Cell–drug conjugates 细胞-药物结合物

By offering spatiotemporal control over the distribution and release of drugs, advanced drug-delivery systems improve the efficacy of treatments and reduce their side effects^1,2,3,4,5. Drug-delivery systems have been formulated as liposomes⁶, lipid nanoparticles⁷, polymeric particles^8,9, albumin nanoparticles¹⁰ and other vehicles and devices^11,12. Drugs can also be conjugated to peptides^13,14, antibodies^15,16 or polymers¹⁷, to enhance the transportation of the drug and the control of its release. In this context, the conjugation of therapeutic drugs to cells has emerged as a new class of drug-delivery system^18,19,20,21.
通过对药物的分布和释放进行时空控制，先进的药物递送系统提高了治疗的有效性并减少了副作用 1,2,3,4,5。药物递送系统已被制备为脂质体 6、脂质纳米颗粒 7、高分子颗粒 8,9、白蛋白纳米颗粒 10 以及其他载体和装置 11,12。药物还可以与肽 13,14、抗体 15,16 或高分子 17 结合，以增强药物的运输和释放控制。在这种背景下，治疗药物与细胞的结合已成为一种新型药物递送系统 18,19,20,21。

The characteristics and functions of cells (such as migration or the secretion of specific factors in response to cues from their microenvironment^{22,23,24,25,26}) have inspired the two main approaches for preparing cell-based drug-delivery systems: cell encapsulation and cell conjugation. Drugs can be encapsulated into cellular Trojan horses through passive diffusion and phagocytosis^27,28,29,30; for instance, doxorubicin (DOX)^31,32, iron oxide nanoparticles³³ and tirapazamine³⁴ can be encapsulated into macrophages for tumour-targeted drug delivery. Moreover, covalent linkers, non-covalent attachment or genetic engineering can be used to anchor small-molecule and macromolecular drugs onto unmodified or engineered cells to form cell–drug conjugates (CDCs) (Fig. 1).
细胞的特征和功能（例如，迁移或对微环境信号的特定因子的分泌 22,23,24,25,26）激发了两种主要的细胞基药物递送系统的制备方法：细胞包封和细胞结合。药物可以通过被动扩散和吞噬作用被包封到细胞特洛伊木马中 27,28,29,30；例如，阿霉素（DOX）31,32、氧化铁纳米颗粒 33 和替拉帕唑 34 可以被包封到巨噬细胞中以实现肿瘤靶向药物递送。此外，化学共价连接剂、非共价结合或基因工程可以用于将小分子和大分子药物锚定到未修饰或工程化的细胞上，以形成细胞-药物结合物（CDCs）（图 1）。

Representative cell types, cell sources, drug formats, drug formulations and applications. CDCs can be administrated by intravenous injection or implantation through a scaffold. HLA, human leukocyte antigen; HSC, haematopoietic stem cell; NK, natural killer; PMP, platelet-derived microparticle.
代表性细胞类型、细胞来源、药物形式、药物配方和应用。CDC 可以通过静脉注射或通过支架植入进行给药。HLA，人类白细胞抗原；HSC，造血干细胞；NK，自然杀伤细胞；PMP，血小板衍生微粒。

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Because CDCs retain the physiochemical plasticity of drugs and the physiological functions of cells, they can be designed for specific treatment needs and disease contexts³⁵. When designing CDCs, the pharmacokinetics of drugs can be improved by leveraging many inherent functions of cells, such as the long circulatory retention of erythrocytes, the wound-targeting capability of platelets and the ability of some types of T cell to traverse the blood–brain barrier (BBB)^23,36. Moreover, by leveraging CDCs, drugs can be released in response to pathological or physiological signals from their cell carriers or from the targeted sites of delivery^37,38. CDCs making use of T cells, natural killer cells, dendritic cells or macrophages can also be designed with drugs that enhance the physiological functions of these cells, to amplify their cell killing or antigen presentation abilities^39,40,41,42. To date, CDCs have been designed to treat cancers, diabetes, cardiovascular diseases and idiopathic pulmonary fibrosis in animal models and are being recognized as a distinct branch of cytopharmaceutical^27,43,44,45,.
由于细胞药物载体（CDCs）保留了药物的物理化学可塑性和细胞的生理功能，因此可以根据特定的治疗需求和疾病背景进行设计。当设计 CDCs 时，可以利用细胞的许多固有功能来改善药物的药代动力学，例如红细胞的长循环滞留、血小板的伤口靶向能力以及某些类型 T 细胞穿越血脑屏障（BBB）的能力。此外，通过利用 CDCs，药物可以根据其细胞载体或靶向输送部位的病理或生理信号释放。利用 T 细胞、自然杀伤细胞、树突状细胞或巨噬细胞的 CDCs 也可以设计成与增强这些细胞生理功能的药物结合，以增强其细胞杀伤或抗原呈递能力。迄今为止，CDCs 已被设计用于在动物模型中治疗癌症、糖尿病、心血管疾病和特发性肺纤维化，并被视为细胞药物学的一个独特分支。

