CYAD-101

引用本文 刘婧禹，尹  桐，吴　玥，等. 转移性结直肠癌三线治疗的研究现状及进展［J］. 中国癌症杂志, 2025, 35 (11): 1056-1066. 基金项目：海军军医大学基础医学研究基金项目（2023MS023）。  利益冲突：作者声明无利益冲突。 伦理批件：不需要。 知情同意：不需要。 第一作者简介 刘婧禹，长海医院肿瘤科博士研究生。专业方向：消化道肿瘤，肿瘤免疫。 通信作者简介 湛先保，临床医学博士、教授、主任医师、博士研究生导师。长海医院肿瘤科主任、肿瘤学教研室主任、国家药物临床试验机构肿瘤专业组组长。2005年曾赴美国南卡罗来纳医科大学访学，2014—2015年赴美国梅奥医学中心访学。擅长消化系统肿瘤的个体化医疗。以课题负责人承担省部级以上基金课题10余项，包括国家自然科学基金课题3项。主要研究领域包括胃肠与胰腺肿瘤和良性疾病的转化医学研究，在国际上率先开展靶向claudin 18.2 CAR-T免疫细胞治疗、消化系统肿瘤mRNA疫苗治疗和胃癌肿瘤类器官体外药敏精准检测技术。在SCI收录期刊上参与发表论著30余篇，获国家科技进步二等奖2项和上海市科技进步一等奖1项、上海市银蛇奖，被评为解放军总后勤部爱军精武标兵、上海市卫生系统先进工作者，行政立大功1次。现兼任海峡两岸医药医生交流协会精准医疗分会主任委员、中国宋庆龄基金会肿瘤区域医疗与产学研联盟胃癌专委会主任委员、上海市抗癌协会胃癌分子靶向与免疫治疗专委会主任委员、中国医师协会胰腺病分会常委、中国医药教育协会腹部肿瘤分会常委、中国临床肿瘤学会免疫治疗专委会委员、中华医学会肿瘤学分会胃肠肿瘤学组成员、中国抗癌协会胰腺癌分会委员等。 作者解读 ▲ 刘婧禹 医师 转移性结直肠癌三线治疗的研究现状及进展 刘婧禹，尹  桐，吴　玥，彭小波，湛先保 海军军医大学第一附属医院肿瘤科，上海 200433 ［摘要］  转移性结直肠癌（metastatic colorectal cancer，mCRC）的三线治疗指在一线、二线治疗失败或患者无法耐受后采用的后续治疗方案，是临床实践中的关键难题，也是近年来转化医学研究的核心领域。随着分子分型技术的普及和新型疗法的涌现，三线治疗策略正从传统化疗向精准靶向治疗联合免疫治疗转变。本研究通过检索PubMed、ClinicalTrials.gov数据库及美国临床肿瘤学会（American Society of Clinical Oncology，ASCO）、欧洲肿瘤内科学会（European Society for Medical Oncology，ESMO）的会议摘要，纳入Ⅲ期随机对照试验、Ⅰ/Ⅱ期前沿临床研究及权威综述，重点关注生存获益、耐药性及生物标志物相关数据。全面梳理了近年来mCRC三线治疗领域的重要进展，包括三线标准药物及治疗［瑞戈非尼、呋喹替尼、曲氟尿苷替匹嘧啶、抗表皮生长因子（epidermal growth factor receptor，EGFR）再挑战治疗］、靶向治疗（如BRAF V600E抑制剂、ERBB2扩增、KRAS G12C抑制剂）、免疫治疗［微卫星高度不稳定（microsatellite instability-high，MSI-H）/错配修复缺陷（deficient mismatch repair，dMMR）、微卫星稳定（microsatellite stable，MSS）/错配修复完整（proficient mismatch repair，pMMR）及靶免联合治疗］的最新临床证据。其中靶向治疗领域取得显著突破：抗EGFR再挑战治疗通过循环肿瘤DNA （circulating tumor DNA，ctDNA）动态监测筛选RAS/BRAF野生型患者，使中位总生存期（overall survival，OS）延长至17.3个月，但耐药机制复杂，继发突变率高，需进一步优化动态监测体系；针对BRAF V600E突变，三联方案（康奈非尼+比美替尼+西妥昔单抗）较传统治疗中位OS延长至9.3个月［风险比（hazard ratio，HR）=0.52］；KRAS G12C抑制剂阿达格拉西布（adagrasib）联合西妥昔单抗的客观缓解率（objective response rate，ORR）提升至34%，中位OS达15.9个月，但肿瘤耐药仍是主要挑战。免疫治疗方面，MSI-H/dMMR患者通过双免疫联合治疗（纳武利尤单抗+伊匹木单抗）获得71%的4年OS率，而MSS型患者依赖免疫-靶向联合治疗策略（如卡博替尼+德瓦鲁单抗），ORR提升至27.6%。新兴治疗领域主要包括人工智能平台的搭建、肠道菌群作为生物标志物与粪便微生物群移植的创新疗法及嵌合抗原受体T（chimeric antigen receptor-T， CAR-T）细胞疗法的最新进展。本综述通过探讨mCRC三线治疗的研究现状及进展，旨在为优化临床决策及未来研究方向提供参考。 ［关键词］ 转移性结直肠癌；三线治疗；靶向治疗；免疫治疗；生物标志物；耐药机制 ［Abstract］  Third-line treatment for metastatic colorectal cancer (mCRC) refers to subsequent therapeutic interventions following the failure or intolerance of first- and second-line treatments. This represents a critical challenge in clinical practice and a core focus of translational medicine research in recent years. With advancements in molecular typing technologies and the emergence of novel therapies, the third-line treatment strategy has evolved from traditional chemotherapy toward precision targeting and immunotherapy. A comprehensive literature search was conducted across PubMed, ClinicalTrials.gov database and American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO) conference abstracts. Phase Ⅲ randomized controlled trials, phase Ⅰ/Ⅱ frontier clinical studies, and authoritative reviews were included, with an emphasis on data related to survival benefits, drug resistance mechanisms, and biomarkers. This review provided an in-depth analysis of significant progress in third-line treatment strategies for mCRC, encompassing standard therapies ［regorafenib, fruquintinib, trifluridine/tipiracil, anti- epidermal growth factor receptor (EGFR) rechallenge therapy］, targeted therapies (e.g., BRAF V600E inhibitors, ERBB2 amplification inhibitors, KRAS G12C inhibitors) and immunotherapies ［microsatellite instability-high (MSI-H)/deficient mismatch repair (dMMR), microsatellite stable (MSS)/proficient mismatch repair (pMMR) and target-immune combination therapies］. Notable breakthroughs have been achieved in targeted therapies. Anti-EGFR rechallenge therapy extended the median overall survival (OS) to 17.3 months in RAS/BRAF wild-type patients identified through dynamic circulating tumor DNA (ctDNA) monitoring. However, drug resistance remains complex, with high secondary mutation rates necessitating further optimization of dynamic monitoring systems. For BRAF V600E mutations, triple therapy (encorafenib+binimetinib+cetuximab) demonstrated a median OS of 9.3 months ［hazard ratio (HR)=0.52］, surpassing conventional treatments. The combination of KRAS G12C inhibitor adagrasib with cetuximab achieved an objective response rate (ORR) of 34% and a median OS of 15.9 months, though tumor resistance continued to pose challenges. In the realm of immunotherapy, dual immunotherapy (nivolumab+ipilimumab) yielded a 4-year OS rate of 71% in MSI-H/dMMR patients. For MSS patients, immune-targeted combination strategies (e.g., cabozantinib+atezolizumab) increased the ORR to 27.6%. Emerging therapies include artificial intelligence platforms for precision medicine, gut microbiota-based biomarkers and fecal microbiota transplantation, as well as advancements in chimeric antigen receptor-T (CAR-T) cell therapy. By summarizing the current status and progress of third-line treatment for mCRC, this review aims to inform clinical decision-making and guide future research directions. ［Key words］ Metastatic colorectal cancer; Third-line treatment; Targeted therapy; Immunotherapy; Biomarkers; Drug resistance mechanism 结直肠癌（colorectal cancer，CRC）是全球第3大常见癌症，每年估计有200万新发病例，近100万人死于结直肠癌，CRC患者的总体5年生存率为64%，约20%的患者在初诊时已处于晚期（Ⅳ期），且多伴有转移［1］。尽管一线和二线治疗可显著延长生存期，但多数患者因耐药仍需进入三线治疗。三线治疗的目标从单纯延长生存期逐渐转向精准控制疾病与改善生活质量并重。近年来，随着分子分型技术的普及和新药研发的突破，三线治疗策略不断革新。本文旨在梳理该领域的临床应用现状及最新研究进展，为临床实践和未来研究方向提供参考。 1 三线标准治疗的研究现状 在转移性CRC（metastatic CRC，mCRC）的临床管理中，三线治疗通常指患者在经历标准化疗方案（如基于5-氟尿嘧啶、奥沙利铂或伊立替康的一线治疗）和二线治疗方案（如交叉使用未用过的化疗方案或靶向药物）均失败后启动的系统性治疗。根据欧洲肿瘤内科学会（European Society for Medical Oncology，ESMO）指南［2］，启动三线治疗的核心标准包括：① 疾病进展的客观证据（如影像学检查确认的肿瘤增大或新发转移）；② 前期治疗方案耐药或无法耐受（如因严重不良反应需终止治疗）；③ 治疗方案实质性改变（需更换不同作用机制的药物，而非仅调整剂量或替换同类药物）［3］。目前临床常用三线治疗药物包括瑞戈非尼（regorafenib）、呋喹替尼（fruquintinib）及曲氟尿苷/替匹嘧啶（trifluridine/tipiracil， FTD/TPI，TAS-102），同时需结合分子分型选择抗EGFR再挑战治疗、抗人表皮生长因子受体2（human epidermal growth factor receptor 2，HER2）治疗或免疫检查点抑制剂（immune checkpoint inhibitor，ICI）［如程序性死亡蛋白-1（programmed death-1，PD-1）抑制剂用于微卫星高度不稳定（microsatellite instability-high，MS I-H ） /错配修复缺陷（deficient mismatch repair，dMMR）型患者］。表1总结了已获批三线治疗药物的机制及相关重要研究。在获批药物的使用情况中值得关注的是，中国mCRC患者三线治疗药物的选择和疗效具有显著的地域特征。一项纳入中国12家三甲医院的回顾性研究（n=1 889）［4］中，瑞戈非尼和呋喹替尼是主要用药，分别占38.6%和32.4%，结果显示，中位无进展生存期（progression-free survival，PFS）为3.2个月，总生存期（overall survival，OS）达8.7个月，显著优于未接受三线治疗组的6.1个月［风险比（hazard ratio， HR）=0.62，P＜0.001］。这些临床数据不仅验证了国际指南在中国的适用性，还凸显了在中国的临床实践中药物的可及性和区域特异性。 三线治疗作为mCRC的“最后防线”，亟待通过精准分型、创新疗法及全程管理打破生存困境。尽管三线获批治疗药物已为部分患者带来生存获益，但近年来针对特定驱动基因突变（如BRAF V600E、ERBB2扩增、KRAS G12C）的靶向治疗，正在通过精准分型和联合策略的优化，逐步突破传统疗法的局限性。 2 抗EGFR再挑战治疗 EGFR是与mCRC临床治疗相关的一个重要分子靶点，其耐药机制主要包括EGFR信号转导通路内的基因突变，以及其他细胞生长因子受体激活的补偿性回路（如HER2、MET）的激活［9］。EGFR属于ErbB受体酪氨酸激酶家族的成员，其中还包括ERBB2（HER2），两者可以配对形成激活的异二聚体，进而诱导RAS/RAF/MEK/MAPK和PI3K/AKT/mTOR等细胞内信号转导通路的激活，导致细胞增殖活跃、延长细胞存活和诱导转移，以此推动肿瘤进展［10］。RAS基因家族（KRAS/NRAS/HRAS）可以被EGFR募集并激活，并刺激下游效应激酶（MAPK/RAF）持续激活，是肿瘤细胞对抗EGFR抑制剂产生耐药性的主要机制［11］。RAS激活后还可以招募下游的丝氨酸-苏氨酸蛋白激酶RAF（其3种同工型包括ARAF、BRAF和CRAF）到细胞内信号级联中［10］。BRAF突变存在于7%~15%的CRC患者中，最常见的发生在第600位氨基酸的热点位点，称为BRAF V600E［12］。该基因突变包含两种不同亚型，BRAF突变1型（BM1）激活KRAS/mTOR/AKT/4EBP1通路，BM2亚型激活细胞周期检查点相关基因［13］。临床数据［14-15］已证实BRAF V600E突变和HER2基因扩增的mCRC患者对EGFR抑制剂的反应率较低，预后较差。EGFR激活触发的第2条主要信号通路是PI3K/AKT/mTOR通路，会导致编码细胞周期调节因子（包括MYC和细胞周期蛋白D1）的多种mRNA翻译增加，与抗EGFR抑制剂的疗效降低相关［16］。 西妥昔单抗和帕尼单抗（两种抗EGFR抑制剂）与化疗联合已被批准用于治疗RAS野生型mCRC，但上述获得性耐药机制会影响其临床获益，最终导致治疗失败［17］。值得关注的是，抗EGFR再挑战治疗在部分耐药的mCRC患者中展现出潜在价值。抗EGFR再挑战治疗指在RAS/BRAF野生型mCRC患者中基于循环肿瘤DNA（circulating tumor DNA，ctDNA）动态监测，对既往从抗EGFR治疗中获益且耐药后恢复敏感性的患者重新启用EGFR抑制剂的治疗策略。其生物学原理是，对抗EGFR抑制剂产生获得性耐药的肿瘤细胞克隆具有短暂的生存期，后续使用化疗联合抗血管生成药物可以有效地杀死对EGFR抑制剂耐药的肿瘤细胞，因此在三线治疗阶段，抗EGFR抑制剂可能再次有效［18］。抗EGFR再挑战治疗已经被列入临床指南推荐。 2020年一项针对中国城市地区的mCRC患者的回顾性研究［19］显示，亚组分析中RAS野生型患者接受抗EGFR再挑战治疗的客观缓解率（objective response rate，ORR）为12.3%，疾病控制率（disease control rates，DCR）达46.8%，提示分子分型对中国患者治疗决策具有重要影响。 2021年，Ⅱ期CAVE试验（NCT04561336）［20］首次探索西妥昔单抗联合程序性死亡蛋白配体-1 （programmed death ligand-1，PD-L1）抑制剂阿维鲁单抗的再挑战疗效，研究纳入77例RAS/BRAF野生型mCRC患者，结果显示，ORR为21%，中位PFS和OS分别为3.6和11.6个月。亚组分析表明，ctDNA清除患者的中位OS显著延长至17.3个月（95% CI：12.5~22.0个月），而突变患者的中位OS为10.4个月（95% CI：7.2~13.6个月；HR=0.49，95% CI：0.27~0.90，P=0.02），初步验证了ctDNA指导抗EGFR再挑战治疗的可行性。 2022年，Ⅱ期VELO试验（NCT05468892）［21］评估帕尼单抗联合TAS-102的疗效，在意向治疗人群中，联合组中位PFS较单药组显著延长（4.0个月 vs 2.5个月，HR=0.48，95% CI：0.28~0.82，P=0.007），且基线ctDNA未检出RAS/BRAF突变的患者中位PFS达6.4个月。同年，中国多中心研究（ChiCTR1900027573）［22］显示，西妥昔单抗联合卡瑞利珠单抗及脂质体伊立替康的ORR为25%，中位OS达15.1个月，且抗EGFR治疗间隔 ＞ 1年的患者获益更好（ORR=33.3%）。 2024年，随机Ⅱ期CITRIC试验（EudraCT  2020-000443-31）［23］进一步验证ctDNA指导的精准再挑战治疗策略，共纳入58例RAS/BRAF/EGFR野生型mCRC患者，抗EGFR再挑战治疗组的ORR较对照组（研究者选择方案）提高近2倍（30% vs 16%），中位OS分别为13.1和9.8个月，且ctDNA动态清除率与疗效呈正相关。 EGFR作为CRC治疗的重要靶点，其耐药问题限制了长期受益。但抗EGFR再挑战治疗通过ctDNA动态筛选RAS/BRAF野生型患者，使中位OS延长至13~17个月，较传统三线治疗有所改善。 ctDNA动态监测作为关键预测工具，为传统化疗耐药患者提供了“二次精准打击”的可能。联合免疫治疗或化疗、治疗间隔和联合方案的优化等临床研究成果已推动抗EGFR再挑战治疗进入临床指南，标志着mCRC治疗向精准化、个体化迈出重要一步。但目前临床证据层级不足，缺乏Ⅲ 期大型随机对照临床试验数据，未来需进一步探索耐药克隆清除的最佳联合方案、ctDNA监测的标准化应用，以及更大规模Ⅲ期临床试验验证长期获益。 3 靶向治疗的新突破 3.1  BRAF V600E突变治疗 BRAF V600E突变在mCRC中发生率为5%~10%，其临床特征包括右半结肠原发、黏液腺癌分化、高MSI比例及肝/腹膜转移倾向，与患者预后不良显著相关［24］。虽然中国患者中BRAF V600E突变率仅为2.8%，远低于西方人群的8%~12%。但这类患者的OS在三线治疗中显著较差（4.1个月 vs 8.9个月，P=0.003），仍然值得关注［25］。针对这一群体的治疗策略历经了单药抑制到多通路联合抑制的演变。 2013年，CORRECT试验（NCT01103323）［5］首次证实多激酶抑制剂瑞戈非尼在三线治疗中具有生存获益（OS：6.4个月 vs 5.0个月；HR=0.77），但BRAF突变亚组疗效有限。2015年，SWOG S1406研究（NCT02164916）［26］探索BRAF抑制剂维莫非尼联合伊立替康和西妥昔单抗（VIC方案）的疗效，结果显示，实验组中位PFS为4.2个月，较对照组（2.0个月）显著延长（HR=0.50，P=0.001），但OS差异无统计学意义（9.6个月 vs 5.9个月），研究揭示单药BRAF抑制剂因EGFR信号反馈激活导致快速耐药，提示需联合靶向治疗。2019年BEACON CRC Ⅲ期研究（NCT02928224）首次对比三联（康奈非尼+比美替尼+西妥昔单抗）与双联方案（康奈非尼+西妥昔单抗），2020年更新数据［27］显示，三联组与双联组的中位OS分别为9.3和8.4个月（对照组为5.4个月），ORR分别为27%和20%，亚组分析表明，双联方案安全性更优（≥3级不良反应发生率分别为58%和65%），而三联方案对C反应蛋白升高患者可能更有优势。 2021年的真实世界研究［28］显示，BRAF V600E突变患者三线治疗中50%接受TAS-102，86%接受瑞戈非尼，但联合化疗±抗VEGF治疗可显著延长患者的进展后生存期（6.5个月 vs 4.4个月， HR=0.41，95% CI：0.17~0.96，P=0.04）。2023年KRYSTAL-1研究（NCT04793958）［29］探索KRAS G12C抑制剂阿达格拉西布（adagrasib）联合西妥昔单抗的疗效，虽然不具有BRAF特异性，但为人们认识旁路激活耐药这一现象提供了新的视角（ORR=34.0%，中位OS为15.9个月）。 针对BRAF V600E突变mCRC，多通路联合阻断疗法能够显著延长中位OS至8~9个月。双联方案因疗效与安全性权衡获益而成为临床优选，真实世界数据提示联合化疗±抗VEGF治疗可进一步改善生存。