药物资讯  >> Vol. 10 No. 3 (May 2021)

CD47分子抑制剂在肿瘤治疗中的应用
Application of CD47 Inhibitor in Tumor Therapy

DOI: 10.12677/PI.2021.103017, PDF, HTML, XML, 下载: 27  浏览: 88 

作者: 李嘉欣, 刘 煜*:中国药科大学生命科学与技术学院,江苏 南京

关键词: CD47SIRPα巨噬细胞免疫治疗CD47 SIRPα Macrophage Immunotherapy

摘要: CD47是一种在多种肿瘤细胞表面高表达的免疫检查点分子。CD47与SIRPα的结合可产生抑制信号,从而降低巨噬细胞的活力并抑制非特异性免疫系统。通过靶向CD47的抑制剂阻断CD47-SIRPα通路的方法是近年来肿瘤免疫治疗的一大热点,包括单克隆抗体、双特异性抗体,核酸类药物与小分子抑制剂等,已在多项研究中表现出显著的抑瘤效果。
Abstract: CD47 is an immune checkpoint molecule overexpressed widely across tumor types. The binding of CD47 to SIRPα can generate inhibitory signals, thereby reducing the activity of macrophages and suppressing the non-specific immunity. Recently, blocking the CD47-SIRPα axis by CD47 inhibitors including monoclonal antibodies, bispecific antibodies, nucleic acid drugs and small molecule inhibitors has been hotspot in tumor immunotherapy and shown significant tumor suppression effects in a number of studies.

文章引用: 李嘉欣, 刘煜. CD47分子抑制剂在肿瘤治疗中的应用[J]. 药物资讯, 2021, 10(3): 129-137. https://doi.org/10.12677/PI.2021.103017

1. 引言

癌症是全球的主要死亡原因,也是阻碍人类预期寿命增长的一大难题。根据世界卫生组织(WHO) 2019年的预估,癌症是人类70岁前死亡的第一或第二位因素 [1]。就总体而言,世界范围内癌症发病率和死亡率正在迅速增长。预计到2040年,全球癌症患者数目将达到2840万,比2020年增加47% [1]。传统的癌症治疗方法主要包括手术,放射疗法和化学疗法。传统疗法无法治疗所有类型和阶段的癌症,并可能降低幸存者的生活质量。由于传统治疗方式的局限性及其不利影响,癌症治疗仍然是一项临床挑战 [2] [3]。

癌症的发生、发展与机体自身的免疫系统运作息息相关。免疫系统通过T细胞、巨噬细胞,自然杀伤细胞(NK细胞)等免疫细胞发挥效应,识别并清除机体中“异己”的肿瘤细胞 [4]。当免疫效应不受控的被激活时,会对机体自身造成损害。免疫检查点是一类负向调节机体免疫应答的分子,能通过维持机体免疫耐受减少免疫应答过程中自身组织的损伤 [5]。但恶性肿瘤也利用这一点,通过在细胞表面过表达免疫检查点分子,逃脱免疫系统的“监视” [6] [7]。近年来学者们开始探索功效高,副作用少的免疫疗法。利用免疫检查点抑制剂治疗肿瘤的相关研究频频传出捷报 [8] [9],使其成为一种治疗肿瘤的重要手段。白细胞分化抗原47 (cluster of differentiation 47, CD47)作为免疫检查点之一,通过与巨噬细胞上的信号调节蛋白α (signal-regulatory protein α, SIRPα)结合从而抑制巨噬细胞的吞噬作用 [10],是继PD-1/PD-L-1之后肿瘤免疫治疗的一大热点。本文将对CD47分子的结构,功能和相关机制以及开发与利用CD47抑制剂的研究进展进行综述,为药物开发和临床研究提供参考。

2. CD47的结构

CD47 (别名IAP, MER6, OA3)是一种在所有类型的细胞中表达的跨膜蛋白,但其表达量因细胞种类而异,在年轻的红细胞和大多数癌细胞中的表达量更高 [11]。CD47属于免疫球蛋白超家族,其大小约为47 kDa,但由于存在高度糖基化结构,在SDS-PAGE中会出现在70 kDa左右的位置 [11]。CD47由位于胞外的1个N端IgV样结构域,5个跨膜螺旋体(transmembrane helices, TMH)和1个位于胞质区的C端短结构域组成(如图1),C端短结构域能被选择性剪接,从而产生四种亚型 [12]。

