase of up to €9.8 million (whose €2.0 million related to the first tranche has been already proceeded as of September 4, 2023). Taking into account the Management’s assumptions regarding estimated cash-flows for the years 2023, 2024 and 2025, the Company believes that following the close of the second tranche subscribed by Fortress which is subject to approval by the extraordinary shareholders’ meeting, its existing cash and cash equivalents should be sufficient, based on the current scope of activities, to fund operating expenses and capital expenditure requirements into the second quarter of 2025. Upcoming Anticipated Milestones More data and evidence in the context of the multi-specific CAR platform and shRNA multiplexing approach will be shared in the first quarter of 2024, with the aim of a clinical evaluation of assets and initiation of clinical trials either by the Company and/or through strategic partnerships afterwards. Upcoming Conferences Celyad Oncology will attend the 7th CAR-TCR Europe summit in London, UK (February 27-29, 2024), the must-attend forum to brainstorm advanced in cell therapies and stay at the forefront of cell therapy innovations. About Celyad Oncology Celyad Oncology is a cutting-edge biotechnology company dedicated to pioneering the discovery and advancement of revolutionary technologies for chimeric antigen receptor (CAR) T-cells. Its primary objective is to unlock the potential of its proprietary technology platforms and intellectual property, enabling to be at the forefront of developing next-generation CAR T-cell therapies. By fully leveraging its innovative technology platforms, Celyad Oncology aims to maximize the transformative impact of its candidate CAR T-cell therapies and redefine the future of CAR T-cell treatments. Celyad Oncology is based in Mont-Saint-Guibert, Belgium. For more information, please visit www.celyad.com. Celyad Oncology Forward-Looking Statement This release may contain forward-looking statements, within the meaning of applicable securities laws, including the Private Securities Litigation Reform Act of 1995, as amended, including, without limitation, statements regarding beliefs about and expectations for the Company’s updated strategic business model, including associated potential benefits, transactions and partnerships, statements regarding the potential value of the Company’s IP, and statements regarding the continuation of the Company’s existence. The words “will,” “potential,” “continue,” “target,” “project,” “should” and similar expressions are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. Any forward-looking statements in this release are based on management’s current expectations and beliefs and are subject to a number of known and unknown risks, uncertainties and important factors which might cause actual events, results, financial condition, performance or achievements of Celyad Oncology to differ materially from those expressed or implied by such forward-looking statements. Such risks and uncertainties include, without limitation, risks related to the material uncertainty about the Company’s ability to continue as a going concern; the Company’s ability to realize the expected benefits of its updated strategic business model; the Company’s ability to develop its IP assets and enter into partnerships with outside parties; the Company’s ability to enforce its patents and other IP rights; the possibility that the Company may infringe on the patents or IP rights of others and be required to defend against patent or other IP rights suits; the possibility