就目前情况而言，获得性耐药和疗效瓶颈（中位OS不足10个月）仍是核心挑战。未来需结合ctDNA动态监测、多组学分析及新型疗法，如抗体药物偶联物（antibody-drug conjugate，ADC），探索序贯治疗策略，以实现精准长期控制。 3.2  ERBB2扩增/高表达治疗 ERBB2（HER2）扩增/高表达在所有CRC中发生率为2%~3%，但在RAS/BRAF野生型中比例升高（5%~6%），而在RAS突变型CRC中较少见（1%~2%），与抗EGFR治疗耐药相关［30］。针对该靶点的治疗策略从双靶向药物联合逐步拓展至ADC药物，显著改善了患者预后。 2015年HERACLES研究（EudraCT 2012-002128-33）［31］首次证实曲妥珠单抗联合拉帕替尼在HER2阳性mCRC中的疗效，ORR为30%，DCR达74%。2021年DESTINY-CRC 01研究（NCT03384940）［32］进一步评估ADC药物德曲妥珠单抗（trastuzumab deruxtecan，T-DXd）的疗效，在既往多线治疗的HER2阳性患者中， ORR达45.3%，但间质性肺病风险值得警惕。2022年DESTINY-CRC 02研究（NCT04744831）［33］对比T-DXd的不同剂量（5.4 mg/kg vs 6.4  mg/kg），结果显示，5.4 mg/ kg组的ORR为37.8%，且安全性更优（≥3级不良反应发生率较低），奠定了其作为标准剂量的地位。2023年图卡替尼（tucatinib）联合曲妥珠单抗的MOUNTAINEER研究（NCT03043313）［34］显示，ORR为38.1%，中位PFS为8.2个月，中位OS为24.1个月，耐受性良好。同期DRUP研究（NCT02925234）［35］表明，曲妥珠单抗（trastuzumab）联合帕妥珠单抗（pertuzumab）的ORR为29%，中位PFS为4.3个月，提示双抗联合的潜在价值。 目前，HER2靶向治疗已从传统双靶向联合（ORR=30%）发展至高效ADC药物（ORR=45.3%）及跨线联合策略（OS为24.1个月）。然而，亚洲人群的分子特征可能影响三线治疗应答。日本一项研究（n=335）［25］发现，亚洲患者中HER2扩增率（2%）低于欧美人群（4%~6%），这可能解释了抗HER2靶向治疗在亚洲人群临床试验中响应率较低的现象。此外，临床仍面临HER2检测标准不统一、耐药机制复杂（如HER2扩增异质性、旁路信号激活）［36］及ADC药物安全性管理（如间质性肺病）［31］的临床挑战。未来需通过动态生物标志物（如ctDNA）监测和新型联合方案及精准剂量优化，进一步提升疗效并降低风险。 3.3  KRAS G12C抑制剂 CRC中KRAS突变的发生率约40%，约85%的KRAS突变发生在3个主要热点密码子（密码子12、13和61）。其中，密码子12突变占主导地位，约占所有KRAS等位基因的65%，其突变与患者不良预后相关［37］。早期研究［38］显示，KRAS G12C突变的不可逆抑制剂单药疗效有限，其耐药机制主要是EGFR信号的反馈激活。 2021年，CodeBreaK100研究（NCT03600 883）首次报道KRAS G12C抑制剂Sotorasib的疗效数据，该Ⅱ期临床试验［39］纳入62例接受过至少两线治疗的KRAS G12C突变mCRC患者，结果显示，ORR为9.7%，中位PFS和OS分别为4.0和10.6个月，虽未达到预设终点，但验证了靶向KRAS G12C的可行性。同年，adagrasib的Ⅰ/ⅠB期研究（NCT03785249）［40］显示，在KRAS G12C突变实体瘤患者中，600 mg每天2次剂量组的CRC亚组部分缓解率为17%，提示单药活性。2022年，KRYSTAL-1研究（NCT04793958）［29］探索adagrasib联合西妥昔单抗的疗效，在32例经多线治疗的KRAS G12C突变mCRC患者中，联合治疗的ORR提升至34.0%，中位PFS和OS分别达6.9和15.9个月，较单药组改善，且耐受性良好（≥3级不良反应发生率为56%）。同年，一项机制研究［41］证实，EGFR抑制剂可阻断KRAS G12C抑制后的ERK信号再激活，为联合治疗提供了理论支持。2023年，Sotorasib联合帕尼单抗的Ⅰb期研究（NCT04185883）［42］进一步验证双靶点抑制策略。在KRAS G12C突变mCRC患者中，联合组的ORR为30%，中位PFS为5.7个月，较单药组（ORR=9.7%）疗效改善。同年，CodeBreaK300研究（NCT05198934）［43］对比Sotorasib联合化疗与标准化疗的效果，初步数据显示，联合化疗组的中位PFS为5.6个月，标准化疗组为2.2个月（HR=0.58），证实联合策略具有临床价值。 KRAS G12C抑制剂联合EGFR单抗（如adagrasib+西妥昔单抗）将ORR提升至34%，中位OS延长至15.9个月，显著优于历史数据。然而KRAS G12C突变检测阈值、克隆性扩增评估缺乏共识，且现有研究多为Ⅰ/Ⅱ期，样本量小，长期生存证据不足。 4 免疫治疗的突破和局限 与靶向治疗聚焦于驱动基因不同，免疫治疗在mCRC中的疗效高度依赖于分子分型。根据微卫星状态和错配修复功能，分为MSI-H/dMMR和MSS/pMMR两种重要亚型，两者在免疫微环境特征、治疗响应及预后方面存在显著差异。根据2024 CSCO指南［44］推荐，基于分子分型、基因状态及既往治疗史，制定了结直肠癌三线个体化治疗的分层流程（图1）。 图1  个体化治疗分层流程图：基于分子分型、基因状态及既往治疗史的mCRC三线个体化治疗的流程分层 Fig. 1 Stratification flowchart for individualized treatment: a stratification flowchart for individualized third-line treatment of mCRC based on molecular subtypes, gene status, and treatment history 4.1  MSI-H/dMMR型的免疫治疗优势 MSI-H/dMMR型患者约占mCRC的5%［45］，其高肿瘤突变负荷（tumor mutation burden，TMB）和免疫细胞浸润特征使其对ICI高度敏感，所以针对该型肿瘤患者多采用单药ICI或双免疫联合的治疗方式。近年来，多项研究验证了ICI在该人群中的突破性疗效。 免疫单药治疗研究中，2019年的KEYNOTE-164研究（NCT02460198）首次证实帕博利珠单抗单药治疗MSI-H/dMMR型mCRC的临床价值，该Ⅱ期试验［46］纳入124例既往接受大于等于二线治疗的患者，ORR为33%，中位PFS和OS分别为2.3和31.4个月，部分患者实现长期生存（5年OS率超40%）。2021年，皮下注射PD-L1抑制剂恩沃利单抗（envafolimab）的 Ⅱ期研究（NCT03667170）［47］显示，在65例MSI-H/dMMR mCRC患者中，ORR为43.1%，中位PFS未达到，12个月PFS率为64.8%，为给药方式优化提供了新选择。2023年，多项研究拓展了免疫治疗的边界，包括GARNET研究（NCT02715284）［48］：多塔利单抗治疗115例dMMR mCRC患者，ORR为43.5%，中位缓解持续时间未达到，2年OS率为63%。另一项研究（NCT03704246）［49］采用国产PD-1抑制剂普特利单抗（pucotenlimab）治疗71例患者，ORR达56.3%，中位PFS未达到，2年OS率为73.5%，且KMT2D突变和高TMB可预测疗效。除此之外，靶向治疗联合免疫治疗也进行了积极探索，2021年LEAP-017 Ⅲ期研究［50］显示，仑伐替尼（多激酶抑制剂）联合帕博利珠单抗（PD-1抑制剂）在pMMR或MSI-H的mCRC患者中，较标准治疗未显著提升OS（9.8个月 vs 9.3个月），但PFS和ORR有所改善。 双免疫联合应用在该类人群中具有较大临床获益，2021年，CheckMate 142研究（NCT02060188）［51］更新数据进一步支持双免疫联合策略，纳武利尤单抗联合伊匹木单抗治疗82例患者，ORR达65%，中位PFS未达到，4年OS率为71%，且安全性可控（3~4级不良反应发生率为32%），成为首个获批的双免疫治疗方案。CheckMate 142长期随访（2023更新）［52］显示，纳武利尤单抗联合瑞拉利单抗（淋巴细胞活化基因3抑制剂）治疗MSI-H/dMMR患者，中位PFS延长至27.5个月，3年OS率为56%，再次证实双免疫靶点的协同效应。 4.2  MSS型肿瘤的免疫治疗困境和策略 MSS/pMMR型CRC约占85%，因存在抑制剂免疫微环境，如低TMB和免疫抑制性细胞富集，ICI对大多数（约85%）患者无效［53］。在双免疫治疗策略探索中，2019年的Ⅱ期CO.26试验（NCT02870920）［54］首次评估双免疫联合治疗（度伐利尤单抗+曲美木单抗）在MSS型mCRC中的疗效，尽管未显著改善PFS，但治疗组OS较对照组延长（6.6个月 vs 4.1个月，HR=0.66，P=0.07），提示双免疫联合的潜在价值。2023年，C-800-01研究（NCT03860272）［55］评估博滕西利单抗+巴尔西利单抗的疗效，在41例难治性MSS型mCRC患者中，ORR为17%，中位PFS为3.7个月，且PD-L1肿瘤免疫评分≥1的患者疗效更优（ORR=25%），支持生物标志物指导的分层治疗。 近年来，免疫检查点抑制剂与靶向药物联合的治疗策略通过协同调控肿瘤微环境与信号转导通路，成为突破MSS/pMMR患者免疫治疗困境的研究焦点。2020年，抗PD-L1抑制剂联合MEK抑制剂（德瓦鲁单抗+Trametinib）的Ⅱ期研究（NCT03428126）［56］显示疗效有限，仅1例部分缓解（ORR=3%），证实单通路抑制难以逆转抑制性免疫微环境。2021年，CAMILLA研究（NCT03539822）［57］探索卡博替尼（多激酶抑制剂）联合德瓦鲁单抗的疗效，29例MSS型mCRC患者中，ORR为27.6%，中位PFS和OS分别为3.7和9.1个月，且RAS野生型患者获益更好（ORR=50%，中位OS为21.5个月），提示抗血管生成联合免疫治疗可能改善肿瘤免疫浸润。该研究的探索性分析进一步揭示了联合治疗反应相关的转录组特征，包括VEGF和原癌基因MET相关信号通路的上调、细胞黏附相关基因表达增加以及细胞外基质活性增强等特征［57］。中国学者在MSS型mCRC免疫联合治疗领域贡献卓著，2024年，中国CAPability-01研究（NCT04724239）［58］首次采用信迪利单抗（抗PD-1抑制剂）+西达本胺（HDAC抑制剂）+贝伐珠单抗三联方案，18周PFS率达40.5%，RNA测序显示，CD8+ T细胞浸润增加，证实表观遗传调控联合抗血管生成治疗存在协同效应，为MSS/pMMR型患者带来新的希望。同年，CEA-CD3双抗（cibisatamab）联合阿替利珠单抗的Ⅰ期研究（NCT02650713）［59］显示，在MSS型患者中， ORR达27.6%，中位PFS为5.2个月，证明了双特异性抗体重塑肿瘤微环境的能力。除此之外， pixatimod（TLR9激动剂）联合纳武利尤单抗的 Ⅰb期研究（NCT05061017）［60］显示，25例MSS型mCRC患者中，ORR为12%，DCR达44%，且免疫炎症标志物（如IP-10）与疗效相关，为“冷肿瘤”转化提供了新思路。新型药物中，伊沃西单抗（ivonescimab）是首个同时靶向PD-1和VEGF-A的人源化四价双特异性抗体，其Ⅰ期研究［61］显示，MSS型患者ORR为11.1%，提示双特异性抗体单药治疗的潜在优势。正在进行的研究也值得关注，SHR-8068（CTLA-4抑制剂）联合阿得贝利单抗（PD-L1抑制剂）及贝伐珠单抗的Ⅰb/Ⅱ期研究（NCT06373133）［62］旨在探索难治性MSS型mCRC的疗效，初步数据显示， DCR超50%。该研究尚未完成，但可能为双免疫治疗联合抗血管生成药物在MSS型CRC（无论有无肝转移）中的临床应用提供有力证据。 总结以上免疫治疗相关研究发现，对于MSI-H/dMMR型患者，ICI单药或双免疫联合治疗（如PD-1/CTLA-4或PD-1/LAG-3抑制剂）展现出较好的临床获益，ORR达30%~65%，部分患者实现长期生存（3年OS率＞50%）。然而，长期生存数据（如超5年生存率）并不完善，关于老年及肝转移等特殊人群的疗效和安全性的证据不足。MSS型mCRC的免疫治疗通过联合靶向（如抗血管生成药物）、表观遗传学调控（HDAC抑制剂）及双免疫策略，将ORR提升至15%~30%，但较MSI-H型患者生存结局仍存在显著差距。针对MSS型患者的临床研究目前仍存在疗效瓶颈，多数联合方案ORR＜30%，中位OS多低于12个月；且生物标志物缺乏，现有标志物（如PD-L1免疫治疗评分、TMB）的预测价值有限，且检测标准不统一；临床试验证据薄弱，关键研究多为 Ⅰ/Ⅱ期，缺乏Ⅲ期研究证据支持。除此之外，联合治疗的安全性存在挑战，双免疫或免疫-靶向联合治疗导致3级以上不良反应发生率升高，限制了临床适用性。未来需解析其耐药机制、开发新型生物标志物（如肠道菌群特征、POLE/POLD1突变）及优化联合策略，以突破“冷肿瘤”转化困境，推动精准治疗。 5 新兴治疗方向 随着人工智能领域研究的不断进展，计算数据整合与合成有望预测靶向治疗的效果和患者预后［63］。一项回顾性研究［64］使用来自癌症基因组图谱结直肠癌（TCGA-CRC-DX）队列中502例患者的499份原发性CRC诊断图像，开发并验证了一种基于深度学习的CRC分子通路预测算法，利用常规HE染色切片的数字化图像来预测关键分子特征和突变状态。该算法在预测MSI-H、染色体不稳定性、BRAF突变及TP53突变方面的表现优于现有方法，可为患者分层提供依据，以便选择靶向药物。 已有研究表明，特定肠道微生物群及其代谢物与CRC患者预后存在关联，可以作为新型的预后相关生物标志物。CAVE试验的后续研究［20，65］中，将应答者和非应答者的粪便样本进行比较，发现产生丁酸的细菌、Agathobacter M104/1和Blautia SR1/5在应答者的粪便样本中富集，并且与生存结果改善相关。除此之外肠道微生物还能改善CRC的免疫治疗效果，已有临床前和临床研究［66-69］确定了肠道菌群在ICI反应中不可或缺的作用。粪便微生物群移植（faecal microbiota transplantation，FMT）是早期的肠道菌群调节策略之一［70］，其与ICI联合应用已在其他癌种中进行了临床试验［71-72］，并被证明可以克服对PD-1抑制剂的耐药性。目前正在对CRC患者进行类似疗法的Ⅰ期临床研究，例如，NCT04729322研究旨在探索FMT联合PD-1抑制剂对经免疫治疗无应答的mCRC患者的效果， NCT04130763研究旨在通过FMT胶囊提高对免疫治疗耐药或难治性的消化系统（食管、胃和肠）癌症患者对PD-1抑制剂的应答率。尽管我们目前对肠道微生物群在mCRC中的作用了解有限，但多项研究结果表明其在临床转化方面存在很大提升空间。 嵌合抗原受体T（ chimeric antigen receptor-T，CAR-T）细胞疗法，最初在血液系统恶性肿瘤治疗中取得成功，近年来在mCRC的治疗领域正进行积极探索。CYAD-101靶向自然杀伤细胞2族成员D（NKG2D）是首个未经基因编辑的同种异体CAR-NK细胞产品， AlloSHRINK Ⅰ期研究（NCT03692429）［73］纳入15例不可切除的mCRC患者，观察到2例部分缓解（含1例KRAS突变患者）、9例疾病稳定（7例持续≥3个月），中位PFS达3.9个月，证实了CYAD-101在晚期mCRC患者中的安全性与初步抗肿瘤活性。一项非随机单臂Ⅰ期临床试验（ChiCTR2000040645）［74］探讨了GCC19 CAR-T细胞（同时靶向CD19和瓜氨酸环化酶C的CAR-T细胞产品）在复发、难治性mCRC患者中的安全性及疗效，结果表明，40%的患者实现部分缓解，中位OS达22.8个月，为mCRC患者的治疗带来了新的希望。除此之外，针对不同靶点的多项临床研究正在进行，其结果值得期待，包括：NCT02349724（CEA）；ChiCTR2100044831、NCT05287165、NCT05319314（鸟苷酸环化酶C）；NCT05759728（LGR5）；NCT05028933 （上皮细胞黏附分子）；NCT06055439（钙粘蛋白17）；联合ICI的研究NCT04991948（NKG2D+帕博利珠单抗）。虽然免疫抑制性微环境、抗原异质性和物理屏障等限制了CAR-T细胞疗法的临床转化，但它代表了CRC治疗中一种变革性的疗法，为靶向治疗和个性化治疗提供了新选择。 6 挑战与未来方向 尽管mCRC三线治疗领域已取得显著进展，但仍面临诸多关键挑战，以下从耐药机制、生物标志物探索以及新型和联合疗法优化3个方面探讨未来突破方向。 6.1  耐药机制解析与应对策略 耐药性仍是靶向治疗和免疫治疗的核心限制因素。例如，抗EGFR再挑战治疗中继发性RAS/BRAF的高突变率［8］、KRAS G12C抑制剂联合抗EGFR抑制剂后发生旁路信号激活导致的耐药［26，38-39］以及MSS型肠癌免疫抑制微环境的困境。未来需结合单细胞测序、空间转录组学及类器官模型，动态追踪耐药克隆的时空异质性，探索针对耐药通路的多重和序贯抑制策略。此外，物的联合应用可能通过重塑肿瘤表观遗传修饰增强疗效［55］，需要更多的研究支持其临床转化。 6.2  生物标志物检测的标准化与动态监测体系优化 当前生物标志物（如HER2扩增阈值、ctDNA清除率）的检测标准尚未统一，且动态监测的临床应用缺乏共识。例如，HERACLES［28］和DESTINY-CRC系列研究［29-30］采用不同免疫组织化学/荧光原位杂交技术标准筛选患者，导致疗效差异显著。未来需建立全球多中心协作平台，制定分子分型和免疫微环境特征的标准化检测流程和阈值，并结合ctDNA检测技术实现治疗反应的实时监测。此外，人工智能可以整合多维度数据并驱动临床决策，肠道菌群可进一步分层临床患者亚型从而实现精准药物选择并预测耐药风险和患者预后，推动mCRC个体治疗精准化。  6.3  联合治疗策略优化和新型治疗模式临床转化 多靶点抑制和靶免联合治疗模式的临床研究结果不一致，且≥3级不良反应发生率较高。未来需要通过计量爬坡研究平衡疗效和不良反应。此外，多学科协作（如放疗联合免疫治疗）可能通过局部微环境调控增强全身抗肿瘤效应，需要更多机制研究的支持。持续新型疗法的开发，ADC、双特异性抗体及CAR-T疗法等新兴技术虽在早期研究中显示出潜力，但临床转化受限，需要大型Ⅲ 期临床研究进一步验证其疗效和安全性。 7 结  论 目前临床指南和临床研究的成果使得mCRC三线治疗格局发生重大改变，显著改善了患者生存结局。对于存在BRAF V600E突变、HER2扩增及KRAS G12C突变等特征性改变的患者群体，靶向治疗成为首要选择，而肿瘤无靶向性改变的患者，瑞戈非尼、呋喹替尼及TAS-102联合贝伐珠单抗在三线治疗中凸显重要价值。经过ctDNA筛选的患者考虑抗EGFR再挑战治疗。对于目前仍存在的争议点，需要通过现有药物及组合的优化、用药顺序的确定和探索安全剂量等方式解决。同时需开展临床前研究探索可以预测免疫检查点抑制剂疗效的生物标志物，提高其在MSS/pMMR型患者中的临床疗效。改善mCRC三线治疗格局对提升患者生存率、生活质量和减轻疾病负担意义重大，相关研究仍存在提升和改善的空间。 第一作者： 　　刘婧禹，博士。 通信作者： 　　湛先保，博士，主任医师，肿瘤科科主任。 作者贡献声明： 　　刘婧禹：文章撰写及修改；尹桐，吴玥：文章技术指导及编译；彭小波：文章修改及审核；湛先保：文章指导及决策。 ［参考文献］ ［1］ DEKKER E, TANIS P J, VLEUGELS J L A, et al. Colorectal cancer［J］. Lancet, 2019, 394(10207): 1467-1480. ［2］ YOSHINO T, CERVANTES A, BANDO H, et al. Pan-Asian adapted ESMO clinical practice guidelines for the diagnosis, treatment and follow-up of patients with metastatic colorectal cancer［J］. ESMO open, 2023, 8(3): 101558. ［3］ SAINI K S, TWELVES C. Determining lines of therapy in patients with solid cancers: a proposed new systematic and comprehensive framework［J］. Br J Cancer, 2021, 125(2): 155-163. ［4］ DENG T, DUAN J J, BAI M, et al. Third-line treatment patterns and clinical outcomes for metastatic colorectal cancer: a retrospective real-world study［J］. Ther Adv Chronic Dis, 2023, 14: 20406223231197311. ［5］ GROTHEY A, VAN CUTSEM E, SOBRERO A, et al. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial［J］. Lancet, 2013, 381(9863): 303-312. ［6］ M A Y E R R J , V A N C U T S E M E , F A L C O N E A , e t a l . Randomized trial of TAS-102 for refractory metastatic colorectal cancer［J］. N Engl J Med, 2015, 372(20): 1909-1919. ［7］ LI J, QIN S K, XU R H, et al. Effect of fruquintinib vs placebo on overall survival in patients with previously treated metastatic colorectal cancer: the FRESCO randomized clinical trial［J］. JAMA, 2018, 319(24): 2486-2496. ［8］ DASARI A, LONARDI S, GARCIA-CARBONERO R, et al. Fruquintinib versus placebo in patients with refractory metastatic colorectal cancer (FRESCO-2): an international, multicentre, randomised, double-blind, phase 3 study［J］. Lancet, 2023, 402(10395): 41-53. ［9］ NAPOLITANO S, MARTINI G, CIARDIELLO D, et al. Targeting the EGFR signalling pathway in metastatic colorectal cancer［J］. Lancet Gastroenterol Hepatol, 2024, 9(7): 664-676. ［10］ CIARDIELLO F, TORTORA G. EGFR antagonists in cancer treatment［J］. N Engl J Med, 2008, 358(11): 1160-1174. ［11］ KARAPETIS C S, KHAMBATA-FORD S, JONKER D J, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer［J］. N Engl J Med, 2008, 359(17): 1757-1765. ［12］ GROTHEY A, FAKIH M, TABERNERO J. Management of BRAF-mutant metastatic colorectal cancer: a review of treatment options and evidence-based guidelines［J］. Ann Oncol, 2021, 32(8): 959-967. ［13］ B A R R A S D , M I S S I A G L I A E , WI R A P A T I P , e t a l . BRAF V600E mutant colorectal cancer subtypes based on gene expression［J］. Clin Cancer Res, 2017, 23(1): 104-115. ［14］ DI NICOLANTONIO F, MARTINI M, MOLINARI F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer［J］. J Clin Oncol, 2008, 26(35): 5705-5712. ［15］ MARTIN V, LANDI L, MOLINARI F, et al. HER2 gene copy number status may influence clinical efficacy to anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients［J］. Br J Cancer, 2013, 108(3): 668-675. ［16］ DAY F L, JORISSEN R N, LIPTON L, et al. PIK3CA and PTEN gene and exon mutation-specific clinicopathologic and molecular associations in colorectal cancer［J］. Clin Cancer Res, 2013, 19(12): 3285-3296. ［17］ MISALE S, DI NICOLANTONIO F, SARTORE-BIANCHI A, et al. Resistance to anti-EGFR therapy in colorectal cancer: from heterogeneity to convergent evolution［J］. Cancer Discov, 2014, 4(11): 1269-1280. ［18］ PARSEGHIAN C M, LOREE J M, MORRIS V K, et al. Anti-EGFR-resistant clones decay exponentially after progression: implications for anti-EGFR re-challenge［J］. Ann Oncol, 2019, 30(2): 243-249. ［19］ SHEN L, LI Q, WANG W, et al. Treatment patterns and direct medical costs of metastatic colorectal cancer patients: a retrospective study of electronic medical records from urban China［J］. J Med Econ, 2020, 23(5): 456-463. ［20］ MARTINELLI E, MARTINI G, FAMIGLIETTI V, et al. Cetuximab rechallenge plus avelumab in pretreated patients with RAS wild-type metastatic colorectal cancer: the phase 2 single-arm clinical CAVE trial［J］. JAMA Oncol, 2021, 7(10): 1529-1535. ［21］ NAPOLITANO S, DE FALCO V, MARTINI G, et al. Panitumumab plus trifluridine-tipiracil as anti-epidermal growth factor receptor rechallenge therapy for refractory RAS wild-type metastatic colorectal cancer: a phase 2 randomized clinical trial［J］. JAMA Oncol, 2023, 9(7): 966-970. ［22］ QUAN M, CHEN J D, CHEN Z Q, et al. China special issue on gastrointestinal tumors-Cetuximab retreatment plus camrelizumab and liposomal irinotecan in patients with RAS wild-type metastatic colorectal cancer: cohort B of the phase Ⅱ CRACK study［J］. Int J Cancer, 2023, 153(11): 1877-1884. ［23］ MONTAGUT C. Circulating tumor (ct) DNA-guided anti-EGFR rechallenge strategy in metastatic colorectal cancer (mCRC): final results of the phase Ⅱ randomized CITRIC trial［C］. ESMO Congress: Barcelona, 2024: abstract LBA33. ［24］ BILLER L H, SCHRAG D. Diagnosis and treatment of metastatic colorectal cancer: a review［J］. JAMA, 2021, 325(7): 669-685. ［25］ PHAM H, DIXON E. Integration of next-generation sequencing in the surgical management of colorectal liver metastasis［J］. Ann Surg Oncol, 2023, 30(11): 6815-6823. ［26］ KOPETZ S, GUTHRIE K A, MORRIS V K, et al. Randomized trial of irinotecan and cetuximab with or without vemurafenib in BRAF-mutant metastatic colorectal cancer (SWOG S1406)［J］. J Clin Oncol, 2021, 39(4): 285-294. ［27］ TABERNERO J, GROTHEY A, VAN CUTSEM E, et al. Encorafenib plus cetuximab as a new standard of care for previously treated BRAF V600E-mutant metastatic colorectal cancer: updated survival results and subgroup analyses from the BEACON study［J］. J Clin Oncol, 2021, 39(4): 273-284. ［28］ GERMANI M M, VETERE G, SANTAMARIA F, et al. Treatment of patients with BRAF V600E-mutated metastatic colorectal cancer after progression to encorafenib and cetuximab: data from a real-world nationwide dataset［J］. ESMO Open, 2024, 9(4): 102996. ［29］ YAEGER R, UBOHA N V, PELSTER M S, et al. Efficacy and safety of adagrasib plus cetuximab in patients with KRAS G12C-mutated metastatic colorectal cancer［J］. Cancer Discov, 2024, 14(6): 982-993. ［30］ RAGHAV K P S, MOASSER M M. Molecular pathways and mechanisms of HER2 in cancer therapy［J］. Clin Cancer Res, 2023, 29(13): 2351-2361. ［31］ SARTORE-BIANCHI A, TRUSOLINO L, MARTINO C, et al. Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2- positive metastatic colorectal cancer (HERACLES): a proof ofconcept, multicentre, open-label, phase 2 trial［J］. Lancet Oncol, 2016, 17(6): 738-746. ［32］ SIENA S, DI BARTOLOMEO M, RAGHAV K, et al. Trastuzumab deruxtecan (DS-8201) in patients with HER2- expressing metastatic colorectal cancer (DESTINY-CRC01): a multicentre, open-label, phase 2 trial［J］. Lancet Oncol, 2021, 22(6): 779-789. ［33］ RAGHAV K, SIENA S, TAKASHIMA A, et al. Trastuzumab deruxtecan in patients with HER2-positive advanced colorectal cancer (DESTINY-CRC02): primary results from a multicentre, randomised, phase 2 trial［J］. Lancet Oncol, 2024, 25(9): 1147-1162. ［34］ STRICKLER J H, CERCEK A, SIENA S, et al. Tucatinib plus trastuzumab for chemotherapy-refractory, HER2 positive, RAS wild-type unresectable or metastatic colorectal cancer (MOUNTAINEER): a multicentre, open-label, phase 2 study［J］. Lancet Oncol, 2023, 24(5): 496-508. ［35］ SPIEKMAN I A C, ZEVERIJN L J, GEURTS B S, et al. Trastuzumab plus pertuzumab for HER2-amplified advanced colorectal cancer: results from the drug rediscovery protocol (DRUP)［J］. Eur J Cancer, 2024, 202: 113988. ［36］ STRICKLER J H, YOSHINO T, GRAHAM R P, et al. Diagnosis and treatment of ERBB2-positive metastatic colorectal cancer: A review［J］. JAMA Oncol, 2022, 8(5): 760-769. ［37］ ZHU G M, PEI L J, XIA H W, et al. Role of oncogenic KRAS in the prognosis, diagnosis and treatment of colorectal cancer［J］. Mol Cancer, 2021, 20(1): 143. ［38］ CANON J, REX K, SAIKI A Y, et al. The clinical KRAS (G12C) inhibitor AMG 510 drives anti-tumour immunity［J］. Nature, 2019, 575(7781): 217-223. ［39］ FAKIH M G, KOPETZ S, KUBOKI Y, et al. Sotorasib for previously treated colorectal cancers with KRAS G12C mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial［J］. Lancet Oncol, 2022, 23(1): 115-124. ［40］ OU S I, JÄNNE P A, LEAL T A, et al. First-in-human phase Ⅰ/ⅠB dose-finding study of adagrasib (MRTX849) in patients with advanced KRAS G12C solid tumors (KRYSTAL-1)［J］. J Clin Oncol, 2022, 40(23): 2530-2538. ［41］ YAEGER R, MEZZADRA R, SINOPOLI J, et al. Molecular characterization of acquired resistance to KRAS G12C-EGFR inhibition in colorectal cancer［J］. Cancer Discov, 2023, 13(1): 41-55. ［42］ KUBOKI Y, FAKIH M, STRICKLER J, et al. Sotorasib with panitumumab in chemotherapy-refractory KRAS G12C mutated colorectal cancer: a phase 1b trial［J］. Nat Med, 2024, 30(1): 265-270. ［43］ FAKIH M G, SALVATORE L, ESAKI T, et al. Sotorasib plus panitumumab in refractory colorectal cancer with mutated KRAS G12C［J］. N Engl J Med, 2023, 389(23): 2125-2139. ［44］ Chinese Society of Clinical Oncology (CSCO) Colorectal Cancer Expert Committee. CSCO guidelines for the diagnosis and treatment of colorectal cancer 2024［J］. Chin J Oncol, 2024, 30(1): 1-50. ［45］ TAIEB J, SVRCEK M, COHEN R, et al. Deficient mismatch repair/microsatellite unstable colorectal cancer: diagnosis, prognosis and treatment［J］. Eur J Cancer, 2022, 175: 136-157. ［46］ LE D T, KIM T W, VAN CUTSEM E, et al. Phase Ⅱ open-label study of pembrolizumab in treatment-refractory, microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: KEYNOTE-164［J］. J Clin Oncol, 2020, 38(1): 11-19. ［47］ LI J, DENG Y H, ZHANG W J, et al. Subcutaneous envafolimab monotherapy in patients with advanced defective mismatch repair/microsatellite instability high solid tumors［J］. J Hematol Oncol, 2021, 14(1): 95. ［48］ ANDRÉ T, BERTON D, CURIGLIANO G, et al. Antitumor activity and safety of dostarlimab monotherapy in patients with mismatch repair deficient solid tumors: a nonrandomized controlled trial［J］. JAMA Netw Open, 2023, 6(11): e2341165. ［49］ ZHANG B, SONG Y, LUO S X, et al. Pucotenlimab in patients with advanced mismatch repair-deficient or microsatellite instability-high solid tumors: a multicenter phase 2 study［J］. Cell Rep Med, 2023, 4(12): 101301. ［50］ KAWAZOE A, XU R H, GARCÍA-ALFONSO P, et al. Lenvatinib plus pembrolizumab versus standard of care for previously treated metastatic colorectal cancer: final analysis of the randomized, open-label, phase Ⅲ LEAP-017 study［J］. J Clin Oncol, 2024, 42(24): 2918-2927. ［51］ LENZ H J, VAN CUTSEM E, LUISA LIMON M, et al. Firstline nivolumab plus low-dose ipilimumab for microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: the phase Ⅱ CheckMate 142 study［J］. J Clin Oncol, 2022, 40(2): 161-170. ［52］ OVERMAN M J, GELSOMINO F, AGLIETTA M, et al. Nivolumab plus relatlimab in patients with previously treated microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: the phase Ⅱ CheckMate 142 study［J］. J Immunother Cancer, 2024, 12(5): e008689. ［53］ LIZARDO D Y, KUANG C Y, HAO S S, et al. Immunotherapy efficacy on mismatch repair-deficient colorectal cancer: from bench to bedside［J］. Biochim Biophys Acta Rev Cancer, 2020, 1874(2): 188447. ［54］ CHEN E X, JONKER D J, LOREE J M, et al. Effect of combined immune checkpoint inhibition vs best supportive care alone in patients with advanced colorectal cancer: the Canadian cancer trials group CO.26 study［J］. JAMA Oncol, 2020, 6(6): 831-838. ［55］ BULLOCK A J, SCHLECHTER B L, FAKIH M G, et al. Botensilimab plus balstilimab in relapsed/refractory microsatellite stable metastatic colorectal cancer: a phase 1 trial［J］. Nat Med, 2024, 30(9): 2558-2567. ［56］ JOHNSON B, HAYMAKER C L, PARRA E R, et al. Phase Ⅱ study of durvalumab (anti-PD-L1) and trametinib (MEKi) in microsatellite stable (MSS) metastatic colorectal cancer (mCRC)［J］. J Immunother Cancer, 2022, 10(8): e005332. ［57］ SAEED A, PARK R, PATHAK H, et al. Clinical and biomarker results from a phase Ⅱ trial of combined cabozantinib and durvalumab in patients with chemotherapy-refractory colorectal cancer (CRC): CAMILLA CRC cohort［J］. Nat Commun, 2024, 15(1): 1533. ［58］ WANG F, JIN Y, WANG M, et al. Combined anti-PD-1, HDAC inhibitor and anti-VEGF for MSS/pMMR colorectal cancer: a randomized phase 2 trial［J］. Nat Med, 2024, 30(4): 1035-1043. ［59］ SEGAL N H, MELERO I, MORENO V, et al. CEA-CD3 bispecific antibody cibisatamab with or without atezolizumab in patients with CEA-positive solid tumours: results of two multiinstitutional phase 1 trials［J］. Nat Commun, 2024, 15(1): 4091. ［60］ LEMECH C, DREDGE K, BAMPTON D, et al. Phase Ⅰb openlabel, multicenter study of pixatimod, an activator of TLR9, in combination with nivolumab in subjects with microsatellitestable metastatic colorectal cancer, metastatic pancreatic ductal adenocarcinoma and other solid tumors［J］. J Immunother Cancer, 2023, 11(1): e006136. ［61］ FRENTZAS S, AUSTRIA MISLANG A R, LEMECH C, et al. Phase 1a dose escalation study of ivonescimab (AK112/SMT112), an anti-PD-1/VEGF-A bispecific antibody, in patients with advanced solid tumors［J］. J Immunother Cancer, 2024, 12(4): e008037. ［62］ ZHANG P, LI X F, WANG X, et al. SHR-8068 combined with adebrelimab and bevacizumab in the treatment of refractory advanced colorectal cancer: study protocol for a single-arm, phase Ⅰb/Ⅱ study［J］. Front Immunol, 2024, 15: 1450533. ［63］ KANN B H, HOSNY A, AERTS H J W L. Artificial intelligence for clinical oncology［J］. Cancer Cell, 2021, 39(7): 916-927. ［64］ BILAL M, RAZA S E A, AZAM A, et al. Development and validation of a weakly supervised deep learning framework to predict the status of molecular pathways and key mutations in colorectal cancer from routine histology images: a retrospective study［J］. Lancet Digit Health, 2021, 3(12): e763-e772. ［65］ MARTINI G, CIARDIELLO D, DALLIO M, et al. Gut microbiota correlates with antitumor activity in patients with mCRC and NSCLC treated with cetuximab plus avelumab［J］. Int J Cancer, 2022, 151(3): 473-480. ［66］ VÉTIZOU M, PITT J M, DAILLÈRE R, et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota［J］. Science, 2015, 350(6264): 1079-1084. ［67］ GOPALAKRISHNAN V, SPENCER C N, NEZI L, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients［J］. Science, 2018, 359(6371): 97-103. ［68］ ROUTY B, LE CHATELIER E, DEROSA L, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors［J］. Science, 2018, 359(6371): 91-97. ［69］ XU X J, LV J, GUO F, et al. Gut microbiome influences the efficacy of PD-1 antibody immunotherapy on MSS-type colorectal cancer via metabolic pathway［J］. Front Microbiol, 2020, 11: 814. ［70］ WONG C C, YU J. Gut microbiota in colorectal cancer development and therapy［J］. Nat Rev Clin Oncol, 2023, 20(7): 429-452. ［71］ BARUCH E N, YOUNGSTER I, BEN-BETZALEL G, et al. Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients［J］. Science, 2021, 371(6529): 602-609. ［72］ DAVAR D, DZUTSEV A K, MCCULLOCH J A, et al. Fecal microbiota transplant overcomes resistance to anti-PD-1 therapy in melanoma patients［J］. Science, 2021, 371(6529): 595-602. ［73］ MICHAUX A, MAUËN S, BREMAN E, et al. Clinical grade manufacture of CYAD-101, a NKG2D-based, first in class, non-gene-edited allogeneic CAR T-cell therapy［J］. J Immunother, 2022, 45(3): 150-161. ［74］ CHEN N F, PU C F, ZHAO L L, et al. Chimeric antigen receptor T cells targeting CD19 and GCC in metastatic colorectal cancer: a nonrandomized clinical trial［J］. JAMA Oncol, 2024, 10(11): 1532-1536. 编辑：莫颖 审核：李广涛 王琳辉

Mont-Saint-Guibert, Belgium; September 25, 2025, 6:30 pm CET; regulated information – Celyad Oncology (Euronext: CYAD) (the “Company”), today announced its financial results for the first half year 2025 ended June 30, 2025. First Half 2025 financial review As of June 30, 2025, the Company’s Treasury position amounted to €0.8 million. After due consideration of detailed budgets and estimated cash flow forecasts for the years 2025 and 2026, the Company projects that its existing cash and cash equivalents will be sufficient to fund its estimated operating and capital expenditures into the fourth quarter of 2025. Key financial figures for first half 2025, compared with the first half of 2024 and full year 2024, are summarized below: Research and Development (R&D) expenses were €2.1 million in June 2025 as compared to €1.5 million during the same period in 2024, an increase of €0.6 million. The increase in the Company’s R&D expenses was primarily driven by the increase of the IP costs related to the licensing activities as well as the increase of the expenses linked to the redevelopment of the catheter. General and Administrative (G&A) expenses remained stable with €1.7 million in June 2025 as in June 2024. Net loss for the first half of 2025, was €3.7 million, or €(0.09) per share, compared to a net loss of €3.0 million, or €(0.07) per share, for the same period in 2024. Net cash used in operations was €3.3 million for the first half of 2025 compared to €2.8 million for the first half of 2024. The increase of €0.5 million was primarily driven by the global increase of IP activities and catheter development costs. As of June 30, 2025, the Company had cash and cash equivalents of €0.8 million. No capital increase occurred in the first half of 2025. The total number of basic shares outstanding was 41.4 million like in December 31, 2024. The interim financial report for first half 2025 of the Company can be found on our website: https://celyad.com/investors/ Financial Calendar 2025 May 20th 2026 : Annual shareholders meeting September 24th 2026 : First Half 2026 Interim results The financial calendar is communicated on an indicative basis and may be subject to change. The Company continues to bolster its intellectual property estate and to pursue options to monetize it The Company’s two main research and development platforms have delivered proof-of-concept and are ready to be incorporated into clinical candidates and/or to be further explored and exploited via potential partnerships The Company actively participated at several key international scientific conferences and published in well-renowned peer-reviewed journals which have raised the interest for the Company’s technologies and developments Operational expenses according to Budget allowing cash runway until third quarter 2025 Mont-Saint-Guibert, Belgium; September 13, 2024, 7:00 am CET; regulated information – Celyad Oncology (Euronext: CYAD) (the “Company”), today announces its financial results for the first half year 2024 ended June 30, 2024, and provides a business update. Michel Lussier, interim Chief Executive Officer of Celyad Oncology, commented: “Celyad Oncology continues to make remarkable progress in developing cutting-edge technologies for chimeric antigen receptor (CAR) T-cell therapy. Our groundbreaking multiplex platform is revolutionizing the potential of CAR T-cells, while our pioneering NKG2D-based multispecific CAR T-cell platform is further paving the way to conquer current limitations of this transformative class of immunotherapy” The Company is pursuing a strategy of continued research and development, with a particular focus on intellectual property (IP). Monetization of its innovative approaches and technologies is a key objective. Celyad Oncology is progressing in this regard and is currently in discussion with potential partners for out-licensing deals; With its research focus, the Company has made concrete progress by providing proof-of-concept of the multiplex short hairpin RNAs (shRNAs) non-gene edited technology platform and the multispecific NKG2D-based CAR T-cell platform, which provide unique options to tackle the major current limitations of CAR T-cell therapies. Options to further explore or validate these data through strategic partnerships, and/or to incorporate these technologies into clinical CAR-T candidates are actively pursued by the Company; The Company continues to share and discuss its latest advances at international scientific conferences throughout the first half of 2024 with updated results provided at the 27th ASGCT [1] Annual Meeting and the Recent insights into Immuno-Oncology VIB conference [2]. The Company is also focusing on sharing data and views with the scientific community and has published a review highlighting the interest of non-gene editing technologies for allogeneic CAR T-cell therapies in Cells [3] and another review providing an overview of all engineering strategies to safely drive CAR T-cells into the future in Frontiers in Immunology [4], two well-renowned peer-reviewed scientific journals; In response to the request expressed by several companies and academic institutions engaged in gene and cell therapies for cardiac applications, the Company has re-initiated the manufacturing and commercialization of C-Cath®, an intra-myocardial injection catheter developed and owned by the Company. Multiplex shRNA non-gene edited technology – The Company developed a chimeric micro-RNA (miRNA) cluster to enable multiplexing of shRNAs, designed for easy, efficient, and tunable downregulation of up to four target genes simultaneously in CAR T-cells. Data successfully demonstrated the feasibility and effectiveness of the multiplex approach to improve allogeneic CAR T-cell viability by avoiding graft-versus-host disease (GvHD) via knocking down of CD3ζ, avoiding host-versus-graft (HvG) reaction and promoting cell persistence via knocking-down of β2M and CIITA, and avoiding CD95L-induced autophagy via knocking-down of CD95; Another multiplex cassette focusing on the knock-down of co-inhibitory receptors (PD-1, LAG-3, TIM-3 and CD95) was also developed to decrease the expression of exhaustion markers at the surface of CAR T-cells. Multispecific NKG2D-based CAR T-cell