FT-538

中文摘要近年来，过继性细胞免疫疗法在肿瘤治疗方面展现出了显著的临床效果。T细胞和自然杀伤（natural killer，NK）细胞是临床常用的过继性免疫细胞，并且在淋巴瘤等恶性肿瘤的治疗中已经取得了良好的效果。通过对免疫细胞进行基因工程改造引入嵌合抗原受体（chimeric antigen receptor，CAR）催生了CAR-T和CAR-NK细胞疗法。CAR-T和CAR-NK细胞可高效识别肿瘤抗原且肿瘤细胞杀伤效果较好，在多种癌症研究中均有满意的结果，已经成为一个炙手可热的领域。此文对基于CAR修饰的免疫细胞疗法的发展与应用进行概述，并针对其挑战与未来发展进行了讨论。正文1细胞免疫治疗肿瘤生物治疗是一种新兴肿瘤治疗模式，主要包括肿瘤免疫治疗、溶瘤病毒治疗、干扰小RNA技术疗法等。肿瘤免疫治疗通过重新启动并维持肿瘤-免疫循环，恢复机体正常的抗肿瘤免疫应答，从而控制与清除肿瘤，比传统的肿瘤治疗方法更加安全并且具有特异性，对晚期肿瘤具有显著的疗效。肿瘤免疫治疗包括4大类：过继性细胞免疫疗法、肿瘤疫苗、非特异性免疫刺激以及免疫检查点单克隆抗体。其中过继性细胞免疫疗法是目前研发热点，也是肿瘤治疗研究中开展最广泛、最深入的领域之一，其具体过程是通过对自体或异体免疫细胞进行体外激活或基因修饰，扩增出足够的具有抗肿瘤活性的免疫细胞后回输给患者。过继性免疫细胞主要包括T细胞、自然杀伤（natural killer，NK）细胞、肿瘤浸润淋巴（tumor infiltrating lymphocyte，TIL）细胞、淋巴因子激活的杀伤（lymphokine-activated killer，LAK）细胞、细胞因子诱导的杀伤（cytokine induced killer，CIK）细胞、巨噬细胞和树突状细胞（dendritic cell，DC）等几大类。将免疫细胞的杀伤活性与抗体的特异性相结合，通过对免疫细胞进行基因工程改造引入嵌合抗原受体（chimeric antigen receptor，CAR）使其靶向表达特定抗原的肿瘤细胞，推动了CAR-T和CAR-NK细胞疗法的发展。CAR是CAR-细胞的核心部分，它使细胞能够不依赖于HLA而通过CAR结构识别更广泛的肿瘤靶抗原。CAR基本结构包括肿瘤相关抗原结合域、通常来自单克隆抗体的抗原结合区域的单链抗体（single chain variable fragment，scFv）、胞外铰链域、跨膜域和胞内信号域。scFv可以与肿瘤细胞表面表达的肿瘤相关抗原紧密结合，这决定了 CAR结构的特异性。铰链域是细胞外区和跨膜域之间的连接序列，它给予CAR充分的定向性和灵活性来与肿瘤抗原结合，会影响CAR-细胞的活性。跨膜域位于铰链和胞内信号域之间，包括CD3 HLA-A2或CD28。胞内信号域的结构决定了CAR-细胞激活信号的强度，其中包含免疫受体酪氨酸激活基序（immunoreceptor tyrosine-based activation motif，ITAM）。目前最经典的胞内结构域是CD3 ITAM的激活区。为了提高CAR修饰的效应细胞的增殖和细胞毒作用，可在胞浆尾部加入共刺激蛋白受体，如CD28、4-1BB、CD134、ICOS等。针对CAR结构的研究除了选取合适的靶向位点scFv，很多研究专注于作为CAR-细胞功能关键区域的胞内信号域，目前已经发展了4代。第1代CAR介导的细胞激活仅通过CD3ζ链完成，抗肿瘤活性有限。第2代CAR在胞内结构域中增加了1个新的共刺激信号，包括1条CD3ζ链和1个共刺激分子对CD3ζ链的信号进行放大，并与其具有相同的抗原特异性，增加了细胞增殖、细胞因子分泌和抗凋亡蛋白的分泌。广泛使用的共刺激分子有 CD28、4-1BB和 CD134（OX40）等。第3代CAR包括1条CD3ζ链和2个或2个以上不同的共刺激分子，使其抗肿瘤效应进一步提高。第4代CAR则通过基因修饰分泌特定的细胞因子或表达额外的共刺激配体增强CAR-细胞的疗效，同时引入了自杀基因系统控制CAR-细胞活性，必要时激活使CAR-细胞凋亡，防止对自体造成损害。CAR结构在免疫细胞中的广泛应用，引导免疫细胞特异性靶向识别肿瘤抗原并介导免疫细胞的激活，从而提高了过继性免疫疗法的适用性和有效性。CAR-细胞对血液肿瘤的治疗具有良好的效果，在实体肿瘤的治疗上也取得了一定的进展，表1中列出了近期CAR-细胞治疗的临床研究。本文对CAR-细胞疗法发展与现阶段应用进行概述，并对不同种类的CAR-细胞治疗的特点及优势进行总结，分别介绍了各自的潜力及应用，并针对其挑战与未来发展进行了讨论。2CAR修饰的T细胞2.1CAR-T细胞概述T细胞在细胞介导的免疫应答中起着关键作用，参与监测和杀死肿瘤细胞或潜在的恶性细胞。基于T细胞的过继免疫疗法治疗恶性肿瘤，特别是血液病癌症效果显著。这种新出现的治疗包括3个模型：TIL、T 细胞受体（T cell receptor，TCR）修饰的T细胞（TCR-T）和CAR-T。与CAR-T疗法相比，前两种技术并没有对T细胞本身进行大的修饰，疗效并不显著，较低的成功率以及对疫苗的依赖也限制了它们的发展。作为一种很有前途的过继免疫治疗方案，CAR-T细胞疗法已接受了超过25年的时间考验，能够识别和消除特定的癌细胞，独立于 MHC。现阶段全球范围已开展的临床试验以第2代CAR-T为主，以CD28或CD137共刺激域构成。一般认为，CD28所传递的活化信号，相较于CD137更强，可使T细胞在较短时间内达到很高的杀伤活性；而CD137所传递的活化信号则会维持得更久。此外，由于CAR-T细胞常常无法进入肿瘤内部，因此主要用于治疗血液系统肿瘤，在实体瘤的治疗中还未见显著的成效。第4代CAR-T细胞在CAR的设计中添加了细胞因子或者趋化因子的受体结构，从而增加T细胞在肿瘤组织中的浸润，以达到增强杀伤实体瘤的效果。2.2CAR-T细胞在抗肿瘤治疗中的应用美国FDA共批准了 5款CAR-T细胞产品上市，分别是瑞士诺华公司的Kymriah用于治疗急性B 淋巴细胞白血病（B-cell acute lymphoblastic leukemia，B-ALL）、美国吉利德公司的Yescarta用于治疗弥漫性大B细胞淋巴瘤（Diffuse large B cell lymphoma，DLBCL）以及Tecartus用于治疗套细胞淋巴瘤（mantle cell lymphoma，MCL）、美国百时美施贵宝公司的Breyanzi用于治疗复发或难治性（ralapsed/refractory，R/R）大 B 细胞淋巴瘤（R/R large B cell lymphoma，R/R LBCL）、美国百时美施贵宝公司和Bluebird Bio公司共同研发的Abecma用于治疗多发性骨髓瘤（multiple myeloma，MM）。中国有2款CAR-T细胞产品获批上市，分别为上海复星凯特生物科技有限公司的阿基仑赛注射液用于治疗成人R/R LBCL、上海药明巨诺生物科技有限公司的瑞基仑赛注射液用于DLBCL。上述产品中除Abecma为APRIL靶点外，其余都是CD19靶点。随着大量临床研究的开展，针对不同靶点的CAR-T细胞还有望应用于急性淋巴细胞白血病（acute lymphoblastic leukemia，ALL）、慢性淋巴细胞白血病（chronic lymphocytic leukemia，CLL）、B细胞淋巴瘤（B cell lymphoma，BCL）、MM及实体瘤等。目前治疗ALL，特别是高病死率的R/R B-ALL最适合的方法就是CAR-T细胞治疗，其中最有效的是抗CD19抗体CAR，CD19是B细胞的重要生物标志物，在B-ALL中有较高的表达。尽管CD19是CAR-T细胞治疗ALL的理想靶点，但研究发现“抗原逃逸”是免疫疗法发展的潜在障碍，当务之急是进行优化，包括识别其他靶点。CD22是CAR-T细胞的另一个潜在靶点，2种不同的抗CD22药物已经在临床试验中用于治疗B-ALL，以弥补抗CD19治疗的不足。由于CLL发病机制导致早期免疫缺陷，因此CAR-T细胞治疗的疗效受到来自CLL患者的T细胞体外扩增困难及体内增殖反应的限制。Ibrutinib是一种不可逆的布鲁顿酪氨酸激酶抑制剂，它不仅可以避免对T细胞的负面影响，还可以提高其抗肿瘤能力。有研究表明，5个周期的Ibrutinib治疗增强了靶向CD19的第2代CAR-T细胞（CTL019）的扩增，并降低了程序性细胞死亡蛋白1在T细胞上的表达；同时增加了 B细胞中CD200的表达，而CD200/CD200受体在调节抗肿瘤免疫方面发挥重要作用。CAR-T细胞是治疗R/R B细胞非霍奇金淋巴瘤（non-Hodgkin lymphoma，NHL）的最新免疫疗法，除抗CD19 CAR-T细胞外，4次跨膜蛋白CD20出现在90%以上的BCL中，是公认的NHL治疗靶点。目前，第1代抗CD20 CAR-T细胞治疗已经在一些研究中使用。CD30是TNF受体（TNF receptor，TNFR）超家族的成员，也可以作为淋巴瘤治疗的潜在靶点。在霍奇金淋巴瘤（Hodgkin lymphoma，HL）中，它是Ki-1抗体的抗原，与里德-斯德伯格细胞结合。CD30阳性淋巴细胞主要分布在淋巴组织的滤泡区周围，而在生发中心不常见。对于淋巴瘤，CD30在经典的HL、间变性大细胞淋巴瘤、DLBCL、原发性纵隔BCL和外周T细胞淋巴瘤中表达。有研究报道，靶向CD30的CAR-T细胞在经高强度淋巴清除预处理后的R/R HL患者中表现出显著的抗肿瘤活性和较低的毒性。MM是一种骨髓来源的难治性恶性肿瘤，由于骨髓瘤的异质细胞遗传学和分子异常原因，这种疾病仍然无法治愈。膜蛋白CD138属于硫酸肝素蛋白聚糖的Syndecan家族，在恶性和分化的浆细胞表达，也存在于肿瘤、成熟的上皮细胞和其他正常组织中。由于CD138在几乎所有MM患者中都有表达，因此被用作主要诊断标志物。临床试验结果显示，使用第2代抗CD138 CAR-T细胞治疗5例难治性MM患者后，4例病情稳定，1例晚期浆细胞白血病患者外周血骨髓瘤细胞减少，这表明CAR-T细胞已经进入骨髓。由此可见，抗CD138 CAR-T细胞治疗MM具有良好的耐受性和潜在的抗肿瘤免疫效果。2.3CAR-T细胞面临的挑战CAR-T细胞治疗面临的主要问题是如何提高过继性细胞的有效性和持久性，以及减少治疗的不良反应。为了增强治疗效果，可以通过添加新的共刺激域或CAR部分来提高CAR-T细胞的有效性和持久性。糖皮质激素诱导的TNFR相关蛋白（glucocorticoid-induced TNFR-related protein，GITR）即CD357和4-1BB都是核因子-κB诱导的TNFR超家族成员，而GITR是组成性表达于T细胞的共刺激分子，添加该结构域或结合CD28和4-1BB可以进一步增强T细胞的作用。