3. CD47的功能

血小板反应蛋白-1 (thrombospondin-1, TSP-1)是第一个被鉴定出的CD47内源性配体,其与CD47的结合可调节多种生物学过程:激活血小板,抑制血管上皮细胞NO-cGMP 信号传导,从而引起血管收缩和血管内皮细胞增生 [13],促进细胞增殖;通过促进血管生成从而促肿瘤生长 [14] 等。

Figure 1. The structure of CD47 and SIRPα: Y represents tyrosine phosphorylation sites

图1. CD47与SIRPα的结构:Y表示酪氨酸磷酸化位点

SIPRα (又名SHPS-1, CD172A, BIT, MFR, P84)是第二个被鉴定出的CD47内源性配体,在所有的髓样细胞上皆有表达。SIRPα是一种跨膜糖蛋白,其胞外N端结构域包括3个免疫球蛋白样结构域,胞质结构域包含4个酪氨酸磷酸化位点,以及2个免疫受体酪氨酸基抑制基序(immunoreceptor tyrosine-based inhibitory motif, ITIM) [11] [15]。正常细胞上所表达的CD47能通过与巨噬细胞上表达的SIRPα N端结构域结合并诱导ITIM磷酸化,从而活化不同的蛋白酪氨酸磷酸酶(如SHP1,SHP2),并激活多个细胞内分子通路,包括免疫受体酪氨酸基激活基序(immunoreceptor tyrosine-based activation motifs, ITAMs)的去磷酸化 [16] [17]。巨噬细胞中的ITAMs去磷酸化可以通过损害肌球蛋白II来防止其收缩吞噬,以避免巨噬细胞损害正常细胞。CD47和SIRPα的相互作用还介导B淋巴细胞与未激活的内皮细胞粘附,调节B细胞的聚集以及参与B淋巴细胞的再生,在免疫系统的调节中发挥着重要的作用 [18]。除了SIRPα,CD47还能与其家族成员SIRPγ结合,SIRPγ的胞外结构域与SIRPα相似,但与CD47的结合亲和力低10倍 [19],且SIRPγ的胞质结构域仅由4个氨基酸组成,没有明显的信号传导功能 [19]。

CD47也被称为整合素相关蛋白(integrin-associated protein, IAP),它可以与整合素αvβ3结合并激活下游信号通路,如几种异源三聚体G蛋白的激活:通过CD47/整联素复合物将CD47与细胞骨架1 (cytoskeleton 1)连接,从而诱导cAMP信号的激活 [20]。

4. CD47抑制剂在肿瘤治疗中的应用

早在1992年就有研究表明OV3 (即CD47)在卵巢癌中大量表达,并在邻近的正常组织中表达量较低 [21]。后续大量研究表明,CD47在多种血液瘤和实体瘤中均存在高表达的情况,包括原发性渗出性淋巴瘤 [22]、胰腺导管腺癌 [23]、非霍奇金淋巴瘤、急性淋巴细胞白血病、胶质母细胞瘤、卵巢癌、乳腺癌、结肠癌、膀胱癌、肝癌和前列腺癌等 [24],并且在多种恶性肿瘤中CD47表达增加与预后不良相关 [10] [25] [26] [27]。肿瘤细胞通过高表达CD47与巨噬细胞上的SIRPα结合,避免被巨噬细胞吞噬达到免疫逃逸的目的;其也能与树突状细胞(DC细胞)上的SIRPα结合,抑制DC细胞的吞噬作用与抗原递呈作用 [11]。综上所述,CD47抑制剂在恶性肿瘤的治疗中具有较大的应用前景。目前靶向CD47疗法在肿瘤治疗中的研究方向主要集中于单克隆抗体、双特异性抗体,核酸类药物与小分子抑制剂等。