platform – Different NKG2D-based multispecific CAR T-cells were developed to provide the proof-of-concept that NKG2D ligands (NKG2DL) are valuable targets in a multispecific CAR approach to counteract relapses due to antigen loss or antigen heterogeneity.PSMA/NKG2DL tandem CAR T-cells, that encompass the extracellular domain of the natural NKG2D receptor fused to an anti-PSMA CAR to overcome antigen heterogeneity and improve anti-tumor efficacy against prostate cancer were developed and demonstrated functionality in vitro against prostate cancer cell lines expressing or not the tumor-associated antigen PSMA; In vivo proof-of-concept of the company’s CD19/NKG2DL tandem CAR T-cell candidate was also provided in a B-ALL relapse model, showing that this multi-specific CAR T-cell candidate has an enhanced anti-tumor efficacy against heterogeneous lymphoma tumors, or to counteract antigen loss, as compared to currently existing treatment options. Data successfully demonstrated the feasibility and effectiveness of the multiplex approach to improve allogeneic CAR T-cell viability by avoiding graft-versus-host disease (GvHD) via knocking down of CD3ζ, avoiding host-versus-graft (HvG) reaction and promoting cell persistence via knocking-down of β2M and CIITA, and avoiding CD95L-induced autophagy via knocking-down of CD95; Another multiplex cassette focusing on the knock-down of co-inhibitory receptors (PD-1, LAG-3, TIM-3 and CD95) was also developed to decrease the expression of exhaustion markers at the surface of CAR T-cells. PSMA/NKG2DL tandem CAR T-cells, that encompass the extracellular domain of the natural NKG2D receptor fused to an anti-PSMA CAR to overcome antigen heterogeneity and improve anti-tumor efficacy against prostate cancer were developed and demonstrated functionality in vitro against prostate cancer cell lines expressing or not the tumor-associated antigen PSMA; In vivo proof-of-concept of the company’s CD19/NKG2DL tandem CAR T-cell candidate was also provided in a B-ALL relapse model, showing that this multi-specific CAR T-cell candidate has an enhanced anti-tumor efficacy against heterogeneous lymphoma tumors, or to counteract antigen loss, as compared to currently existing treatment options. As of June 30, 2024, the Company’s Treasury position amounts to €6.2 million. After due consideration of detailed budgets and estimated cash flow forecasts for the years 2024 and 2025, the Company projects that its existing cash and cash equivalents will be sufficient to fund its estimated operating and capital expenditures into the third quarter of 2025. Key financial figures for first half 2024, compared with the first half of 2023 and full year 2023, are summarized below: Research and Development (R&D) expenses were €1.5 million in June 2024 as compared to €2.1 million during the same period in 2023, a decrease of €0.6 million. The decrease in the Company’s R&D expenses is primarily driven by the Company’s strategic decision in 2022 and beginning of 2023 to discontinue clinical development and prioritization of most promising research programs. General and Administrative (G&A) expenses were €1.7 million in June 2024 as compared to €3.7 million during the same period in 2023, a decrease of €2.0 million. This decrease is mainly related to the decrease in employee expenses related to headcount reduction and management changes to support the Company’s reorganization (notably resulting from Nasdaq delisting and SEC deregistration of the Company) and to a decrease in insurance costs and consulting fees. As of June 30, 2024, Management has determined that there has been no event (such as a firm sublicense or collaboration contract) that led to a change in fair value of the contingent consideration and other financial liabilities. The Company’s other income is mainly associated with grants received and some insurance compensations. Net loss for the first half of 2024, was €3.0 million, or € (0.07) per share, compared to a net loss of €3.7 million, or € (0.17) per share, for the same period in 2023. As noted above, the decrease in net loss between periods was primarily due to the decrease of R&D and General and administrative expenses in 2024 partly compensated by lower amounts of grants revenues from public institutions. Net cash used in operations was €2.8 million for the first half of 2024 compared to €8.3 million for the first half of 2023. The decrease of €5.5 million is primarily driven by the global decrease in preclinical and clinical activities, insurance costs and headcount. In 2023 the deviation between Net cash used in operations and Loss of the period was mainly explained by the change in the working capital (Trade payables and other liabilities decrease). The decrease of these costs is in line with the Company’s decision to adopt and implement over the last few months of the year 2022 the new business strategy to focus on early-stage discovery research in areas of expertise where it can leverage the differentiated nature of its platforms. More data and evidence in the context of the multispecific CAR and shRNA multiplex platforms, with the possibility of a clinical evaluation of assets and initiation of clinical trials either by the Company and/or through strategic partnerships afterwards; Celyad Oncology will attend and present updated data on its programs at the 9th CAR-TCR Summit in Boston, US (Sep. 17-20), the Advanced Therapies Europe in Estoril (Sep. 10-12) and present two posters at the 39th Annual Meeting of the Society for Immunotherapy of Cancer (SITC) in Houston (Nov. 6-10); The Company anticipates the appointment of a new CEO in the second half of 2024. April 2nd 2025 : Full Year 2024 Financial Statements May 5th 2025 : Annual shareholders meeting September 25th 2025 : First Half 2025 Interim results The financial calendar is communicated on an indicative basis and may be subject to change. [1] American Society of Gene & Cell Therapy Annual meeting, Baltimore, US (May 7-11, 2024) [2] Recent insights into Immuno-Oncology VIB (Flemish Institute for Biotechnology) conference, Antwerp (May 30-31, 2024) [3] Cells 2024;13(2):146 [4] Front Immunol. 2024:15:1411393 Georges Rawadi was appointed Chief Executive Officer as from April 27, 2023 Celyad Oncology has received approximately EUR 9.8m in private placement commitments from historical shareholders Encouraging progress in multiplex shRNA platform development, which allows now targeting of up to four genes simultaneously, were presented at international meetings In vitro validation of NKG2D-based multi-specific CAR T-cell platform with a first candidate targeting both NKG2D ligands and CD19was also presented Mont-Saint-Guibert, Belgium; September 4, 2023, 10:00 pm CET; regulated information – Celyad Oncology (Euronext: CYAD) (the “Company” or “Celyad Oncology”), today announces its financial results and recent business developments for the first half year, ended June 30, 2023. “Celyad Oncology is now fully focused on maximizing the potential of its proprietary technology platforms and intellectual property, enabling the Company to be at the forefront of developing next-generation CAR T-cell therapies. We are eager to see the impact of our research efforts on the future of CAR T-cell treatments, with the goal to broaden the range of cancer indications and tackle the main limitations of current CAR T-cell therapies” commented Georges Rawadi, Celyad Oncology’s Chief Executive Officer. First Half 2023 and recent corporate highlights: Georges Rawadi was appointed Chief Executive Officer of the Company as from April 27, 2023. Georges Rawadi is a seasoned executive with over 20 years of experience in pharma/biotech, as research director, business developer, CEO, and board member. He also has insightful knowledge of both the company and the CAR-T space as he spent four years at Celyad Oncology (2014-2018) as Vice-President Business Development & Intellectual Property (“BD & IP”). Georges Rawadi has a genuine passion for seeking and creating new business opportunities. On May 5th, 2023, the Company announced voluntary delisting of its American Depositary Shares representing ordinary shares (“ADSs”) from the Nasdaq Global Market. Delisting was effective as of July 20, 2023. The Company continues to be listed on Euronext Brussels and Euronext Paris. 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; and A 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 end of the fourth quarter of 2024. A first tranche of 2.0 million was disbursed in the context of authorized capital as of September 4, 2023; and A 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 end of the fourth quarter of 2024. First Half 2023 and recent operational highlights: Short hairpin ribonucleic acid (shRNA) non-gene edited technology – During this first half of 2023, we have collected and presented data validating our shRNA multiplexing approach:We developed a micro-RNA (miRNA)-based multiplex shRNA platform designed for easy, efficient, and tunable downregulation of up to four target genes simultaneously; We showed that the downregulation of each target gene could be fine-tuned, from a moderate downregulation up to a functional knock-out, without the need of gene editing thereby avoiding associated potential safety issues; The plug-and-play design of our platform is designed to allow swapping of each target sequence without affecting the performance of the technology and streamlining of the generation of engineered adoptive T-cell therapies; To demonstrate the effectiveness of our approach, we have been able to simultaneous knock-down in CAR T-cells several genes involved in different cellular processes such as alloreactivity (CD3ζ), cell persistence (β2M, CIITA), T-cell exhaustion (PD-1, LAG-3), or ligand-induced apoptosis (CD95); Data were presented at the World Oncology Cell Therapy Congress in Boston, US (April 25-26, 2023) and at the CAR-TCR Summit in Boston, US (August 29 – September 1). NKG2D-based CAR T-cells and multi-specific CAR T-cell platform – During this first half of 2023, we have published data validating our NKG2D-based CAR T-cell approach and presented data from our multi-specific CAR T-cell platform:Results from 16 patients treated in the dose-escalation segment of the hematological arm of the Phase I THINK trial were published in The Lancet Haematology Journal (Lancet Haematol. 