对于淋巴瘤、MM和其他类型的恶性肿瘤中存在的抗原逃逸或多种肿瘤抗原共存现象，重建CAR部分是一种新的方法。双特异性CAR-T细胞是很有前途的治疗方法，它包括2种形式：（1）1个CAR由2个不同的scFv组成（TanCAR），或者2个不同的CAR在1个T细胞上连接不同的scFv（双信号CAR）；（2）依次或同时注入不同类型的CAR-T细胞池。此外，使用带有趋化因子基因的溶瘤病毒招募CAR-T细胞，可延长它们的持久性并直接攻击肿瘤细胞。这是一种很有前景的方法，可用于血液系统恶性肿瘤的检测。溶瘤病毒可以感染宿主，复制并破坏某些肿瘤细胞，而不伤害正常细胞。有证据表明，肿瘤细胞可以通过MHCⅠ在其细胞质中整合呈递新病毒蛋白，被CD8+ T细胞识别和摧毁。使用黏液瘤病毒、5型腺病毒和柯萨奇病毒A组21型等病毒杀灭这种类型的肿瘤细胞的应用已在MM中报道。在CAR-T细胞治疗过程中，避免失控的T细胞增殖、细胞因子风暴或靶向/非靶向肿瘤效应至关重要，可以激活自杀基因（也称为消除基因）系统来破坏注入的CAR-T细胞，调节CAR-T细胞的扩增并限制毒性。目前，单纯疱疹病毒胸苷激酶/更昔洛韦自杀基因治疗在同种异体造血干细胞背景下已经显示了其安全性和有效性；其他非免疫原性自杀系统，如经过修饰的内在凋亡途径中的半胱氨酸天冬氨酸蛋白酶9（caspase 9，Cas9）也已经被开发出来。然而，这种自杀策略是通过彻底消除CAR-T细胞来缩短治疗时间。“可转换的”CAR-T细胞弥补了这个缺陷，通过在scFv和T细胞表面之间插入一个小的分子复合物来启动细胞功能并通过停止添加它来关闭细胞功能。另一种切换细胞开启和关闭的方法是设计可溶性细胞间分子，包含肿瘤抗原特异性抗体，通过改变中间分子的浓度来改变细胞功能。CAR-T细胞治疗在治疗恶性血液病，特别是ALL、CLL和淋巴瘤取得了很大的进展，而有效性、细胞持久性和不良反应成为广泛应用的瓶颈，需要更加创新的实验室研究来加强该疗法的有效性。3CAR修饰的NK细胞3.1CAR-NK细胞概述NK细胞是机体内天然免疫系统的核心细胞。与T细胞不同，NK细胞对靶细胞的识别不依赖于MHC的表达，而是依赖于其表面杀伤细胞激活受体和杀伤细胞抑制受体（killer inhibitory receptor，KIR）间的动态平衡。当NK细胞表面的KIR识别出自身MHCⅠ的时候，NK细胞会主动失活，保护宿主细胞不受误攻击；在MHCⅠ改变、缺失或表达较低的肿瘤细胞中，KIR介导的抑制作用减弱，NK细胞以“缺失自我标记”为靶点对肿瘤细胞造成杀伤。NK细胞的杀伤作用主要体现在，NK细胞可以直接释放穿孔素和颗粒酶B，执行诱导靶细胞凋亡的作用；同时，NK细胞可以分泌大量细胞因子，如TNF-a、粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony stimulating factor，GM-CSF）、IFN-γ、IL-3等，对靶细胞起到直接作用，或激活其他种类的免疫细胞对靶细胞进行攻击；NK细胞还能表达Fas配体和TNF相关调亡诱导配体，使靶细胞进入程序性凋亡；最后，NK细胞通过其表面CD16与靶细胞表面的抗原决定簇特异性结合从而发挥抗体依赖的细胞毒性（antibody-dependent cell-mediated cytotoxicity，ADCC）作用。除了具有强大的杀伤功能外，NK细胞还具有很强的免疫调节功能，分泌多种共刺激分子，募集和激活其他免疫细胞，如DC和巨噬细胞，间接帮助消灭靶细胞，从而调节机体的免疫状态和免疫功能。目前NK细胞所用CAR结构大多沿用自CAR-T细胞。大多数CAR-NK细胞使用CD3-ITAM作为信号转导区域进行信号转导，并利用CD3-ITAM启动NK细胞杀伤。CD3ζ二聚体可以传递来自CD16的信号，从而有助于ADCC作用。除单一的CD3型ζ链之外，胞内信号区还包括共刺激结构域，如CD28、CD137（4-1BB）或CD244（2B4）等分子，具有很好的信号转导效应，有助于CAR-NK细胞的杀伤作用。不同的共刺激分子对NK细胞的作用也不同，如在ALL异种移植小鼠模型中，CD244对CAR-NK92细胞的刺激作用强于CD137，可以募集并激活酪氨酸激酶启动激活信号，可能更适合作为CAR-NK细胞的胞内信号域。与CAR-T细胞相比，CAR-NK细胞具有多种优势。首先，NK细胞易分离，寿命较短，体内转移的CAR-NK细胞不易过度增殖，导致移植物抗宿主病（graft versus-host disease， GVHD）的风险低。其次，NK细胞分泌的细胞因子主要包括IFN-γ和GM-CSF，比活化CAR-T细胞释放的细胞因子如TNF-а、IL-6、IL-2、IL-8 等更加安全，这些由CAR-T细胞产生的促炎细胞因子可能导致细胞因子释放综合征（cytokine release syndrome，CRS）和神经毒性。除了以CAR依赖的方式杀伤肿瘤靶细胞外，CAR-NK细胞还能以CAR非依赖的方式消除癌细胞，发挥自身天然的杀伤肿瘤细胞的活性，如通过激活NK细胞受体NCR、NKG2D、共刺激受体DNAM-1（CD226）和某些激活的KIR（KIR2DS1、KIR2DS4和KIR2DL4 ）等上调被激活。其次，CAR-NK细胞可以通过表面的CD16介导ADCC作用清除肿瘤细胞。CAR-NK细胞拥有CAR依赖和NK细胞受体依赖的双重杀伤机制，可以有效根除一些不表达CAR所靶向抗原的异种肿瘤。最后，NK细胞可从PBMC、NK细胞系和诱导性多能干细胞（induced pluripotent stem cell，iPSC）等中直接获取，价格更加便宜。CAR-NK细胞中的NK细胞可以从多种来源大规模生产，包括NK-92细胞系、外周血单个核细胞（peripheral blood mononuclear cell, PBMC）、脐带血（umbilical cord blood，UCB）、CD34+ 造血祖细胞（hematopoietic progenitor cells，HPC）和iPSC。目前大多数CAR-NK细胞的临床试验使用的NK92细胞系在体外具有无限的增殖能力，对反复冻存、解冻的敏感性降低，使生产时间更短，成本更低。然而，NK92细胞系为肿瘤细胞系，具有潜在致瘤风险，缺乏CD16和NKp44的表达，且在输注前需要辐照而失去体内扩增潜力。PBMC是NK细胞的重要来源，GVHD风险低，可以从健康捐赠者PBMC中分离出足够数量的NK细胞，其中90%是成熟表型的CD56dimCD16+ NK细胞，细胞毒作用更强但增殖能力下降，且这种来源的CAR-NK细胞无需辐照处理。同样的方法也适用于从UCB分离NK细胞。UCB的优势是可以选择具有特定HLA类型和特定NK细胞受体特征的供者。然而，UCB单位体积有限，NK细胞数量较少、表型不成熟，黏附分子CD16、穿孔素和颗粒酶B的表达减少，NKG2A等抑制受体表达增加，使获取足够数量的临床级NK细胞成为挑战。同时，PBMC和UCB来源的CAR-NK细胞由不同供者提供，使产品很难标准化。从CD34+ HPC中分化NK细胞是获得大量临床级成熟NK细胞的另一种途径。CD34+HPC可以从骨髓、胚胎干细胞或UCB中分离，在培养系统中扩增和分化为成熟的NK细胞。除上述来源外，iPSC由于可以更有效地设计以稳定表达CAR而成为新兴的CAR-NK细胞来源，从单个CAR-iPSC细胞出发就足以分化为大量高度同源的CAR-NK细胞产品用于临床，为CAR-NK细胞提供了一种可再生的来源。然而，与UCB NK细胞类似，iPSC来源的NK细胞通常表型不成熟。3.2CAR-NK细胞在抗肿瘤治疗中的应用使用非CAR工程化的NK细胞进行免疫治疗已显示出良好的效果，而用基因工程化的CAR-NK细胞可以提高临床疗效。在恶性淋巴肿瘤的临床研究中，CD33和CD19是CAR-NK细胞疗法最常见的靶点。Tang等首次将CAR-NK-92细胞应用于临床试验。他们构建了包含CD28和4-1BB共刺激分子在内的第3代慢病毒载体，并在CD33的scFv和CD28之间插入Fc片段来检测转导的CAR-NK细胞。用上述构建的CD33-CAR载体转导经改造的能够表达IL-2的NK-92-MI细胞，并确认转导效率达90%以上。经过符合GMP级生产要求的细胞转导与培养后，CD33-CAR-NK-92细胞在体外对人早幼粒白血病细胞系HL-60展现了良好的杀伤作用。接着，他们对3例R/R急性髓系白血病（acute myloid lymphoma，AML）患者进行挽救化疗，没有观察到明显的不良反应，证明以5×109个CD33-CAR-NK-92细胞剂量进行输注并辐照是安全的。输注后，患者的血液中能够检测到CAR-NK-92细胞。Liu等对11例R/R NHL或CLL患者进行了UCB来源的HLA不相合的抗CD19-CAR-NK细胞治疗。将携带抗CD19、CD28跨膜区和CD28ζ信号内域的scFv的逆转录病毒载体与人IL-15基因和可诱导的Cas9自杀基因结合，导入UCB来源的NK细胞。体外扩增CAR-NK细胞，并在淋巴清除化疗后对患者进行一次性输注，未观察到明显的GVHD和CSF。在接受治疗的11例患者中，8例恶性肿瘤有一定程度的缓解，其中7例得到完全缓解。输注后的CAR-NK细胞可以扩增并维持一定水平至少12个月。这些已应用于临床的CAR-NK细胞治疗方案展现了 CAR-NK细胞对B细胞系恶性肿瘤的抗癌作用是有效且安全的，且疗效均强于非CAR-NK细胞治疗。临床前研究表明CAR-NK细胞具有很强的抗肿瘤功能，但在实体瘤中的临床应用尚处于起步阶段。目前有多项临床前试验研究CAR-NK细胞疗法治疗多发性实体瘤的疗效，如神经母细胞瘤、前列腺癌、胶质母细胞瘤和乳腺癌等。Liu等用表皮生长因子受体（epidermal growth factor receptor，EGFR）作为治疗三阴性乳腺癌（triple-negative breast cancer，TNBC）的靶点，研究了EGFR-CAR-NK细胞在体内外对TNBC细胞的杀伤作用。将抗EGFR scFv的DNA片段克隆至带有CD8较链、CD28跨膜区域、4-1BB和CD3ζ胞内信号域的第3代CAR慢病毒载体中，并转导NK细胞。结果表明，EGFR-CAR-NK细胞可以被表达上调的TNBC细胞激活，并在体外特异性地触发TNBC细胞的裂解，并对小鼠体内乳腺癌细胞来源的异种移植和患者来源的异种移植肿瘤均有抑制作用。除此之外，EGFR-CAR-NK细胞可通过同时触发TNBC细胞凋亡来提高疗效，表明EGFR-CAR-NK细胞对治疗TNBC是有潜力的治疗策略。