that the Company may not successfully defend itself against claims of patent infringement or other IP rights suits, which could result in substantial claims for damages against the Company; the possibility that the Company may become involved in lawsuits to protect or enforce its patents, which could be expensive, time-consuming, and unsuccessful; the Company’s ability to protect its IP rights throughout the world; the potential for patents held by the Company to be found invalid or unenforceable; and other risks identified in Celyad Oncology’s U.S. Securities and Exchange Commission (SEC) filings and reports, including in the latest Annual Report on Form 20-F filed with the SEC and subsequent filings and reports by Celyad Oncology. These forward-looking statements speak only as of the date of publication of this document and Celyad Oncology’s actual results may differ materially from those expressed or implied by these forward-looking statements. Celyad Oncology expressly disclaims any obligation to update any such forward-looking statements in this document to reflect any change in its expectations with regard thereto or any change in events, conditions or circumstances on which any such statement is based, unless required by law or regulation. Celyad Oncology Contacts: Investor Contact:Media Contact:David Georgesinvestors@celyad.comcommunications@celyad.com Source: Celyad Oncology SA

近日，有关于加州大学戴维斯分校发现的“癌细胞生死开关”的消息不胫而走，其中被认为是生死开关的是CD95（Fas）受体上的一个关键表位，这个表位的激活将导致癌细胞细胞程序性死亡。不过能程序性死亡的或许不止癌细胞？同样受到影响，且杀伤肿瘤的T细胞，NK细胞也被凋亡了，那又怎么办？这样一看，Fas或许也能致癌……虽然此前新闻对于该表位的发现报道甚多，但核心主要证明了这一靶点可成药，可成药并不意味着能够做到临床转化，而且对于这个关键表位具体是如何发现，发现了这个表位将如何实践和应用，报道相对较少。本文将对此进行简单解析。Fas受体表位的灵感来源和实际意义受体聚集是通过属于TNFα（肿瘤坏死因子）超家族的死亡受体激活外源性细胞凋亡的最关键步骤，通过这种机制制作激动性抗体来激动程序性死亡受体诱导癌细胞死亡是一种治疗思路。但是一直以来，这种思路在临床上并不成功。其中在癌症治疗领域研究比较广的是这一超家族中的死亡受体-5 (DR5)，而关于Fas激动性抗体的临床前的抗体探索，大都没有表现出亲和力而失败了。死亡受体-5 (DR5)和Fas受体(Fas或FasR或CD95)都属于TNF-α受体超家族，他们都属于胞质尾区包含死亡结构域（DD）来诱导外源凋亡的TNF-α受体群体，机理在于它们均包含三个富含半胱氨酸的外部结构域(CRD1-3 )，而通过外部结构域招募受体，受体聚集，然后激活外源性凋亡信号。但是到底具体到是哪个外部受体结构域的基序，受体聚集后怎么去激活外源性凋亡信号的研究其实相当缺乏。直到近年来，通过DR5的一系列研究，如临床的二价或多价单特异性抗体、双特异性抗体、受体诱变研究、功能测定和NMR结构分析，最后才表征出了DR5的胞外结构域(ECD)中的受体聚集构象自动抑制基序。最后发现的这个构象自动抑制基序是一个在外部结构域上，带正电荷，带有精氨酸，赖氨酸氨基酸残基的半胱氨酸稳定块状物，也被称为是高度显著的带正电荷的残基表位（PPCR）。而与DR5类似的Fas虽然在此前的研究中被远远低估，但是最近的发现却给Fas赋予了更重要的地位。与DR5不同，Fas信号对于维持T淋巴细胞的活化诱导的细胞死亡(AICD)以实现健康的免疫稳态至关重要。而T淋巴细胞的活化诱导又和近年来的各类免疫疗法相关。其中更是以CAR-T的耐药问题为最。关于CAR-T的一系列研究还发现了Fas相关的死亡受体通路受损是CD19 CAR-T出现耐药的一种原因——CAR-T的疗效会高度依赖于Fas-FasL通路的旁观者信号的传导和杀伤。另外自身免疫性淋巴细胞增生综合征(ALPS)是一种Fas基因介导的凋亡受损导致淋巴细胞增殖的疾病。由于淋巴细胞凋亡障碍，患者出现慢性淋巴细胞增殖、自身免疫反应和致瘤倾向。研究Fas的基因还有可能解决ALPS的治疗问题。因此探究Fas的成药性是有必要的，特别是探究和DR5类似的关键表位。Fas表位所在和未来应用与DR5的PPCR不同，Fas PPCR存在于CRD2（第二个外部结构域）而不是CRD3的中间，与DR5高度可变的CRD3的特征不同，仅来自两个已经被发表的Fas结构的叠加，而其高度保守的序列CRRCR在灵长类中是完全相同的。而在卵巢肿瘤模型中，PPCR（必须有R87，如上图的87号）参与对高级Fas激动剂信号和CAR-T旁观者功能是不可或缺的。而如果出现干扰PPCR参与的FasL或Fas单点突变R87，则会抑制肿瘤细胞和T细胞中的凋亡信号。这里采用的CAR-T用的是靶向卵巢癌靶点NaPi2b和FRα的CAR-T，对于这类难以穿透的实体瘤如果采用双特异性抗原靶向CAR-T，并同时在异源肿瘤床中激活旁观者Fas/caspase-8的亲和力介导的杀伤，具有更高的穿透潜力。而之所以目前临床前所采用的Fas抗体的有限活性是由于它们不能与Fas PPCR结合，这其实为后续的Fas抗体的开发埋下了基础。但是值得注意的是，去激动Fas本身也具有致癌性，因为FAS能调节肿瘤凋亡，也能调节T细胞凋亡，并具有癌症干细胞调节功能。研究人员需要了解TCR和FAS之间的相互作用才能了解并开发出下一代的选择性FAS激动性抗体，这样就能做到差异化结合PPCR，更多去结合肿瘤而不是T细胞。因此这也是研究人员所述下一步的研究方向。在肿瘤中，这种靶向对于实体癌免疫疗法来说是一把双刃剑。然而，像DR5一样，能够靶向分布在整个Fas胞外域的表位的额外Fas抗体可以为其在肿瘤和T细胞中的差异活化提供进一步的线索。总结总的来看，虽然此次开关的具体发现对可成药性有了一定的依据，但是距离实践中FAS与TCR之间的作用尚未阐明，距离临床前研究都尚且很远，临床转化可能需要花费更长的时间。目前的研究仅能初步支持开发Fas激动抗体在卵巢癌双靶点CAR-T中的应用。参考来源：Mondal, T., Gaur, H., Wamba, B.E.N. et al. Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer. Cell Death Differ (2023). https://doi-org.libproxy1.nus.edu.sg/10.1038/s41418-023-01229-7