In this Review, we first classify CDCs according to cell type and designed functions, and describe their main preparation methods: covalent modification, non-covalent attachment and genetic engineering. We then summarize the rationale for the design of CDCs for the treatment of cancer, autoimmune diseases and other pathologies. We end by discussing opportunities and challenges in the further development of CDCs.
在本综述中，我们首先根据细胞类型和设计功能对 CDC 进行分类，并描述其主要制备方法：共价修饰、非共价附着和基因工程。然后，我们总结了设计 CDC 用于治疗癌症、自身免疫疾病和其他病理的理由。最后，我们讨论了 CDC 进一步发展的机遇和挑战。

Erythrocytes, platelets, stem cells, leukocytes, tissue-resident immune cells and other cell types have been used to construct CDCs. In some CDCs, the cell has a central therapeutic role and the conjugated drug serves as an adjuvant to the cell’s function. Typically, the cell serves as a vehicle to deliver the drug, which renders CDCs three main capabilities: prolonged retention in circulation, permeation through certain physiological barriers and targeted delivery to specific organs or tissues^25,44 (Fig. 1).
红细胞、血小板、干细胞、白细胞、组织驻留免疫细胞及其他细胞类型已被用于构建细胞药物复合物（CDCs）。在某些 CDCs 中，细胞具有中心治疗作用，而结合的药物则作为细胞功能的辅助剂。通常，细胞作为药物的载体，从而赋予 CDCs 三种主要能力：在循环中延长滞留时间、穿透某些生理屏障以及定向输送到特定器官或组织 25,44（图 1）。

Prolonged retention in circulation
在循环中延长滞留时间

For most pharmacological agents, short periods in circulation often impair treatment efficacy and amplify any off-target toxicity. Also, to attain sustained effects (as may be required for the treatment of chronic diseases), a limited circulation time implies frequent and repetitive dosing. Prolonging a drug’s circulation time can therefore improve its therapeutic index by increasing the drug’s exposure to target tissues⁴⁶.
对于大多数药理学药物，短时间的循环往往会降低治疗效果并加剧任何非靶向毒性。此外，为了获得持续的效果（这可能是治疗慢性疾病所需的），有限的循环时间意味着需要频繁和重复给药。因此，延长药物的循环时间可以通过增加药物对靶组织的暴露来改善其治疗指数。

Erythrocytes, platelets and monocytes have been used to prolong the circulation of drugs. The lifespan of erythrocytes in circulation is about 120 d in the human body and 40 d in mice^45,46. For platelets, it is approximately 8–10 d in humans⁴⁷ and 3–5 d in mice⁴⁸. The biconcave shape of mature erythrocytes (~7 μm in diameter)⁴⁵ and disk-like shape of platelets⁴⁷ provide sufficient accessible area for drug conjugation. Erythrocytes have been used to boost the retention of encapsulated enzymes⁴⁹ or coupled antigens and nanocarriers^50,51,52,53 in blood. Platelet-conjugated antibodies have displayed longer circulation time than native antibodies⁵⁴. Human classical monocytes have a lifespan of about 1 d⁵⁵ and mouse monocytes have a half-life of 20 h⁵⁶ in circulation before differentiating into macrophages, whose lifespan ranges from several months to years. Drugs have also been conjugated to macrophages to prolong the drug’s circulation time⁵⁷.
红细胞、血小板和单核细胞已被用于延长药物的循环时间。红细胞在人体内的循环寿命约为 120 天，而在小鼠中为 40 天。对于血小板，人体内约为 8-10 天，小鼠中为 3-5 天。成熟红细胞的双凹形状（直径约 7 微米）和血小板的盘状形状提供了足够的可接触面积用于药物结合。红细胞已被用于增强包封酶或耦合抗原和纳米载体在血液中的保留。与原生抗体相比，血小板结合的抗体显示出更长的循环时间。人类经典单核细胞的寿命约为 1 天，而小鼠单核细胞在分化为巨噬细胞之前的循环半衰期为 20 小时，巨噬细胞的寿命范围从几个月到几年。药物也已与巨噬细胞结合，以延长药物的循环时间。

Permeation through physiological barriers
通过生理屏障的渗透

The BBB and other physiological barriers are obstacles to pharmacotherapeutic interventions. The BBB plays a central role in protecting the central nervous system from environmental interferences; it guarantees homeostasis^58,59 and prevents the entry of 98% of small-molecule drugs and almost all of the macromolecules into the brain. The reduced permeability of the BBB is orchestrated by tightly junctional endothelial cells, the basal lamina, pericytes and astrocytic endfeet⁵⁸. Another large contributor to the reduced transportation of drugs into brain tissue is the low expression of efflux transporters by endothelial cells and tumour cells⁶⁰. Similarly, other tissues throughout the body (such as the bone marrow) are protected by other physiological barriers (for the bone marrow, the blood–bone marrow barrier⁶¹).
血脑屏障（BBB）及其他生理屏障是药物治疗干预的障碍。血脑屏障在保护中枢神经系统免受环境干扰方面发挥着核心作用；它保证了体内稳态，并防止 98%的小分子药物和几乎所有大分子进入大脑。血脑屏障的低通透性是由紧密连接的内皮细胞、基底膜、周细胞和星形胶质细胞的足突共同调控的。内皮细胞和肿瘤细胞对药物外排转运蛋白的低表达也是导致药物进入脑组织运输减少的重要因素。同样，身体其他组织（如骨髓）也受到其他生理屏障的保护（对于骨髓而言，是血-骨髓屏障）。