Cao等也以间皮素（mesothelin，MSLN）作为治疗胃癌的靶点，将抗MSLN 的 scFv （NKG2D跨膜区，结合 2B4-CD3 胞内信号结构域）克隆到慢病毒载体中，并转导NK-92细胞得到MSLN-CAR-NK细胞。结果表明，MSLN-CAR-NK细胞在体外对MSLN阳性的胃癌细胞具有特异性杀伤作用。此外，与非CAR工程化的NK-92细胞和CD19-CAR-NK细胞相比，MSLN-CAR-NK细胞可分泌更强的细胞因子，并可以有效清除小鼠体内皮下和腹膜肿瘤模型的胃癌细胞，对异种移植的胃癌具有较强的抗肿瘤和NK细胞浸润能力，显著延长了小鼠的存活时间，证明针对胃癌的MSLN靶点是非常具有潜力的。CAR-NK细胞具有很大的临床应用潜力，进一步的研究正在将CAR-NK细胞治疗的临床前结果转化至临床。3.3CAR-NK细胞面临的挑战CAR-NK细胞疗法是近年来发展起来的一种治疗肿瘤的免疫治疗方法，尽管在临床前研究中取得了不错的进展，但CAR-NK疗法的临床应用仍处于起步阶段，并面临着一些挑战。我们希望通过对当前挑战进行阐述，并讨论未来的改进方法，以改善临床环境下的CAR-NK的治疗结果。NK细胞的扩增是CAR-NK细胞生产的重要步骤之一，需要大量的CAR-NK细胞才能达到最佳的临床疗效，因此产生足够数量的NK细胞仍然是CAR-NK疗法的主要挑战。多种细胞因子，如IL-2、IL-15、IL-12、IL-21和IL-18等已被用于体外扩增原代NK细胞，但效果有限，而在开发出合适的人造APC（artificial APC，aAPC）后可以特异性并大量扩增NK细胞。Shah等使用以K562为基质表达膜结合型IL-21的aAPC在14 d的NK细胞共培养中，使UCB NK细胞的平均扩增倍数达到了2 389倍，而仅添加IL-2的NK细胞在14 d的培养中平均扩增倍数只有20倍，表明aAPC在NK细胞扩增中具有显著的促进作用。而在Denman等的研究中，膜结合型IL-21的K562还可以延缓NK细胞的端粒缩短，而端粒缩短情况也被认为是限制NK细胞分裂能力的重要因素之一。为获得足够数量的NK细胞用于CAR转导，需要开发更多、更有效的临床级别体外扩增方法。除了扩增的挑战外，CAR-NK细胞的CAR结构以及转导过程仍面临诸多问题，同时也有很多研究正在努力解决这些挑战。由于CAR结构最先用于T细胞，而针对NK细胞的CAR最优结构的设计尚未被系统地研究。CAR结合表位的位置及其与NK细胞表面的距离可能影响与抗原的结合、免疫突触的最佳形成和CAR-NK细胞的激活，同时不同来源的NK细胞对共刺激信号要求也不尽相同。Li等通过表达特制的CAR结构，能促进由iPSC分化的NK细胞的激活，这种含跨膜NKG2D和胞内2B4结构域的构建体比CAR-T构建体性能更好。因此，针对NK细胞独特的激活受体和信号激活蛋白而定制的基于NK细胞的CAR结构潜力巨大。除此之外，目前所开发的CAR大多针对癌细胞上差异表达的非突变自身抗原，导致CAR可能与正常组织上表达的抗原非特异性结合，对健康组织也造成损害。针对这一问题，可以导入自杀基因，如诱导型Cаs9，控制CAR活性剂量；也有研究指出，如果2个单独的结构或2个胞外结构域以串联形式结合，产生双特异性CAR，细胞毒性会有效降低；split CAR构造，使用小分子的二聚化以形成功能单元。而在转导过程中，目前主流的导入CAR的方式为逆转录病毒转染。但这种方法依赖宿主细胞的分裂，NK细胞在未激活时转导效率会下降。同时，逆转录病毒转染后可能存在遗传毒性，并可能触发相关机制使NK细胞凋亡。针对这一问题，有研究指出可以使用RNA工程，NK细胞的存活率和转染效率会更高。CAR-NK细胞治疗作为一种新的过继免疫治疗手段具有巨大的潜力和可行性，但仍存在许多问题需要解决。如果这些问题得到解决，CAR-NK细胞疗法将成为对抗肿瘤的强大手段。4以iPSC为平台生产CAR-T/NK细胞4.1iPSC概述2006年，山中伸弥团队在小鼠胚胎和成人成纤维细胞中诱导多能性的4个转录因子一Oct3/4、Sox2、Klf4和cMyc，实现了体细胞向多能干细胞的重编程。iPSC可在体外无限扩增，并且理论上可分化成任何细胞类型，在形态、基因和蛋白表达、表观遗传修饰状态、细胞倍增能力、类胚体和畸形瘤生成能力、分化能力等方面都与胚胎干细胞相似，同时避开了伦理问题，在疾病模拟、药物筛选和细胞治疗中有着巨大的应用前景，是细胞疗法的理想细胞来源。iPSC体外培养时如果通过构建合适的环境，模拟体内发育过程，理论上可分化成任何细胞类型，包括T细胞和NK细胞。iPSC衍生的T/NK细胞能够为临床治疗提供同质且基因明确的细胞群体，消除了使用外周血或UCB来源的T/NK细胞时供者间的差异。此外，通过使用各种基因编辑技术，如病毒载体、转座子或成簇规律间隔短回文重复序列Cas9系统，可以对未分化的iPSC进行遗传操作以产生均一的目标细胞群细胞如CAR-T/NK细胞，这些优势使iPSC衍生的细胞成为“现货型”细胞疗法的理想细胞群。4.2iPSC衍生的CAR-T/NK细胞在抗肿瘤治疗中的应用2013年，Sadelaint团队用编码二代抗CD19-CAR的慢病毒载体转导外周血淋巴细胞来源的iPSC，收获的iPSC-CAR-T细胞表现出与CAR-TCR-γẟ外周血淋巴细胞类似的抗肿瘤活性。Kanfman团队用iPSC生产的NK细胞表达的激活和抑制受体与外周血NK细胞相似，并且在体外和体内都表现出强大的抗肿瘤和抗病毒活性。为了提高CAR-NK细胞对肿瘤细胞的杀伤活性，2018年，Kanfman团队构建了具有NK细胞特异性激活域（NKG2D-2B4ζ）的CAR-NK细胞并以PiggyBac转座子为载体将其编码基因整合至iPSC，所收获的NK-CAR（NKG2D-2B4ζ）-iPSC-NK 细胞显示出与外周血NK细胞和未修饰的iPSC-NK细胞相似的表型，并且比 T-CAR（CD28-41BBζ）-iPSC-NK细胞表现出更高的抗肿瘤活性。美国Fate Therapeutic公司以iPSC为平台生产的CAR-NK细胞和CAR-T细胞产品均已进入Ⅰ期临床研究。FT596是由iPSC衍生的CAR-NK细胞产品，除了表达靶向B细胞抗原CD19的CAR，FT596还表达高亲和力且不可清除的CD16受体以增强ADCC，及IL-15受体融合蛋白以增加CAR-NK细胞的体内存活时间。目前公布的Ⅰ期临床试验结果显示，在接受FT596治疗的14例R/R BCL患者中，10例（71%）患者获得客观缓解，其中7例（50%）获得完全缓解。安全性方面，数据显示FT596疗法耐受性良好，未观察到神经毒性综合征、GVHD等。除了FT596外，该公司的FT500、FT516、FT538和FT576均为由iPSC衍生的NK细胞产品且都已进入Ⅰ期临床研究。FT819是首个由iPSC衍生的CAR-T细胞产品，除了表达靶向B细胞抗原CD19的CAR，FT538完全消除了内源性TCR的表达以及GVHD。临床数据显示，FT819在体内对表达CD19的白血病和淋巴瘤细胞系具有抗原特异性的细胞毒性，与健康的供体来源的CAR-T细胞相当；骨髓评估显示，FT819能够在体内持续存在并继续表现出肿瘤清除能力。iPSC细胞系作为一种可再生细胞来源，能够大规模生产高质量的同种异体CAR-T/NK细胞，具有更高的产品一致性、现货供应和更广泛的患者可及性，有望成为未来细胞疗法领域的重要方向。4.3iPSC衍生的CAR-T/NK细胞面临的挑战iPSC衍生的NK细胞和T细胞在近几年发展迅速，提供了可以即取即用的产品，能够极大降低肿瘤免疫治疗的成本，形成一种可以提前生产、储备，具有可预期疗效的免疫细胞治疗产品。iPSC衍生的细胞治疗产品从科研端走向临床仍面临着诸多新的挑战。其中，能够在临床应用需要克服的重要挑战之一是确立符合临床使用要求的有效细胞分化策略。虽然在基础科学研究中，已经有多项实验报道可以将iPSC成功分化为T细胞和NK细胞，然而很多分化方案仍然使用了血清和滋养层细胞，存在外源因子污染的安全性风险，不符合各国药典规则。iPSC的分化策略直接影响到最后生成的T细胞或者NK细胞的表型是否与人体中自然产生的细胞相同，不同企业或机构最后生产出的细胞产品性状很可能大不相同。此外，iPSC具有无限增殖的潜力，如果未分化的iPSC被输入到患者体内，可能具有产生肿瘤的潜力。因此，需要设定安全检查或者基因工程改造策略，将这一风险最小化。目前，对iPSC衍生的细胞治疗的功能陈述还没有建立统一的标准，如何规范基于iPSC衍生的细胞治疗产品工艺的稳定性也是目前亟需解决的问题。5CAR修饰的其他免疫细胞由于肿瘤微环境（tumor micro-environment，TME）和缺乏特异性靶向肿瘤抗原，CAR-T细胞在实体瘤中的应用仍然受到严重限制，除了 NK细胞，目前针对许多实体瘤的新策略是将CAR应用到巨噬细胞、DC和CIK细胞中。巨噬细胞M1表型具有促炎和抗肿瘤活性，在2类实体瘤异种移植小鼠模型中，CAR-巨噬细胞靶向MSLN或人表皮生长因子受体2，在人红白血病细胞中表现出抗肿瘤吞噬活性，降低了肿瘤负荷，并延长了总生存期；TME中的另一个关键作用细胞是起APC作用的DC，它可以启动幼稚T细胞并重新激活记忆应答，并与细胞毒性CD8+细胞交叉呈递，促进更强的抗肿瘤应答，Suh等用4-1BB抗CD33的CAR转导了 DC，在AML模型中对其进行了测试，经CAR-T/CAR-DC治疗的小鼠AML的负担明显减少，存活率也随之增加；CIK细胞也是备受关注的一类效应细胞，其具有经典NK细胞以及CD4+和CD8+ T细胞的共同特征，可以通过多种受体识别病原体，人表皮生长因子2-CAR-CIK细胞可完全消除人肺泡横纹肌肉瘤，而野生型CIK细胞仅能实现部分肿瘤生长抑制，而且使用CAR-CIK细胞没有严重CRS或GVHD的迹象。癌症的免疫编辑和耐药性在抗癌免疫疗法中十分常见，许多癌症通过失去MHCⅠ抗原呈递机制来逃避免疫控制，高达50%的ALL和LBCL患者中都发现CD19丢失引发的免疫逃逸，这些患者在抗CD19 CAR-T细胞治疗后复发。与自体CAR-T细胞产品中的主要细胞类型αβ T细胞不同，γẟ T细胞具有先天和适应性免疫系统的特性，它们可以以不依赖MHC的方式发挥作用，具有多种杀伤机制，并将抗原交叉呈递给αβ T细胞，靶向二唾液酸神经节苷脂2（ disialoganglioside 2，GD2）的γẟ CAR-T细胞能够向肿瘤细胞迁移和抗原交叉呈递，并增强了GD2在癌细胞系中的特异性杀伤，在另一项研究中，Rozenbaum等证明γẟ CAR-T细胞能够靶向那些失去C19（即抗原阴性）的CD19白血病细胞。记忆性T细胞和Treg作为免疫细胞，因其不会引起GVHD，也被CAR修饰后用于免疫治疗。