4.1. 抗CD47单克隆抗体

研究表明,抗CD47单克隆抗体(Anti-CD47 monoclonal Antibody, Anti-CD47 mAb)通过阻断CD47-SIRPα相互作用,以增强肿瘤中巨噬细胞和其他吞噬细胞的吞噬活性 [28] [29] [30]。如小胶质细胞与中枢神经系统中的先天免疫效应细胞在使用Anti-CD47 mAb后被激活,并抑制肿瘤生长 [3]。抗体依赖性细胞介导的细胞毒效应(Antibody-dependent cell-mediated cytotoxicity, ADCC)和阻断非Fc依赖性的CD47内在功能是最为公认的抗CD47治疗机制,但谁占主导地位尚未可知。已知CD47在体内广泛表达,如果ADCC效应占主导地位,Anti-CD47 mAb疗法对正常细胞造成脱靶毒性的可能性将增高 [3]。如果由阻断非Fc依赖性的CD47内在功能占主导地位,Anti-CD47 mAb的研发应当致力于去除其ADCC效应。还有研究表明Anti-CD47 mAb能通过阻断CD47-SIRPα信号通路,增强巨噬细胞介导的抗体依赖性的细胞吞噬(antibody-dependent cellular phagocytosis, ADCP)作用,促进巨噬细胞对肿瘤细胞的吞噬 [31]。而在缺失Fc受体的情况下,Anti-CD47 mAb的F(ab') 2片段仍然具有类似的促进巨噬细胞吞噬的效果 [32]。此外,在Anti-CD47 mAb介导巨噬细胞吞噬肿瘤细胞后,肿瘤主要组织相容性复合体(major histocompatibility complex, MHC)-I特异性抗原由巨噬细胞递呈给T细胞并激活T细胞免疫反应,从而引发CD8+ T细胞毒性反应 [28] [33]。

研究表明,Anti-CD47 mAb的使用可能成为治疗血液系统恶性肿瘤的一种新方法。CD47在人急性淋巴细胞白血病(Acute lymphoblastic leukemia, ALL),急性髓样白血病(acute myeloid leukemia, AML)和多发性骨髓瘤中表达升高,并被认为与这些疾病的进展和恶化相关 [34] [35]。目前白血病主要通过骨髓移植法进行治疗,但骨髓移植疗法存在骨髓资源稀缺、自身排异性、高复发率等棘手问题 [3],因此Anti-CD47 mAb治疗白血病的研究得到广泛的关注。Mark P Chao等发现Anti-CD47 mAb消除了移植人原发性ALL小鼠模型的外周血、骨髓、脾脏和肝脏中的ALL细胞 [35];Y Wang等发现在移植人AML的小鼠模型中,Anti-CD47 mAb刺激巨噬细胞优先吞噬白血病干细胞,与阿糖胞苷联用能介导靶向性的白血病干细胞与白血病细胞的耗竭 [36]。

鉴于CD47在多种癌症类型中广泛表达,Anti-CD47 mAb治疗实体瘤的研究也层出不穷。小细胞肺癌(Small-cell lung cancer, SCLC)是一种高度侵袭性的肺癌亚型,目前可供其选择的医疗手段十分有限。Kipp Weiskopf等发现CD47在人SCLC细胞的表面高度表达。在体内试验中,与阴性对照组相比,Anti-CD47 mAb抑瘤率高达80.88%,可显著抑制肿瘤生长 [29]。三阴性乳腺癌(Triple negative breast cancers, TNBC)极易复发与转移,是乳腺癌四种亚型中最易致死的一种,并且TNBC的CD47表达明显高于其他亚型的乳腺癌 [37]。Sukhbir Kaur等发现Anti-CD47 mAb可以抑制TNBC肿瘤干细胞(cancer stem cells, CSCs)的增殖与不对称细胞分裂,并在mRNA和蛋白水平下调表皮生长因子受体(Epidermal Growth Factor Receptor, EGFR)的表达,抑制EGFR的磷酸化,故提出单独使用Anti-CD47 mAb或与EGFR抑制剂联合可能对高表达EGFR的肿瘤有效 [37]。食管鳞状细胞癌(Esophageal squamous cell cancer, ESCC)是中国最常见的一种食道癌亚型,由于其侵袭性扩散且缺乏明确的临床症状,患者死亡率非常高。CD47在ESCC细胞表面高表达并且巨噬细胞在肿瘤内部高度浸润,Zhao CL等通过体外实验发现Anti-CD47 mAb以剂量依赖性的方式促进巨噬细胞吞噬肿瘤细胞 [38],为Anti-CD47 mAb治疗ESCC的潜在应用提供了理论基础。