Cells used in CDCs can bypass some physiological barriers. For example, leukocytes can migrate along a chemoattractant gradient through amoeboid movement⁶². This process is initiated with the strapping and rolling of leukocytes on the membranes of endothelial cells, followed by interaction of membrane integrins: leukocyte function-associated antigen 1 interacts with intercellular adhesion molecule 1, and very late antigen 4 interacts with vascular cell adhesion molecule 1. Stem cells, which also possess specific adhesion molecules (CD44, CD99 and the integrins α₄ and β₁) and chemokine receptors (such as the C–X–C motif chemokine receptor 4 and the C–C motif chemokine receptor 2), can also undergo rolling, adhesion and transmigration⁶³. The specific molecular mechanisms behind these processes are not yet fully understood.
在 CDC 中使用的细胞可以绕过一些生理屏障。例如，白细胞可以通过变形虫运动沿着趋化因子梯度迁移。这个过程始于白细胞在内皮细胞膜上的粘附和滚动，随后膜整合素之间发生相互作用：白细胞功能相关抗原 1 与细胞间粘附分子 1 相互作用，极晚抗原 4 与血管细胞粘附分子 1 相互作用。干细胞也具有特定的粘附分子（CD44、CD99 以及整合素α4 和β1）和趋化因子受体（如 C–X–C 基序趋化因子受体 4 和 C–C 基序趋化因子受体 2），也可以经历滚动、粘附和穿越。这些过程背后的具体分子机制尚未完全理解。

Such active cell migration can be applied to transport cargo across physiological barriers. The ability of leukocytes and stem cells to cross the BBB under pathological conditions or disease states offers the potential to extend the effectiveness of drugs conjugated to them, as these cells can recognize inflammatory signals secreted by pathological brain cells and cross the BBB via paracellular and transcellular pathways⁶⁰. In fact, some cells can permeate the BBB to access brain tumours^64,65,66. Also, senescent neutrophils with increased expression of C–X–C motif chemokine receptor 4 and decreased expression of C–X–C motif chemokine receptor 2 return to the bone marrow for apoptosis and thus can hitchhike drugs to it⁶⁷.
这种主动细胞迁移可以用于跨越生理屏障运输货物。白细胞和干细胞在病理条件或疾病状态下穿越血脑屏障的能力为延长与它们结合的药物的有效性提供了潜力，因为这些细胞能够识别病理性脑细胞分泌的炎症信号，并通过细胞旁和细胞内途径穿越血脑屏障。事实上，一些细胞可以穿透血脑屏障以接触脑肿瘤。此外，具有增加的 C–X–C 基序趋化因子受体 4 表达和减少的 C–X–C 基序趋化因子受体 2 表达的衰老中性粒细胞返回骨髓进行凋亡，从而可以搭载药物到达骨髓。

Importantly, the retention of drugs on the cell membrane of CDCs, especially in CDCs using hyperphagocytic macrophages (these cells can internalize conjugated therapeutics effectively⁶⁸) needs to be extensively characterized so that a drug cargo with appropriate size and shape and suitable orientations can be designed^69,70,71.
重要的是，需要对 CDC 细胞膜上药物的保留进行广泛表征，特别是在使用超吞噬巨噬细胞的 CDC 中（这些细胞可以有效地内化结合的治疗药物 68），以便设计出具有适当大小和形状以及合适取向的药物载体 69,70,71。

Targeted delivery to specific organs or tissues
靶向递送至特定器官或组织

CDCs can be constructed to facilitate the spatiotemporal migration, metabolism and elimination pathways of the conjugated drug, so as to reduce the drug’s off-target effects⁷². For example, erythrocyte–drug conjugates can be used to target the spleen and liver, particularly local macrophages^50,73 in these tissues, as erythrocytes are cleared from the circulation by splenic and hepatic phagocytes. Other CDCs can be used to specifically target other tissues; in particular, mesenchymal stem cells can actively migrate towards injured tissues or home to bone marrow^74,75,76, whereas platelet–drug conjugates prefer to accumulate at wound sites or inflammatory sites because of the associated collagen exposure^77,78,79; T cells and monocytes are attracted by specific tumour-related chemokines, cytokines and growth factors^80,81,82; neutrophils, monocytes and other immune cells migrate towards inflammatory sites^83,84; and dendritic cells can migrate to lymph nodes and have been used as vaccines for cancer immunotherapy⁸⁵. Additionally, bacteria may migrate towards aerobic or hypoxic environments, or may be attracted by an externally applied magnetic field; they may also migrate along chemoattractant gradients surrounding tumours^{86,87,88,89,90}.
CDC 可以构建以促进结合药物的时空迁移、代谢和消除途径，从而减少药物的脱靶效应。例如，红细胞-药物结合物可以用于靶向脾脏和肝脏，特别是这些组织中的局部巨噬细胞，因为红细胞被脾脏和肝脏的吞噬细胞清除。其他 CDC 可以用于特定靶向其他组织；特别是间充质干细胞可以主动迁移到受损组织或归巢到骨髓，而血小板-药物结合物则倾向于在伤口或炎症部位积聚，因为与之相关的胶原暴露；T 细胞和单核细胞受到特定肿瘤相关趋化因子、细胞因子和生长因子的吸引；中性粒细胞、单核细胞和其他免疫细胞向炎症部位迁移；树突状细胞可以迁移到淋巴结，并已被用作癌症免疫治疗的疫苗。 此外，细菌可能会向有氧或缺氧环境迁移，或可能受到外部施加的磁场吸引；它们也可能沿着肿瘤周围的化学吸引物梯度迁移。