Fernandez等构建表达NKG2D的CAR-记忆性T细胞，可以靶向清除表达NKG2D配件的骨肉瘤细胞，也是一种很有前途的骨肉瘤免疫治疗方法；Treg是一种能抑制传统T细胞和其他免疫细胞功能的T细胞亚群，针对其免疫抑制特性，目前也已开发CAR-Treg疗法治疗移植排斥以及一系列自身免疫性疾病。6展望过继性细胞免疫治疗是近年来发展迅速的一种个性化免疫治疗方法。而通过基因工程改造的方式对过继性免疫细胞进行CAR修饰，进一步提高了其识别肿瘤抗原的能力和对肿瘤细胞的杀伤效果。其中CAR-T细胞治疗在治疗恶性血液病特别是ALL、CLL和淋巴瘤方面取得了很大的进展，但有效性、细胞持久性和不良反应成为这种方法广泛应用的瓶颈。因此很多研究尝试用CAR修饰其他免疫细胞来代替T细胞，从而提高对实体瘤的治疗效果，同时减轻不良反应。其中，CAR-NK细胞由于杀伤能力强，导致移植物抗宿主病的风险低，同时只会引起较轻的细胞因子风暴和神经毒性不良反应，是除CAR-T细胞外研究最多的CAR修饰的免疫细胞。其他相关技术如以iPSC为平台生产T/NK细胞，CAR修饰应用到巨噬细胞、DC细胞和CIK细胞中以治疗实体瘤等方面均取得了较大突破。CAR修饰的免疫细胞治疗研发具有广泛的应用前景，但有很多问题需要解决才能用于临床治疗，需要进行更多的创新工作研究。作者徐瑞升  陈玉莹  李奇林  谭文松  蔡海波华东理工大学生物反应器工程国家重点实验室，上海 200237通信作者：蔡海波，Email：caihaibo@ecust.edu.cn引用本文：徐瑞升，陈玉莹，李奇林，等.  基于嵌合抗原受体修饰的免疫细胞疗法发展与应用的研究进展 [J]. 国际生物制品学杂志, 2023, 46(5): 273-282.  DOI: 10.3760/cma.j.cn311962-20221125-00081识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

Dose Escalation Ongoing in Landmark Phase 1 Study of FT819 CD19-targeted 1XX CAR T-cell Program; Interim Clinical Data Demonstrated Favorable Safety Profile and Complete Responses in Aggressive Large B-cell Lymphoma FT576 BCMA-targeted CAR NK Cell Program Accruing Patients in Multi-dose Escalation Cohorts for Multiple Myeloma; Initial Translational Data Support Potential of Combination Regimen to Induce Differentiated Immune Reconstitution Profile and Extend FT576 Functional Persistence Clinical Initiation of FT522 ADR-armed, CD19-targeted CAR NK Cell Program for B-cell Lymphoma Anticipated in 2H23; Preclinical Studies Ongoing to Extend Clinical Reach to Autoimmune Diseases Completed Corporate Restructuring and Strategic Assessment of Pipeline Assets; Ended 1Q23 with $426 Million in Cash, Cash Equivalents, and Receivables Supporting Runway into 2H25 SAN DIEGO, May 03, 2023 (GLOBE NEWSWIRE) -- Fate Therapeutics, Inc. (NASDAQ: FATE), a clinical-stage biopharmaceutical company dedicated to bringing a first-in-class pipeline of induced pluripotent stem cell (iPSC)-derived cellular immunotherapies to patients with cancer and autoimmune disorders, today reported business highlights and financial results for the first quarter ended March 31, 2023. “Over the first months of 2023, we have sharpened our clinical focus and significantly reduced our operating expenses, creating the necessary cash runway to achieve key milestones across our multiplexed-engineered CAR NK and CAR T-cell pipeline. We sincerely thank our employees whose patience and perseverance have allowed us to emerge through this transition period with a renewed sense of energy, commitment, and drive to bring first-in-class, iPSC-derived cellular immunotherapies to patients with cancer and autoimmune disorders,” said Scott Wolchko, President and Chief Executive Officer of Fate Therapeutics. “We are now well-positioned to clinically assess higher therapeutic exposures for our FT576 BCMA-targeted CAR NK cell program in multiple myeloma and our FT819 CD19-targeted CAR T-cell program in B-cell malignancies. In addition, we aim to bring our FT522 CD19-targeted CAR NK cell program, which incorporates our proprietary ADR technology designed to enhance NK cell potency, extend functional persistence, and resist host immune cell rejection, into clinical development in the second half of 2023 for B-cell lymphoma, and intend to expand its clinical reach to include severe autoimmune disorders. Finally, we are excited to be jointly developing our clinical strategy with ONO Pharmaceutical for FT825/ONO-8250, our HER2-targeted CAR T-cell collaboration program for solid tumors for which we plan to submit an IND application in the second half of 2023.” NK Cell Programs FT576 BCMA-targeted CAR NK Cell Program Accruing Patients in Multi-dose Escalation Cohorts for Multiple Myeloma. The Company’s Phase 1 study of FT576, its multiplexed-engineered, BCMA-targeted chimeric antigen receptor (CAR) NK cell product candidate for relapsed / refractory multiple myeloma, is currently enrolling two-dose treatment cohorts as monotherapy and in combination with CD38-targeted monoclonal antibody (mAb) therapy at 300 million cells per dose. Upon clearance of the current treatment cohorts, the Company plans to open and assess three-dose treatment cohorts starting at 1 billion cells per dose. At the 2022 American Society of Hematology (ASH) Annual Meeting in December, the Company presented interim Phase 1 clinical data from nine heavily pre-treated patients in the single-dose cohorts, which showed encouraging clinical evidence of BCMA-targeted activity and a favorable safety profile indicating the potential for administration in the outpatient setting. Translational data from the