2023 Mar;10(3):e191-e202) and provided proof-of-concept for targeting NKG2D ligands (NKG2DL) with CAR T-cell therapy; We have developed different CD19/NKG2DL multi-specific CAR T-cells, utilizing both tandem and dual NKG2D-based CARs that encompass the extracellular domain of the natural NKG2D receptor fused to an anti-CD19 scFv, or co-expressed with an anti-CD19 CAR, respectively; The majority of our CD19/NKG2DL multi-specific CAR T-cell candidates were able to secrete cytokines, proliferate, and eliminate acute lymphoblastic leukemia tumor cells lacking the CD19 antigen in vitro. Interestingly, some of these multi-specific CAR T-cells displayed a better in vitro functionality against wild-type leukemia tumor cells expressing the CD19 antigen as compared to CD19-specific single targeting CAR T-cells, highlighting the potential of our approach against both CD19 positive and CD19 negative cancer cells; First in vivo data suggest that our CD19/NKG2DL multi-specific CAR T-cell candidates have an enhanced anti-tumor efficacy against heterogeneous lymphoma tumors as compared to currently existing treatment options; We are currently developing several NKG2D-based multi-specific CAR T-cells for the treatment of diverse solid cancers where there is a high heterogeneity in antigen expression; Data were presented at the Immuno-Oncology Summit Europe 2023 held in London, UK (June 20-22, 2023). We developed a micro-RNA (miRNA)-based multiplex shRNA platform designed for easy, efficient, and tunable downregulation of up to four target genes simultaneously; We showed that the downregulation of each target gene could be fine-tuned, from a moderate downregulation up to a functional knock-out, without the need of gene editing thereby avoiding associated potential safety issues; The plug-and-play design of our platform is designed to allow swapping of each target sequence without affecting the performance of the technology and streamlining of the generation of engineered adoptive T-cell therapies; To demonstrate the effectiveness of our approach, we have been able to simultaneous knock-down in CAR T-cells several genes involved in different cellular processes such as alloreactivity (CD3ζ), cell persistence (β2M, CIITA), T-cell exhaustion (PD-1, LAG-3), or ligand-induced apoptosis (CD95); Data were presented at the World Oncology Cell Therapy Congress in Boston, US (April 25-26, 2023) and at the CAR-TCR Summit in Boston, US (August 29 – September 1). Results from 16 patients treated in the dose-escalation segment of the hematological arm of the Phase I THINK trial were published in The Lancet Haematology Journal (Lancet Haematol. 2023 Mar;10(3):e191-e202) and provided proof-of-concept for targeting NKG2D ligands (NKG2DL) with CAR T-cell therapy; We have developed different CD19/NKG2DL multi-specific CAR T-cells, utilizing both tandem and dual NKG2D-based CARs that encompass the extracellular domain of the natural NKG2D receptor fused to an anti-CD19 scFv, or co-expressed with an anti-CD19 CAR, respectively; The majority of our CD19/NKG2DL multi-specific CAR T-cell candidates were able to secrete cytokines, proliferate, and eliminate acute lymphoblastic leukemia tumor cells lacking the CD19 antigen in vitro. Interestingly, some of these multi-specific CAR T-cells displayed a better in vitro functionality against wild-type leukemia tumor cells expressing the CD19 antigen as compared to CD19-specific single targeting CAR T-cells, highlighting the potential of our approach against both CD19 positive and CD19 negative cancer cells; First in vivo data suggest that our CD19/NKG2DL multi-specific CAR T-cell candidates have an enhanced anti-tumor efficacy against heterogeneous lymphoma tumors as compared to currently existing treatment options; We are currently developing several NKG2D-based multi-specific CAR T-cells for the treatment of diverse solid cancers where there is a high heterogeneity in antigen expression; Data were presented at the Immuno-Oncology Summit Europe 2023 held in London, UK (June 20-22, 2023). Upcoming anticipated milestones More data and evidence in the context of the multi-specific CAR platform and shRNA multiplexing approach in H2 2023, with the aim of a clinical evaluation of assets and initiation of clinical trials either by the Company and/or through strategic partnerships afterwards; Relocation, in H2 2023, into a new research facility which fits better its current needs after the strategic shift. The Company will remain headquartered at the Axis Parc, Mont-Saint-Guibert, Belgium but with its new business location at Dumont 9. Upcoming Conferences The Company will take part in the 4th International Conference on Lymphocyte Engineering (ICLE) in Munich (September 12-14) and the annual congress of the Society for Immunotherapy of Cancer (SITC) in San Diego (November 1-5), as well at several business conferences in the second half of 2023. First Half 2023 Financial Results Key financial figures for the first half of 2023, compared with the first half of 2022 and full year 2022, are summarized below: The Company’s license and collaboration agreements generated no revenue in the first half of 2023 similar to the first half of 2022. The Research and Development (R&D) expenses have decreased primarily due to the Company’s decision to discontinue some of preclinical programs and manufacturing and clinical study activities after the Company’s decision to adopt and implement a new business strategy. Furthermore, there has been a decrease of employee expenses and related travel costs which is mainly related to headcount reduction through 2022, to support the Group’s reorganization around preclinical and clinical programs, as well as a decrease of the expenses associated with share-based payments (non-cash expenses) related to the warrant plan offered to the Company’s employees, managers and directors. General and Administrative (G&A) expenses were €3.7 million in 2023 as compared to €6.2 million in 2022. This decrease is primarily related to lower insurances costs, the decrease of employee expenses due to headcount reduction and management changes through 2022 to support the Company’s reorganization and the decrease of the expenses associated with the share-based payments (non-cash expenses) related to the warrants plan offered to the Company’s employees, managers and directors. As of June 30, 2023, there was no change in fair value of the contingent consideration and other financial liabilities as Management has determined that there have been no event (such as a firm sublicense or collaboration contract) that increases the probability of the projected future cash outflow due to Celdara Medical, LLC and Dartmouth College, indicating that the probability is remote, similar to December 31, 2022. Regarding the other income/other expenses, the Company recorded €2.1 million in net other income for the first half of 2023 compared to a net other income of €1.6 million for the first half of 2022. The net other income for the first half of 2023 is primarily due to the gain on the sale of certain fixed assets to Cellistic for €1.1 million and grant income from the Walloon Region of €0.8 million. Net loss was €3.7 million, or €(0.17) per share, for the first half of 2023 compared to a net loss of €14.1 million, or €(0.62) per share, for the same period of 2022. Net cash used in operations, was €8.3 million for the first half of 2023 compared to €16.3 million for the first half of 2022. The decrease of €8.0 million is primarily driven by the sale of the manufacturing activities in 2022 combined with global decrease on preclinical and clinical activities, insurance costs, headcount, management changes costs and associated impact on the change in working capital. As of June 30, 2023, the Company had cash and cash equivalents of €5.0 million. No capital increase has occurred in the first half of 2023. As of June 30, 2023, the total number of basic shares outstanding were 22.6 million similar to December 31, 2022. Conference Call and Webcast Details A conference call will be held on Tuesday 5th of September at 1:00 p.m. CET / 7:00 a.m. EDT discuss half year 2023 financial results and provide an update on the Company’s recent changes and upcoming milestones. Participants may access the conference call by dialing +1-877-407-9716 or +1-201-493-6779 (United States, International), +32 (0) 800-73-904 (Belgium Fixed) or +32 (0) 800-73-566 (Belgium Mobile). Participants may ask for instant telephone access to the event via the “Call me” link or attend the conference live webcast. Archived recording will be available in the “Events” section of the Celyad website after the event. Financial Calendar 2023 The financial calendar is communicated on an indicative basis and may be subject to change. Starting in 2023, Celyad Oncology is now entirely focused on its new business strategy with one clear objective: help to overcome the current limitations of CAR-T approaches. It plans to do this via (i) strengthening of its research focus centered around NKG2D, B7-H6 and shRNA platforms; (ii) maximizing the value of its IP estate and (iii) driving innovation through strategic collaborations. Mont-Saint-Guibert, Belgium – Celyad Oncology (Euronext & Nasdaq: CYAD) (the “Company”), a biotechnology company focused on innovative technologies for chimeric antigen receptor (CAR) T-cell therapies, today announces its financial results for the fiscal