Cells exerting therapeutic functions
具有治疗功能的细胞

In addition to their transportation and targeting capabilities, cells in CDCs may intrinsically serve as pharmacological agents. In particular, stem cells have been used to promote engraftment, to repair wounds and to treat ischaemic diseases^91,92,93. For example, a stem cell conjugated to an inflammation modulatory stimulator (such as tumour necrosis factor α (TNFα)) can act as the primary therapeutic entity (the release of TNFα can trigger the stem cells to secrete multiple muscle repair factors⁹⁴).
除了它们的运输和靶向能力，CDC 中的细胞可能本质上也充当药理学剂。特别是，干细胞已被用于促进移植、修复伤口和治疗缺血性疾病 91,92,93。例如，结合了炎症调节刺激物（如肿瘤坏死因子α（TNFα））的干细胞可以作为主要治疗实体（TNFα的释放可以触发干细胞分泌多种肌肉修复因子 94）。

CD8⁺ T cells and natural killer cells can eliminate exogenous cells, tumour cells or cells infected by bacteria or viruses^{95,96,97,98,99,100}. Regulatory T cells (T_reg cells) can suppress the activity of autoreactive CD4⁺ and CD8⁺ T cells to prevent autoimmune diseases^101,102, and tumour-associated macrophages with the M2 phenotype promote tumour progression and metastasis¹⁰³. Bacteria-induced inflammation can contribute to antitumour immunity^104,105 (notably, Salmonella typhimurium can kill tumour cells directly, or indirectly by activating CD8⁺ T cells and natural killer cells^106,107). Bacteria can promote the capability of dendritic cells to cross-present tumour cell antigens and to inhibit T_reg cells and suppress the expression of immunosuppressive enzymes^87,108. Therefore, CDCs that incorporate any of these cells can be designed to release stimulatory factors that can enhance the cells’ proliferation, persistence or activity, or to foster a phenotype transformation that leads to therapeutic function (Fig. 1).
CD8+ T 细胞和自然杀伤细胞可以消灭外源细胞、肿瘤细胞或被细菌或病毒感染的细胞 95,96,97,98,99,100。调节性 T 细胞（Treg 细胞）可以抑制自反应性 CD4+和 CD8+ T 细胞的活性，以防止自身免疫疾病 101,102，而具有 M2 表型的肿瘤相关巨噬细胞促进肿瘤进展和转移 103。细菌诱导的炎症可以促进抗肿瘤免疫 104,105（值得注意的是，沙门氏菌可以直接杀死肿瘤细胞，或通过激活 CD8+ T 细胞和自然杀伤细胞间接杀死肿瘤细胞 106,107）。细菌可以促进树突状细胞交叉呈递肿瘤细胞抗原的能力，并抑制 Treg 细胞，抑制免疫抑制酶的表达 87,108。因此，结合这些细胞的 CDC 可以被设计为释放刺激因子，以增强细胞的增殖、持久性或活性，或促进导致治疗功能的表型转变（图 1）。

CDCs incorporating small-molecule drugs, peptide drugs or protein drugs have been constructed by tethering the drug to proteins, lipids or carbohydrates at the cell’s membrane. The retention of the drug at the cell’s surface is challenging because the plasma membrane is dynamic; it undergoes constant internalization, replacement and degradation^109,110,111. Techniques for producing CDCs must therefore be performed in an environment that preserves the integrity of the cell membrane and of the cell’s inherent physiological functions and pharmacological activity. Functional CDCs are typically created via covalent modification, non-covalent attachment or genetic engineering (Fig. 2).
通过将小分子药物、肽药物或蛋白药物连接到细胞膜上的蛋白质、脂质或碳水化合物，构建了 CDC。由于质膜是动态的，经历着不断的内化、替换和降解，因此药物在细胞表面的保留是具有挑战性的。因此，生产 CDC 的技术必须在保持细胞膜完整性及细胞固有生理功能和药理活性的环境中进行。功能性 CDC 通常通过共价修饰、非共价结合或基因工程创建（图 2）。