CD38-targeted mAb combination regimen showed rapid and selective depletion of CD38-positive patient immune cells in the peripheral blood and bone marrow that extended through the first month of therapy, indicating that the regimen may uniquely serve to attenuate reconstitution of activated T cells, extend functional persistence of FT576, and enable dual-antigen targeting of myeloma cells.Initiation of Clinical Assessment of FT522 ADR-armed, CD19-targeted CAR NK Cell Program Anticipated in 2H23. FT522 is the Company’s first product candidate to incorporate its proprietary alloimmune defense receptor (ADR) technology, which has been shown in preclinical studies to increase NK cell potency, enhance functional persistence, and confer resistance to host immune cell allo-reactivity. The Company has recently submitted an Investigational New Drug (IND) application to the U.S. Food and Drug Administration (FDA) to investigate the safety and activity of FT522 in combination with CD20-targeted mAb therapy in patients with B-cell lymphoma, including without prior administration of intensive conditioning chemotherapy. In addition, the Company is currently conducting preclinical studies to support clinical assessment of FT522 in autoimmune disease, including in combination with CD20- and CD38-targeted mAb therapy, to selectively target and durably deplete pathogenic B cells, plasma cells, and auto-reactive T cells. T-cell Programs First-of-kind FT819 Program Advancing in Single-dose Escalation Cohorts for B-cell Malignancies. The Company’s landmark Phase 1 clinical trial of FT819, which is the first-ever clinical investigation of a T-cell product candidate manufactured from a clonal master iPSC line, is currently enrolling patients in single-dose escalation cohorts at 540 million cells in B-cell lymphoma and at 180 million cells in chronic lymphocytic leukemia. At the 2022 ASH Annual Meeting, the Company presented interim Phase 1 clinical data from eight patients with relapsed / refractory aggressive large B-cell lymphoma treated with a single dose of FT819 ranging from 90 million cells to 360 million cells, which demonstrated a favorable safety profile and objective responses including in patients who were not eligible for or had previously failed autologous CD19-targeted CAR T-cell therapy. FT819 incorporates several novel features including the integration of a novel CD19-targeted 1XX CAR construct into the T-cell receptor alpha constant (TRAC) locus, which is intended to promote uniform CAR expression, enhance T-cell potency, and prevent graft-versus-host disease.2023 IND Submission Planned for HER2-targeted CAR T-cell Program for Solid Tumors. Under the Company’s collaboration with ONO Pharmaceutical Co., Ltd. (ONO), the companies are co-developing FT825/ONO-8250, an iPSC-derived CAR T-cell product candidate targeting human epidermal growth factor receptor 2 (HER2)-expressing solid tumors. IND-enabling activities for FT825/ONO-8250 are currently ongoing, and the Company plans to submit an IND application to the FDA in 2023 to jointly conduct with ONO a Phase 1 study for the treatment of patients with HER2-positive solid tumors. The multiplexed-engineered, iPSC-derived CAR T-cell product candidate incorporates seven novel synthetic controls designed to enhance effector cell function and overcome unique challenges in treating solid tumors, including a novel HER2-targeted binding domain with a differentiated targeting profile, a synthetic CXCR2 receptor to promote cell trafficking, a synthetic TGFβ receptor to redirect immunosuppressive signals in the tumor microenvironment, and a synthetic interleukin-7 receptor fusion protein to induce T-cell activation. Strategic Pipeline Prioritization & Corporate RestructuringDuring the first quarter of 2023, in connection with the termination of its collaboration with Janssen Biotech, Inc. (Janssen), the Company discontinued all collaboration activities, including withdrawing an IND application previously allowed by the FDA for a first collaboration product for the treatment of B-cell lymphoma. In addition, following a strategic review of its wholly-owned iPSC-derived NK cell and T-cell programs, the Company focused its operations on advancing its most innovative and differentiated programs and initiated the discontinuation of its FT516, FT596, FT538, and FT536 NK cell product candidates. As part of its corporate restructuring, the Company reduced its workforce to approximately 220 employees. First Quarter 2023 Financial Results & 2023 Guidance