year 2022 ended December 31, 2022 and provides a business update. Michel Lussier, Interim Chief Executive Officer of Celyad Oncology, said: “2022 was a crossroad year for Celyad Oncology, with important changes and turning points. We strongly believe all those changes, together with our prior accomplishments, significantly strengthened our position. Having dealt with the 2022 challenges, Celyad has now positively reinvented itself as a leaner, more agile organization. We believe that Celyad is well prepared and has the relevant unique assets and know how to create significant shareholder value in the next few years.” Operational highlights Since late 2022, the Company has implemented a differentiated and innovative strategy, increasing its R&D efforts in areas of expertise where it believes it can leverage the differentiated nature of its platform technology and continue to bolster its intellectual property (IP) estate; and The Company will continue to explore options to tackle the major current limitations of CAR T-cell therapies through development of its dual targeting CARs with NKG2D capabilities, B7-H6 targeting immunotherapies and multiplexing approach of short hairpin RNAs (shRNAs). Upcoming Anticipated Milestones The Company will provide an update on its shRNA multiplexing and dual CAR platforms and business development in the first half of 2023; The Company will take part in the World Oncology Cell Therapy Congress in Boston, US (25-26 April 2023), as well as in the Immuno-Oncology summit in London (20-22 June 2023); The Company anticipates the arrival of a new CEO in the first half of 2023; and We anticipate fundraising in the first half of 2023. Full Year 2022 Financial Review As of December 31, 2022, the Company had cash and cash equivalents of €12.4 million ($13.3 million). The Company projects that its existing cash and cash equivalents should be sufficient to fund operating expenses and capital expenditure requirements into the fourth quarter of 2023. After due consideration of detailed budgets and estimated cash flow forecasts for the years 2023 and 2024, the Company projects 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 the financial statements are issued. The Company is currently evaluating different financing options to obtain the required funding to extend the Company’s cash runway beyond 12 months from the date the financial statements are issued. Key financial figures for full-year 2022, compared with full-year 2021, are summarized below: (1) “Treasury position” is an alternative performance measure determined by adding Short-term investments and Cash and cash equivalents from the statement of financial position prepared in accordance with IFRS. The purpose of this measure by Management is to identify the level of cash available internally (excluding external sources of financing) within 12 months. The Company’s license and collaboration agreements generated no revenue in 2022 and in 2021. Research and Development (R&D) expenses were €18.9 million in 2022 as compared to €20.8 million in 2021, a year-over-year decrease of €1.8 million. The decrease in the Company’s R&D expenses is primarily driven by the Company’s decision to discontinue some of preclinical and in process development costs after the Company’s decision to adopt and implement a new business strategy over the last few months of 2022, as well as a decrease of the expenses associated with share-based payments (non-cash expenses) related to the warrant plan offered to our employees and directors. General and Administrative (G&A) expenses were €10.5 million in 2022 as compared to €9.9 million in 2021, an increase of €0.6 million. This increase is primarily related to higher insurances costs and consulting fees partially compensated by the decrease of the expenses associated with the share-based payments (non-cash expenses) related to the warrants plan offered to our employees and directors. The fair value adjustment (€14.7 million) relating to the contingent consideration and other financial liabilities as of December 31, 2022, is mainly driven by the full reversal of the liability. This liability is a result of business combination accounting (IFRS3) which requires the liability to be recorded unless the possibility of any outflow is remote. This impairment comes as a result of the Company’s strategic shift in focus away from clinical development and the early stage nature of the implementation of the Celyad 2.0 strategy, which involves shifting from an organization focused on clinical development to one prioritizing R&D discovery and the monetization of its intellectual property (IP) portfolio through partnerships, collaborations and license agreements. To date, no effective sublicence contract nor collaboration contract has been entered into, and as a result there is uncertainty as to the timing and amount of associated short, medium and long term revenues. Given this uncertainty, and per accounting standards, the Company recognizes a full impairment loss on the remaining value of goodwill, In Process Research and Development, and Horizon Discovery’s shRNA platform, resulting in a non-cash impairment of €35.1 million on a consolidated basis for the financial year ended December 31, 2022. This accounting conclusion, which reflects the Company’s financial situation as of December 31, 2022, does not affect Management’s commitment to continue in its efforts to pursue the potential monetization of the Company’s IP. If and when such a firm sublicense or collaboration contract occurs and hence increases the probability of revenue, the Management will estimate the reversal of the impairment which will be limited so that the carrying amount of the asset does not exceed its recoverable amount along with the remeasurement of the related contingent liability. The Company’s other income is principally associated with grants received from the Walloon Region mainly in the form of recoverable cash advances (RCAs) and R&D tax credit income as well as the gain on sale of the CTMU activities: Net loss for the year ended December 31, 2022 was €40.9 million, or €1.81 per share, compared to a net loss of €26.5 million, or €1.70 per share, for the same period in 2021. As noted above, the increase in net loss between periods was primarily due to the non-cash impairment adjustment on the Oncology intangible assets. Net cash used in operations for the year ended December 31, 2022, which excludes non-cash effects, amounted to €28.0 million, which is in line with net cash used in operations of €26.6 million for the year ended December 31, 2021. The net assets of the Company as of December 31, 2022, on a BE-GAAP non-consolidated basis, have fallen below half of the Company’s capital. As a result, in accordance with Article 7:228 of the Belgian Code for Companies and Associations, the Board of Directors plans to submit for a vote, at its May 5, 2023 shareholders’ meeting, its business plan including a proposal to continue the Company’s activities. The Board of Directors will publish a detailed report regarding this proposal on or around April 3, 2023, together with the convocation with proposed resolutions for the shareholders’ meeting. Annual Report 2022 The Annual Report for the year ended December 31, 2022 will be published on March 23, 2023, and will be available on the Company’s website, www.celyad.com. The Company’s statutory auditor, EY Bedrijfsrevisoren/Réviseurs d’Entreprises BV/SRL (EY), has confirmed that the completed audit has not revealed any material misstatement in the consolidated financial statements. EY also confirmed that the accounting data reported in the press release are consistent, in all material respects, with the consolidated financial statements from which it has been derived. Conference Call and Webcast Details A conference call will be held on Friday, March 24th at 1:00 p.m. CET / 8:00 a.m. EDT to review the financial and operating results for full year 2022. Please dial into the call five to ten minutes prior to start time using the appropriate number below and ask to join the “Celyad Oncology SA call”: United States / International: +1-877-407-9716 or +1-201-493-6779 Belgium: +32 (0) 800-73-904 (Fixed) or +32 (0) 800-73-566 (Mobile) Participants may also access to the live webcast link for instant telephone access to the event. Archived recording will be available in the “Events” section of the Celyad website after the event. Financial Calendar 2023 The financial calendar is communicated on an indicative basis and may be subject to change. Celyad Oncology has implemented a strategic shift from an organization focused on clinical development to one fully harnessing the true potential of its proprietary technology platforms and intellectual property The company is now prioritizing internal discovery endeavors to tackle the major current limitations of CAR T-cell therapies As of December 31, 2022, the Company ended the year with an unaudited treasury position of €12.4 million ($13.3 million) Mont-Saint-Guibert, Belgium – Celyad Oncology (Euronext & Nasdaq: CYAD) (the “Company”), a biotechnology company focused on the discovery and development of innovative technologies for chimeric antigen receptor (CAR) T-cell therapies, today provides a fourth quarter 2022 business update and an outlook for 2023. Michel Lussier, interim Chief Executive Officer of Celyad Oncology, said: “The second half of 2022 has been a pivotal time for the Company as we have engaged in a new Celyad 2.0 strategy to leverage our innovative technologies and R&D platforms and focus on IP partnering transactions. We’ve stretched our cash runway by divesting our manufacturing business unit, and discontinued our clinical programs to focus on selected, critical R&D efforts to mitigate the current limitations of CAR T-cell therapy. We believe we are now well-positioned to unleash the power of our IP estate and