由于CD47在红细胞和大多数肿瘤细胞的表面高度表达,Anti-CD47 mAb发挥抑制肿瘤细胞功能的同时也会损害红细胞,导致血凝现象 [39],进而出现贫血和血小板降低的副作用。在体内实验中,大量的红细胞将成为肿瘤细胞的最佳“覆盖物”,Anti-CD47 mAb在结合肿瘤细胞之前会被红细胞耗尽。因此,如何在最大限度杀伤肿瘤细胞的同时保护红细胞是一个有待解决的问题。第一种解决方案是通过控制CD47-SIRPα通路抑制的时间来提高抗体特异性,如先对患者进行低剂量的Anti-CD47 mAb治疗以去除更容易表达CD47的老化红细胞,从而诱导机体代偿性造血 [40]。第二种解决方案是通过开发IgG4型的Anti-CD47 mAb [41] 降低抗体对红细胞和血小板的作用,但同时也会使Anti-CD47 mAb针对肿瘤细胞的杀伤能力大幅下降。第三种方案是完全放弃Anti-CD47 mAb的ADCC效应,只使用CD47-SIRPα本身的生物效应来释放巨噬细胞的抗肿瘤潜力 [42]。在这种情况下,Anti-CD47 mAb必须与其他药物联合使用,特别是那些具有强ADCC效应的药物或调节免疫力的系统性PD-1/PD-L1抑制剂药物 [43]。

4.2. 双特异性抗体

双特异性抗体(Bispecific Antibody, BsAb)是含有两种特异性抗原结合位点的合成抗体,能在靶细胞和功能分子/细胞间架起桥梁,激发具有导向性的抗肿瘤反应,在肿瘤的免疫治疗中具有广阔的应用前景。由于CD47在正常细胞上也表达,降低抗CD47抗体对正常细胞造成损伤的方法之一是构建CD47低亲和力和另一种肿瘤抗原高亲和力的双特异性抗体。

CD20/CD47 BsAb:利妥昔单抗是一种抗CD20抗体,能与免疫细胞的Fc受体结合,激活抗体诱导的促吞噬信号。由于CD20与CD47是非霍奇金淋巴瘤的治疗靶标,Piccione E. C.等构建了一种CD20/CD47 BsAb NI-1701,该双抗对CD47的亲和力较低,能降低对正常细胞的杀伤。动物实验发现NI-1701对淋巴瘤具有与联合疗法相当的抗肿瘤功效,并佐证了抗CD47单抗和利妥昔单抗获得的体内治疗协同作用 [44]。

CD19/CD47 BsAb:在非霍奇金淋巴瘤的分型中,B细胞淋巴瘤比T细胞淋巴瘤更常见,其患者人数约占所有非霍奇金淋巴瘤的85%。抗CD20抗体与化疗联用是B细胞淋巴瘤的一线治疗方法,但多达50%的患者在接受抗CD20治疗后复发并产生相应耐药 [45] [46]。CD19在B细胞恶性肿瘤中广泛表达,是一个有希望克服抗CD20耐药的靶标。Buatois, V.等在人IgG1 Fc骨架上构建了一种BsAb,包含高亲和力的CD19靶向臂与低亲和力的CD47靶向臂,以赋予完整的效应子机制:所得的双抗能靶向CD19+的癌细胞并阻断CD47与巨噬细胞表面的SIRPα相互作用,通过ADCP和ADCC效应诱导杀死肿瘤细胞 [47]。

GPC3/CD47 BsAb:GPC3是一种肝癌(hepatocellular carcinoma, HCC)相关抗原,但在临床试验中抗GPC3治疗HCC效果并不理想 [48]。将GPC3与广泛表达的免疫检查点CD47联合构建GPC3/CD47 BsAb,体外试验发现其对表达双抗原的HCC细胞诱导增强了ADCC效应,体内试验发现GPC3/CD47 BsAb优先针对表达双抗原的肿瘤细胞发挥强大的抗肿瘤活性。并且在CD47/SIRPα人源化小鼠中,该双抗具有更长的血清半衰期,且不会引起全身毒性 [49]。