Cash & Investment Position: Cash, cash equivalents and investments as of March 31, 2023 were $412.8 million. In addition, as of March 31, 2023, cash receivables from collaborations were $13.5 million. The Company expects its cash, cash equivalents, and investments to exceed $300 million at year-end 2023.Total Revenue: Revenue was $59.0 million for the first quarter of 2023, of which $52.3 million was associated with the termination of its collaboration with Janssen and $6.7 million was derived from its ongoing collaboration with ONO. Under the ONO collaboration, a one-time amount of $6.2 million was recorded as revenue for the first quarter of 2023 associated with the Company’s conduct of IND-enabling activities for FT825/ONO-8250, for which ONO exercised its development and commercialization option in November 2022. For each of the remaining three quarters of 2023, the Company expects to recognize approximately $0.8 million in revenue under the ONO collaboration in connection with its conduct of preclinical development activities for a second collaboration candidate targeting an undisclosed solid tumor antigen.Total Operating Expenses: For the first quarter of 2023, GAAP operating expenses were $87.6 million, including research and development expenses of $65.6 million and general and administrative expenses of $21.9 million. Such amounts included $11.0 million of non-cash stock-based compensation expense and a one-time charge of $12.9 million for severance and other employee termination-related costs associated with the Company’s corporate restructuring. For the full year ending December 31, 2023, the Company expects its GAAP operating expenses to be between $265 million to $285 million.Shares Outstanding: Common shares outstanding were 98.2 million, and preferred shares outstanding were 2.8 million, as of March 31, 2023. Each preferred share is convertible into five common shares. Today's Conference Call and WebcastThe Company will conduct a conference call today, Wednesday, May 3, 2023 at 5:00 p.m. ET to review financial and operating results for the quarter ended March 31, 2023. In order to participate in the conference call, please register using the conference link here. The live webcast can be accessed under "Events & Presentations" in the Investors section of the Company's website at www.fatetherapeutics.com. The archived webcast will be available on the Company's website beginning approximately two hours after the event. About Fate Therapeutics’ iPSC Product PlatformThe Company’s proprietary induced pluripotent stem cell (iPSC) product platform enables mass production of off-the-shelf, multiplexed-engineered cell products that are selectively designed, incorporate novel synthetic controls of cell function, and are intended to deliver multiple mechanisms of therapeutic importance to patients. Human iPSCs possess the unique dual properties of unlimited self-renewal and differentiation potential into all cell types of the body. The Company’s platform combines multiplexed engineering and single-cell selection of human iPSCs to create clonal master iPSC lines. Analogous to master cell lines used to mass produce biopharmaceutical drug products such as monoclonal antibodies, the Company utilizes its clonal master iPSC lines as a renewable cell source to manufacture multiplexed-engineered cell products which are well-defined and uniform in composition, can be stored in inventory for off-the-shelf availability, can be combined and administered with other therapies, and can potentially reach a broad patient population. As a result, the Company’s platform is uniquely designed to overcome numerous limitations associated with the manufacture of cell therapies using patient- or donor-sourced cells. Fate Therapeutics’ iPSC product platform is supported by an intellectual property portfolio of over 400 issued patents and 450 pending patent applications. About FT576FT576 is an investigational, universal, off-the-shelf natural killer (NK) cell cancer immunotherapy derived from a clonal master induced pluripotent stem cell (iPSC) line engineered with four functional components: a proprietary chimeric antigen receptor (CAR) optimized for NK cell biology that targets B-cell maturation antigen (BCMA); a novel high-affinity 158V, non-cleavable CD16 (hnCD16) Fc receptor, which has been modified to prevent its down-regulation and to enhance its binding to tumor-targeting antibodies; an IL-15 receptor fusion (IL-15RF) that augments NK cell activity; and the deletion of the CD38 