to help making the cell therapy approach a success.” Operational highlights The Company announced a strategic shift in October 2022 to prioritize discovery research in areas of expertise where it can leverage the differentiated nature of its platforms. The Company has implemented a differentiated and innovative strategy, which it believes has the potential to tackle the major current limitations of CAR T-cell therapies. This strategy includes a multiplexing approach of the short hairpin RNA (shRNA) platform, a dual CAR development of a next-generation NKG2D-based CAR, and the development of B7-H6-targeting immunotherapies. In October 2022, the Company decided to discontinue the development of CYAD-101, the allogeneic TIM-based, NKG2D-based CAR T-cell candidate for metastatic colorectal cancer (mCRC), based on a strategic, financial and medical review, taking into account the costs associated with the pursuit of the program. There were no new safety concerns leading to this decision. The clinical hold announced in March 2022 on the CYAD-101-002 Phase 1b trial had been lifted in July 2022 by the FDA. Data collected in the IMMUNICY-1 trial of the clinical program CYAD-211, the allogeneic shRNA-based, anti-BCMA CAR T candidate for relapsed or refractory multiple myeloma (r/r MM), which was developed to validate shRNA technology in the clinic, have shown a favorable safety profile for CYAD-211 across all dose-levels and cohorts, with 19 patients treated in total. The lack of observed graft-versus-host disease (GvHD) despite engraftment of CYAD-211 provided proof-of-concept for the use of shRNA as a technology to control GvHD of allogeneic CAR T-cells. In December 2022, the Company decided to discontinue the development of its remaining clinical program CYAD-211 based on a strategic and financial review. There were no safety concerns leading to this decision and all patients previously treated with CYAD-211 still continue to receive their protocol-defined follow-up. Corporate highlights In September 2022, the Company entered into a €6 million asset purchase agreement with Cellistic, the cell therapy development and manufacturing business of Ncardia BV, whereby Cellistic acquired Celyad Oncology’s Good Manufacturing Practice (GMP) grade cell therapy manufacturing business unit. Since October 2022, the Company has implemented a strategic shift from an organization focused on clinical development to one prioritizing R&D discovery and leveraging its IP estate through partnerships, collaborations and license agreements. The Company has compiled a foundational and broad IP estate that controls key aspects of developing therapies in the allogeneic cell therapy space. The patents around allogeneic CAR T-cell therapies and NKG2D-based therapies provide an avenue to develop intellectual property programs and to partner with outside parties around the licensing of these patents. Financial highlights As of December 31, 2022, the Company had cash and cash equivalents of €12.4 million ($13.3 million). Net cash burn during the fourth quarter of 2022 amounted to €1.0 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 fourth quarter of 2023. After due consideration of detailed budgets and estimated cash flow forecasts for the year 2023, which reflect the new strategy of the Company and include expenses and cash outflows estimations in relation to the development of its proprietary technology platforms and intellectual property, 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. Outlook for 2023 Celyad Oncology is increasing its R&D efforts on areas of expertise where it believes it can leverage the differentiated nature of its platform technology and continue to bolster its IP estate. The Company will continue to leverage the dynamic potential of the shRNA platform, and to explore options to tackle the major current limitations of CAR T-cell therapies through its dual targeting CARs with NKG2D capabilities and B7-H6 targeting cell therapies. The Company will provide updates on the potential proof-of-concept of the dual CAR and multiplexing research programs and on business development in the course of 2023 and will take part in several conferences to share these data. Financial Calendar 2023 The financial calendar is communicated on an indicative basis and may be subject to change. Company continues to transition business focus to monetizing unique cell therapy intellectual property and prioritizing R&D discovery Clinical updates expected by end of year for the Phase 1 dose-escalation IMMUNICY-1 trial for lead shRNA-based allogeneic CAR T candidate, CYAD-211, for relapsed/refractory multiple myeloma (r/r MM) Mont-Saint-Guibert, Belgium – Celyad Oncology SA (Euronext & Nasdaq: CYAD) (the “Company”), a biotechnology company focused on the discovery and development of chimeric antigen receptor T cell (CAR T) therapies for cancer, today provided an update on its financial results and recent business developments for the fiscal quarter ended September 30, 2022. “This past quarter has been a pivotal moment for the Company as we focus on a new Celyad 2.0 strategy as we seek to monetize our valuable IP estate and leverage our dynamic shRNA technology for our R&D programs. We’ve bolstered our cash runway with an asset purchase agreement for our manufacturing business unit and we believe we are well-positioned to unleash the power of our IP estate and potentially redefine the cell therapy space,” said Michel Lussier, interim Chief Executive Officer of the Company. Recent Highlights The Company entered into a €6 million asset purchase agreement with Cellistic whereby Cellistic acquired Celyad Oncology’s Good Manufacturing Practice (GMP) grade cell therapy manufacturing facility Based on a strategic, financial and medical review, taking into account the costs associated with the pursuit of the program and the delays to reach key medical milestones following the resolution of the previously announced clinical hold, the Company has decided to discontinue the development of CYAD-101 Update on Business Model and Research Programs As previously announced, with Celyad 2.0, Celyad Oncology is implementing a strategic shift from an organization focused on clinical development to one prioritizing R&D discovery and the monetization of its IP estate through partnerships, collaborations and license agreements. The Company intends to focus its R&D efforts on areas of expertise where it believes it can leverage the differentiated nature of its platform technology and continue to bolster its IP estate. The Company possesses key technology and controls IP which covers the potential development of next-generation therapies, including those using short hairpin RNA (shRNA) and T cell receptor Inhibitor Molecule (TIM). Celyad Oncology has expanded the IP estate in-licensed from Dartmouth College with additional patents to broadly cover aspects of allogeneic cell therapy.Current discovery programs have the potential to create additional independent IP. The Company is developing a potential next-generation NKG2D Type I receptor CAR T candidate and a technology to potentially utilize this receptor as a basis for dual CAR technology. The Company is also considering the potential to focus R&D efforts on either B7-H6 CAR T or bispecific antibody candidates for a powerful new antigenic target in the oncology field. In addition, the Company is seeking to advance its shRNA platform through multiplexing technology that allows it to modulate multiple genes simultaneously. This technology is potentially complementary to the Company’s All-in-One Vector approach, which allows for the expression of multiple shRNAs in a single construct within a single transduction step. Combining multiplexed shRNAs with CARs and additional genes of choice provides potential for broad therapeutic functionality. Update on Clinical Programs CYAD-211 – Allogeneic shRNA-based, anti-BCMA CAR T candidate for r/r MM The dose-escalation Phase 1 IMMUNICY-1 trial is evaluating the tolerability and clinical activity of a single infusion of CYAD-211 following preconditioning with CyFlu (cyclophosphamide and fludarabine) in patients with relapsed / refractory multiple myeloma (r/r MM). CYAD-211 was developed to demonstrate potential proof of concept of shRNA technology in the clinic. Our other clinical studies of shRNA disclosed to date have demonstrated encouraging safety and bioactivity signals, and its use as a technology to avoid Graft-versus-Host disease of allogeneic CAR Ts could be a viable approach Clinical updates are expected by year end CYAD-211 was developed to demonstrate potential proof of concept of shRNA technology in the clinic. Our other clinical studies of shRNA disclosed to date have demonstrated encouraging safety and bioactivity signals, and its use as a technology to avoid Graft-versus-Host disease of allogeneic CAR Ts could be a viable approach Clinical updates are expected by year end Third Quarter 2022 Financial Review As of September 30, 2022, the Company had cash and cash equivalents of €13.4 million ($13.1 million). Net cash burn during the first quarter of 2022 amounted to €1.0 million ($1.0 million), in line with expectations. The Company confirms its previous guidance that its existing cash and cash equivalents should be sufficient to fund operating expenses and capital expenditure requirements up to mid-2023. This guidance does not include any potential proceeds from the equity purchase agreement established with Lincoln Park Capital Fund, LLC. After due consideration of detailed budgets and estimated cash flow forecasts for the years 2022 and 2023 which reflect the current strategy of the Company and include expenses and cash outflows estimations in relation to the development of discretionary research programs and pipeline of products candidates, 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 the release is issued.