a–c, Schematics of covalent modification (a), non-covalent attachment (b) and genetic engineering (c), including (1) N-hydroxysuccinimide and primary amine reaction; (2) maleimide–thiol reaction; (3) azido–dibenzocyclooctyne reaction; (4) ketone–oxyamine reaction; (5) phenylboronic acid–diol reaction; (6) HaloTag-mediated reaction (the blue pea-like icon represents the HaloTag protein); (7) sortase-mediated reaction (the purple pea-like icon represents the enzyme); (8) electrostatic interaction; (9) biotin–avidin interaction; (10) interaction between the glucose transporter and a ligand; (11) receptor–ligand recognition; (12) membrane fusion; (13) aliphatic chain and hydrophobic molecule insertion; (14) supramolecular host–guest interaction; (15) expression of membrane anchors for further modification mediated by genetic engineering; and (16) expression of protein therapeutics mediated by genetic engineering.
a–c，共价修饰的示意图（a）、非共价结合的示意图（b）和基因工程的示意图（c），包括（1）N-羟基琥珀酰亚胺与初级胺的反应；（2）马来酰亚胺与硫醇的反应；（3）叠氮基与二苯并环辛烯的反应；（4）酮与氧胺的反应；（5）苯硼酸与二醇的反应；（6）HaloTag 介导的反应（蓝色豌豆状图标代表 HaloTag 蛋白）；（7）sortase 介导的反应（紫色豌豆状图标代表酶）；（8）静电相互作用；（9）生物素与抗生物素的相互作用；（10）葡萄糖转运蛋白与配体之间的相互作用；（11）受体与配体的识别；（12）膜融合；（13）脂肪链与疏水分子的插入；（14）超分子主客体相互作用；（15）通过基因工程介导的膜锚定蛋白的表达以进行进一步修饰；（16）通过基因工程介导的蛋白治疗剂的表达。

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Covalent modification 共价修饰

Covalent bonds between a drug and the plasma membrane of a cell are generally stable. The efficiency of binding of a drug to a cell membrane primarily relies on the efficiency of the relevant chemical reactions (Fig. 3), which typically involve the functional chemical groups thiol, amine and hydroxyl, which are natively present at the cell membrane^36,39. Other reactions can be based on non-native groups introduced via metabolic reactions¹¹².
药物与细胞膜之间的共价键通常是稳定的。药物与细胞膜结合的效率主要依赖于相关化学反应的效率（图 3），这些反应通常涉及在细胞膜上天然存在的功能性化学基团，如巯基、氨基和羟基 36,39。其他反应可以基于通过代谢反应引入的非天然基团 112。

a–g, Schematics of methods for leveraging covalent reactions for the preparation of CDCs, including amine-based reaction (a), thiol-based reaction (b), ketone–oxyamine reaction (c), diol-based reaction (d), metabolic reaction (e), Halo/SNAP/CLIP tagging (f) and enzyme-mediated reaction (g). HaloTag is a modified haloalkane dehalogenase and SNAP-tag and CLIP-tag are proteins derived from human O⁶-alkylguanine-DNA alkyltransferase. ATP, adenosine triphosphate.
a–g，利用共价反应制备 CDC 的方案，包括基于胺的反应（a）、基于硫醇的反应（b）、酮-氧胺反应（c）、二醇基反应（d）、代谢反应（e）、Halo/SNAP/CLIP 标记（f）和酶介导反应（g）。HaloTag 是一种改良的卤烷脱卤酶，SNAP-tag 和 CLIP-tag 是源自人类 O6-烷基鸟嘌呤-DNA 烷基转移酶的蛋白质。ATP，腺苷三磷酸。

Full size image 完整图像

Conjugation methods are largely focused on minimizing the toxicity of the initial conjugation species and side products of the chemical reaction, and on preserving the structure of the cell membrane. Yet, improvements in the specificity of conjugation towards specific membrane components (such as proteins and polysaccharides) and in characterization of the stability of the bond in vivo are needed to ensure the integrity of CDCs under physiological environments.
结合方法主要集中在最小化初始结合物种和化学反应副产物的毒性，以及保持细胞膜的结构。然而，需要在结合对特定膜成分（如蛋白质和多糖）的特异性以及在体内结合稳定性表征方面进行改进，以确保 CDC 在生理环境下的完整性。