gene (CD38KO), which promotes persistence and function in high oxidative stress environments. In preclinical studies, FT576 has demonstrated that the high-avidity binding of the BCMA-targeted CAR construct enables sustained tumor control against various multiple myeloma cell lines, including in long-term in vivo xenograft mouse models. Additionally, in combination with daratumumab, FT576 has shown complete tumor clearance and improved survival compared to primary BCMA-targeted CAR T cells in a disseminated xenograft model of multiple myeloma. FT576 is being investigated in a multicenter Phase 1 clinical trial for the treatment of relapsed / refractory multiple myeloma as a monotherapy and in combination with daratumumab (NCT05182073). About FT819FT819 is an investigational, universal, off-the-shelf, T-cell receptor (TCR)-less CD19 chimeric antigen receptor (CAR) T-cell cancer immunotherapy derived from a clonal master induced pluripotent stem cell (iPSC) line, which is engineered with the following features designed to improve the safety and efficacy of CAR19 T-cell therapy: a novel 1XX CAR signaling domain, which has been shown to extend T-cell effector function without eliciting exhaustion; integration of the CAR19 transgene directly into the T-cell receptor alpha constant (TRAC) locus, which has been shown to promote uniform CAR19 expression and enhanced T-cell potency; and complete bi-allelic disruption of TCR expression for the prevention of graft-versus-host disease. FT819 demonstrated antigen-specific cytolytic activity in vitro against CD19-expressing leukemia and lymphoma cell lines comparable to that of primary CAR T cells, and persisted and maintained tumor clearance in the bone marrow in an in vivo disseminated xenograft model of lymphoblastic leukemia. FT819 is being investigated in a multicenter Phase 1 clinical trial for the treatment of relapsed / refractory B-cell malignancies, including B-cell lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia (NCT04629729). About Fate Therapeutics, Inc.Fate Therapeutics is a clinical-stage biopharmaceutical company dedicated to bringing a first-in-class pipeline of induced pluripotent stem cell (iPSC)-derived cellular immunotherapies to patients with cancer and autoimmune disorders. Using its proprietary iPSC product platform, the Company has established a leadership position in creating multiplexed-engineered iPSC lines and in the manufacture and clinical development of off-the-shelf, iPSC-derived cell products. The Company’s effector cell pipeline includes multiplexed-engineered, iPSC-derived natural killer (NK) cell and T-cell product candidates, which incorporate novel synthetic controls of cell function, such as chimeric antigen receptors (CARs) to target tumor-associated antigens, and are intended to deliver multiple mechanisms of therapeutic importance to patients including in combination with well-established cancer therapies. Fate Therapeutics is headquartered in San Diego, CA. For more information, please visit www.fatetherapeutics.com. Forward-Looking StatementsThis release contains "forward-looking statements" within the meaning of the Private Securities Litigation Reform Act of 1995 including statements regarding the progress of and plans related to the Company's product candidates, clinical studies and preclinical research and development programs, the therapeutic and market potential of the Company’s product candidates, the Company’s clinical and product development strategy, the Company’s plans to submit IND applications for its FT522 CD19-targeted CAR NK cell program and its FT825/ONO-8250 HER2-targeted CAR T-cell solid tumor program under its collaboration with ONO, the Company's expectations regarding its receipt of future payments for milestones achieved under its collaboration agreement with Janssen prior to the termination of the agreement, the anticipated effects of the Company’s workforce reduction and reprioritization of preclinical and clinical development activities, including its projected cash runway, and the timing of such events. These and any other forward-looking statements in this release are based on management's current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to, the risk that the Company’s product candidates may not demonstrate the requisite safety or efficacy to warrant further development or to achieve regulatory approval, the risk that results observed in prior studies of the Company’s product candidates, including preclinical studies and clinical trials, will