PD-L1/CD47 BsAb:PD-L1是一种重要的免疫抑制分子,其在肿瘤细胞上表达,通过与免疫细胞表面的PD-1结合来抑制抗肿瘤活性。有研究表明巨噬细胞PD-1表达与吞噬活性降低有关,而阻断PD-1/PD-L1信号传导可与抗CD47抗体发挥协同作用,恢复巨噬细胞吞噬作用,以抑制肿瘤的生长 [50]。Wang Y等构建了一种具有三种优点的PD-L1/CD47 BsAb IBI322:可有效靶向肿瘤细胞;选择性阻断肿瘤细胞CD47而非正常细胞;双重抑制CD47和PD-L1信号以协同激活抗肿瘤免疫应答。该双抗为“1 + 2”结构,即抗CD47臂为F(ab)形式,抗PD-L1臂由两个单域抗体串联在一起,其对PD-L1CD47+细胞的亲和力比CD47单抗弱约10~30倍 [51]。目前IBI322已经进入治疗晚期恶性肿瘤的Ⅰ期临床试验阶段(NCT04328831) [51]。

4.3. 核酸类药物

MicroRNA (miRNA)是长度为20~25个核苷酸的非编码RNA,通过与3'非翻译区(UTR)中的互补序列结合,负调控基因的表达并参与多种生物学过程 [52]。有研究表明,miRNA可通过调节影响免疫系统的关键基因调节免疫网络 [53]。miR-200a是miR-200家族的重要成员,在多种癌症中起着抑癌作用 [19] [20] [21]。Zhao Y等证明了miR-200a通过下调CD47阻止免疫逃逸来促进鼻咽癌细胞吞噬作用,并提出miR-200a在转录后水平上阻断CD47,可以与抗CD47抗体协同作用来影响蛋白质水平,进一步提高其功效并减少并发症 [53]。此外,多种肿瘤中的CD47表达还受到包括miR-133a、miR-155,miR-708和miR-141在内的miRNA调控 [54] [55] [56] [57]。但开发安全有效的序列特异性miRNA拮抗剂,并选择合适的给药途径仍然面临很大的挑战 [58]。

4.4. 小分子抑制剂

与抗体治疗相比,小分子化合物具有代谢半衰期短,灵活的体内分布与分子量小而更易进入细胞等优点 [59]。目前针对CD47/SIRPα轴的小分子抑制剂包括直接阻断CD47与SIRPα结合,在转录、翻译和翻译后修饰(Post-Translational Modifications, PTM)水平破坏CD47/SIRPα的抑制剂。NCGC00138783,pep-20和D4-2是三种直接抑制CD47和SIRPα之间相互作用的抑制剂;RRx-001、二甲双胍、4-甲基伞形酮,JQ1和吉非替尼可在转录或翻译水平抑制CD47表达,其中RRx-001已进入III期临床试验 [59] [60];SEN177和PQ912在可PTM水平调节CD47的表达。尽管靶向CD47的小分子抑制剂研究前景辽阔,小分子抑制剂调节CD47在癌症以及其他疾病中的具体机制仍然未知。对于上述的许多小分子抑制剂,仍缺乏体内和临床数据。

5. 总结与展望

肿瘤细胞表面的CD47可与巨噬细胞上的SIRPα结合,从而抑制巨噬细胞对其进行吞噬。靶向CD47的抑制剂可通过激活巨噬细胞来激活先天免疫,同时激活特异性CD8+ T细胞以激活适应性免疫系统,发挥双重作用。目前靶向CD47的抑制剂类型主要有单克隆抗体、双特异性抗体、核酸类药物与小分子抑制剂等,在肿瘤治疗领域有着令人看好的前景。但CD47在红细胞、血小板表面同样高表达,导致CD47抑制剂可能具有贫血与血小板降低的毒副作用。如何在降低CD47抑制剂毒性的同时保持甚至其提高抗肿瘤的能力,是CD47抑制剂应用于临床肿瘤治疗急需攻克的难题。目前可通过构建CD47低亲和力+肿瘤特异性抗原高亲和力双抗,与低剂量抗CD47单抗清除老化红细胞等方法降低抗CD47疗法对正常细胞的损伤。尽管至今尚未有一款靶向CD47的产品获得上市许可,但据NextPharma数据库显示,目前全球有超过50个在研抗CD47疗法,CD47抑制剂在肿瘤治疗中的应用具有极大潜力。

NOTES

*通讯作者。

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