Primary amines can react with activated carboxylic esters to form amide bonds under physiological or weakly basic conditions (pH = ~7.2–9.0)^113,114,115. Drugs containing benzotriazolyl carbonate¹¹⁶, cyanuric chloride¹¹⁷ or aldehyde groups¹¹⁸ can also be used. Naturally, the cytotoxicity of the drug, reaction side products and any residual unreacted drug in the mixture must be considered. Maleimide–thiol Michael addition (which occurs efficiently in physiological buffer at pH 7.2 and has emerged as a strategy for the preparation of CDCs^39,119) and the thiol/disulfide exchange reaction are used for thiol-based cell-surface engineering^120,121. However, the thiol/disulfide exchange reaction generates a fragile disulfide bond in the linker, which may break down under reductive conditions (hence allowing for the glutathione-controlled detachment of a drug¹²²). For hydroxyl groups, a boronic ester bond can be formed between diol groups of the carbohydrate residues and the phenylboronic acid of the conjugated drug. This reaction takes only a few minutes to complete in phosphate buffer saline (pH = ~7.0) and can be affected by the concentration of glucose in the surrounding solution¹²³.
初级胺可以在生理或弱碱性条件下（pH ≈ 7.2–9.0）与活化的羧酸酯反应形成酰胺键。含有苯并三唑基碳酸酯、氰尿酸氯化物或醛基的药物也可以使用。自然地，必须考虑药物的细胞毒性、反应副产物以及混合物中任何未反应的残留药物。马来酰亚胺-硫醇迈克尔加成（在 pH 7.2 的生理缓冲液中高效发生，并已成为制备 CDC 的策略）和硫醇/二硫化物交换反应用于基于硫醇的细胞表面工程。然而，硫醇/二硫化物交换反应在连接体中生成脆弱的二硫化物键，该键可能在还原条件下断裂（因此允许谷胱甘肽控制药物的脱离）。对于羟基，可以在碳水化合物残基的二醇基团与结合药物的苯硼酸之间形成硼酸酯键。该反应在磷酸盐缓冲盐水中（pH ≈ 7.0）仅需几分钟即可完成，并可能受到周围溶液中葡萄糖浓度的影响。

In addition to native reactive groups, external reactive groups can facilitate the conjugation of a drug to cell membranes. For example, ketones selectively react with oxyamine groups to yield an oxime bond, generating water as the only reaction by-product¹²⁴. Thus, ketones and oxyamine groups have been functionalized on lipid vesicles and can be further immobilized on the plasma membrane via a membrane fusion method for subsequent conjugation^125,126,127. Moreover, a glycoengineering metabolic pathway can introduce sugar analogues into membrane-anchored glycoproteins, which serve as chemical tags on the cell membrane^112,128. To introduce glycoprotein tags, a two-step reaction is generally performed: first, cells are incubated with an azide- or alkyne-modified monosaccharide derived from trehalose, mannosamine, mannose, galactose or glucosamine. After glycan biosynthesis, glycoproteins and glycolipids integrated with the azide- or alkyne-modified monosaccharide group are expressed on the cell’s surface. Drugs bearing diabenzocyclooctyne, bicyclononyne and azide are then conjugated to the cell through a click reaction¹²⁹. These reactions are attractive because they conjugate a drug to a target cell without notably affecting other bystander cells that do not present glycoprotein tags. These reactions also have higher specificity and efficiency^130,131.
除了原生反应基团，外部反应基团可以促进药物与细胞膜的结合。例如，酮类选择性地与氧胺基团反应，生成一个肟键，水作为唯一的反应副产物。因此，酮类和氧胺基团已在脂质囊泡上功能化，并可以通过膜融合方法进一步固定在质膜上，以便后续结合。此外，糖工程代谢途径可以将糖类类似物引入膜锚定的糖蛋白中，这些糖蛋白作为细胞膜上的化学标签。为了引入糖蛋白标签，通常进行两步反应：首先，将细胞与来源于海藻糖、甘露胺、甘露糖、半乳糖或葡萄糖胺的叠氮或炔烃修饰的单糖孵育。经过糖苷生物合成后，整合了叠氮或炔烃修饰单糖基团的糖蛋白和糖脂在细胞表面表达。然后，带有二苯并环辛烯、双环壬烯和叠氮的药物通过点击反应与细胞结合。 这些反应具有吸引力，因为它们将药物结合到目标细胞上，而不会显著影响其他不呈现糖蛋白标签的旁观细胞。这些反应还具有更高的特异性和效率 130,131。

Sortase^132,133, biotin ligase¹³⁴, transglutaminase^135,136 and other enzymes can also be used to conjugate drugs to cells. A self-labelling enzyme system such as HaloTagging¹³⁷ has been employed to prepare CDCs. For example, the HaloTag protein (a bacterial dehalogenase) can be genetically encoded to be displayed on the cell membrane, where it covalently reacts with chloroalkane substrates⁴¹. These conjugations are often performed under a physiologically compatible environment.
Sortase132,133、生物素连接酶 134、转谷氨酰胺酶 135,136 及其他酶也可以用于将药物连接到细胞上。自标记酶系统如 HaloTagging137 已被用于制备 CDC。例如，HaloTag 蛋白（一种细菌脱卤素酶）可以通过基因编码显示在细胞膜上，在那里它与氯烷烃底物 41 发生共价反应。这些连接通常在生理兼容的环境下进行。

Non-covalent attachment 非共价结合

Although covalently anchored drugs on cellular surfaces are generally stable, covalent modification can cause a decrease in the therapeutic activity of the bound drug. CDCs can also be produced by non-covalent attachment, via electrostatic interactions, receptor–ligand conjugation, avidin–biotin conjugation, antibody–antigen recognition, host–guest complexation or hydrophobic insertion. Compared with covalent conjugation, non-covalent binding is easier and gentler to the cell (Fig. 2 and Table 1).
尽管共价锚定的药物在细胞表面通常是稳定的，但共价修饰可能会导致结合药物的治疗活性降低。CDC 还可以通过非共价结合产生，方式包括静电相互作用、受体-配体结合、亲和素-生物素结合、抗体-抗原识别、宿主-客体复合或疏水插入。与共价结合相比，非共价结合对细胞来说更容易且更温和（图 2 和表 1）。