not be observed in ongoing or future studies involving these product candidates, the risk of a delay or difficulties in the manufacturing of the Company’s product candidates or in the initiation and conduct of, or enrollment of patients in, any clinical trials, the risk that the Company may cease or delay preclinical or clinical development of any of its product candidates for a variety of reasons (including requirements that may be imposed by regulatory authorities on the initiation or conduct of clinical trials, changes in the therapeutic, regulatory, or competitive landscape for which the Company’s product candidates are being developed, the amount and type of data to be generated or otherwise to support regulatory approval, difficulties or delays in patient enrollment and continuation in the Company’s ongoing and planned clinical trials, difficulties in manufacturing or supplying the Company’s product candidates for clinical testing, and any adverse events or other negative results that may be observed during preclinical or clinical development), the risk that results observed in preclinical studies of its product candidates may not be replicated in ongoing or future clinical trials, the risk that its product candidates may not produce therapeutic benefits or may cause other unanticipated adverse effects, the risk that the Company may not comply with its obligations under and otherwise maintain its collaboration agreement with ONO Pharmaceutical, Ltd. or other parties with which the Company may enter into future collaborations on the agreed upon terms, the risk that research funding and milestone payments received by the Company under its collaborations may be less than expected, and the risk that the Company may incur operating expenses in amounts greater than anticipated. For a discussion of other risks and uncertainties, and other important factors, any of which could cause the Company’s actual results to differ from those contained in the forward-looking statements, see the risks and uncertainties detailed in the Company’s periodic filings with the Securities and Exchange Commission, including but not limited to the Company’s most recently filed periodic report, and from time to time in the Company’s press releases and other investor communications. Fate Therapeutics is providing the information in this release as of this date and does not undertake any obligation to update any forward-looking statements contained in this release as a result of new information, future events or otherwise. Condensed Consolidated Statements of Operations and Comprehensive Loss(in thousands, except share and per share data)(unaudited) Three Months Ended March 31, 2023 2022 Collaboration revenue$58,980 $18,414 Operating expenses: Research and development 65,629 72,139 General and administrative 21,943 20,742 Total operating expenses 87,572 92,881 Loss from operations (28,592) (74,467)Other income (expense): Interest income 3,694 418 Change in fair value of stock price appreciation milestones 1,718 8,359 Other Income 4,299 — Total other income (expense), net 9,711 8,777 Net loss$(18,881) $(65,690)Other comprehensive income (loss): Unrealized gain on available-for-sale securities, net 1,208 (2,088)Comprehensive loss$(17,673) $(67,778)Net loss per common share, basic and diluted$(0.19) $(0.68)Weighted–average common shares used to compute basic and diluted net loss per share 98,054,687 96,343,529 Condensed Consolidated Balance Sheets(in thousands)(unaudited) March 31, December 31, 2023 2022 Assets Current assets: Cash and cash equivalents$42,020 $61,333 Accounts receivable 13,500 38,480 Short-term investments 366,878 374,894 Prepaid expenses and other current assets 15,490 27,367 Total current assets 437,888 502,074 Long-term investments 3,912 4,942 Operating lease right-of-use asset 64,682 66,069 Other long-term assets 123,854 132,476 Total assets$630,336 $705,561 Liabilities and stockholders’ equity Current liabilities: Accounts payable and accrued expenses$41,368 $62,197 Deferred revenue, current portion 1,738 42,226 CIRM award liability, current portion — 4,000 Operating lease liability, current portion 5,545 5,628 Total current liabilities 48,651 114,051 Operating lease liability, net of current portion 102,070 103,710 Stock price appreciation milestones, net of current portion 2,143 3,861 Stockholders’ equity 477,472 483,939 Total liabilities and stockholders’ equity$630,336 $705,561 Contact:Matthew GuidoStern Investor Relations, Inc.212.362.1200matthew.guido@sternir.com