The sialic acid residues of glycoproteins endow plasma membranes with native negative charges^138,139,140. As a result, cationic materials such as poly-l-lysine³⁷ and poly-diallyldimethyl ammonium chloride¹⁴¹, as well as cationic nanoparticles¹⁴², are electrostatically attracted towards cell surfaces. Also, polyphenol-functionalized nanocomplexes can assemble on cell surfaces mainly owing to hydrogen bonding¹⁴³. Receptor–ligand recognition arising from hydrogen bonding, van der Waals forces, electrostatic interactions, π–π interactions or hydrophobic interactions has high specificity and is fundamental in cell communication. Thus, various ligands have been used to produce ligand-modified drugs that can bind specifically to their corresponding receptors on cell membranes^144,145. Also, the avidin–biotin system has high specificity, binding affinity (K_d = 1 × 10^–15 M^–1) and stability^{146,147,148,149}. Antibodies can recognize specific antigens with high selectivity¹⁵⁰. Anti-immunoglobulin G (anti-IgG)¹⁵¹, anti-CD45 (ref. ³⁷), anti-NK1.1 (ref. ¹⁵²), anti-CR1 (ref. ¹⁵³) and hyaluronic acid⁷⁰ have all been used as drug-loaded vehicles. Host–guest interactions also provide a stable and biocompatible alternative. For example, β-cyclodextrin can bind adamantane with affinities on the order of 10⁴ to 10⁵ M^–1 and has thus been used to construct supramolecular cell conjugates¹⁵⁴.
糖蛋白的唾液酸残基赋予细胞膜固有的负电荷。因此，阳离子材料如聚-L-赖氨酸和聚二烯丙基二甲基氯化铵，以及阳离子纳米颗粒，都会通过静电作用被吸引到细胞表面。此外，功能化的多酚纳米复合物主要由于氢键的作用可以在细胞表面组装。由氢键、范德瓦尔斯力、静电相互作用、π–π相互作用或疏水相互作用引起的受体-配体识别具有高特异性，并在细胞通信中起着基础性作用。因此，各种配体已被用于生产配体修饰药物，这些药物可以特异性地结合到细胞膜上的相应受体。此外，链霉亲和素-生物素系统具有高特异性、结合亲和力（Kd = 1 × 10–15 M–1）和稳定性。抗体可以以高选择性识别特定抗原。抗免疫球蛋白 G（抗-IgG）、抗-CD45、抗-NK1.1、抗-CR1 和透明质酸均已被用作药物载体。 宿主-客体相互作用还提供了一种稳定且生物相容的替代方案。例如，β-环糊精可以与金刚烷结合，其亲和力在 104 到 105 M–1 的数量级，因此被用于构建超分子细胞结合物。

Different from the aforementioned strategies, aliphatic chains and hydrophobic ligands can also be inserted readily into the lipid bilayer of plasma membranes, therefore bypassing irreversible protein degradation caused by covalent conjugation^155,156. Because hydrophobic insertion does not alter the chemical structure of proteins and glycans, nor does it interfere with receptor–ligand interactions, it has been associated with lesser cytotoxicity than that caused by other cell modification approaches^157,158. However, drugs anchored onto cell membranes via hydrophobic insertion may detach from cell membranes¹⁵⁹ and long hydrophobic chains may slow down dissociation rates¹⁶⁰.
不同于上述策略，脂肪链和疏水配体也可以轻易地插入到细胞膜的脂质双层中，从而绕过由共价结合引起的不可逆蛋白降解 155,156。由于疏水插入不会改变蛋白质和糖类的化学结构，也不会干扰受体-配体相互作用，因此与其他细胞修饰方法相比，其细胞毒性较小 157,158。然而，通过疏水插入锚定在细胞膜上的药物可能会从细胞膜上脱落 159，而长疏水链可能会减缓解离速率 160。

Genetic engineering 基因工程

Genetic engineering can be leveraged to incorporate therapeutic proteins (via DNA or messenger RNA) into cells via electroporation or through various gene delivery carriers, such as viral vectors, lipidic vectors and polymeric vectors. The desired protein is then expressed on the cell membrane¹⁶¹. Gene carriers can overcome the physical barriers associated with the plasma membrane and subcellular compartments and thus deliver genes into subcellular sites. Lentiviral and retroviral vectors have high transfection efficiencies in vitro and can integrate genes of interest into the host cell genome for sustained protein expression; however, they can result in insertional mutagenesis and hence increase the risk of unexpected diseases¹⁶². Adenovirus-mediated transfection cannot lead to the integration of genes into the host cell genome and is mostly used for transient transfection^163,164. Emerging non-viral vectors such as lipid nanoparticles and polymer nanoparticles can diminish cytotoxicity against host cells, but they usually have a lower transfection efficiency than viral vectors^165,166. Also, cationic polymers and non-ionic polymeric surfactants are used with viral carriers to reduce the repulsion between viruses and cells, to augment the gene transfection efficiency into stem cells and macrophages, which are difficult to transfect¹⁶⁷. Electroporation has also been used to facilitate gene delivery into cells. For example, electroporation combined with chloroquine can increase the transient expression of enhanced green fluorescent protein in human marrow stromal cells from 12 to 50% (ref. ¹⁶⁸). However, damage to the cells during electroporation is always a concern.
基因工程可以利用电穿孔或通过各种基因递送载体（如病毒载体、脂质载体和聚合物载体）将治疗性蛋白（通过 DNA 或信使 RNA）引入细胞。然后，所需的蛋白在细胞膜上表达。基因载体可以克服与质膜和亚细胞区室相关的物理障碍，从而将基因递送到亚细胞位点。慢病毒和逆转录病毒载体在体外具有高转染效率，并且可以将感兴趣的基因整合到宿主细胞基因组中，以实现持续的蛋白表达；然而，它们可能导致插入突变，从而增加意外疾病的风险。腺病毒介导的转染无法将基因整合到宿主细胞基因组中，主要用于瞬时转染。新兴的非病毒载体，如脂质纳米颗粒和聚合物纳米颗粒，可以减少对宿主细胞的细胞毒性，但它们通常具有比病毒载体更低的转染效率。 此外，阳离子聚合物和非离子聚合物表面活性剂与病毒载体一起使用，以减少病毒与细胞之间的排斥，增强基因转染效率，尤其是在难以转染的干细胞和巨噬细胞中。电穿孔也被用于促进基因传递到细胞中。例如，电穿孔结合氯喹可以将人骨髓间充质细胞中增强型绿色荧光蛋白的瞬时表达从 12%提高到 50%。然而，在电穿孔过程中对细胞的损伤始终是一个关注点。

Many functional proteins (such as human leukocyte antigen E (ref. ¹⁶⁹), human leukocyte antigen G (ref. ¹⁷⁰) and CD47 (ref. ¹⁷¹)) have been genetically expressed on the surface of pluripotent stem cells to decrease their immunogenicity and thus enhance their therapeutic efficacy. Human embryonic stem cells expressing cytotoxic T lymphocyte-associated antigen 4-Ig and programmed death ligand 1 (PD-L1) have been shown to simultaneously disrupt the T cell co-stimulatory pathway and activate the T cell suppressing pathway, thereby reducing allogeneic immune rejection¹⁷². More recent studies have also shown that haematopoietic stem and progenitor cells¹⁷³ and platelets¹⁷⁴ that are genetically engineered to express a high level of PD-L1 may reverse autoimmune diseases. Briefly, lentiviruses encoding PD-L1 were collected from HEK293T cells transfected with PD-L1 plasmid, packaging plasmid and envelope plasmid and then the target cells were infected with lentiviruses encoding PD-L1 (ref. ¹⁷⁴) (the representative gene sequences of the proteins are summarized in Supplementary Table 1). Moreover, methods of genetic engineering can introduce specific membrane proteins as drug-loaded anchors for further conjugation⁴¹. To ensure that the genetically engineered functional proteins are settled on the cell membrane, additional genetic sequences for the signal peptide and transmembrane region should be incorporated in the gene sequences (an example is the Igκ signal peptide (METDTLLLWVLLLWVPGSTGD) and the transmembrane region (amino acids Ala513–Arg561) of the platelet-derived growth factor receptor used in the pDisplay vector¹⁷⁵. Moreover, the signal peptides and transmembrane regions from CD3, CD4, CD8 and CD28 have been used to generate chimaeric antigen receptors^176,177. Genetic engineering may lead to the transient or long-term expression of specific proteins, depending on the requirements of the treatment; for instance, the continuous expression of proteins can substantially elevate the dose of protein drugs and extend the duration of proteins on the cell’s surface¹⁷⁸.
许多功能性蛋白（如人类白细胞抗原 E（参考文献 169）、人类白细胞抗原 G（参考文献 170）和 CD47（参考文献 171））已在多能干细胞表面进行基因表达，以降低其免疫原性，从而增强其治疗效果。表达细胞毒性 T 淋巴细胞相关抗原 4-Ig 和程序性死亡配体 1（PD-L1）的人类胚胎干细胞已被证明能够同时破坏 T 细胞共刺激通路并激活 T 细胞抑制通路，从而减少异体免疫排斥。最近的研究还表明，经过基因工程改造以高水平表达 PD-L1 的造血干细胞和祖细胞以及血小板可能逆转自身免疫疾病。简而言之，编码 PD-L1 的慢病毒是从转染了 PD-L1 质粒、包装质粒和包膜质粒的 HEK293T 细胞中收集的，然后将编码 PD-L1 的慢病毒感染目标细胞（参考文献 174）（这些蛋白的代表性基因序列汇总在补充表 1 中）。 此外，基因工程的方法可以引入特定的膜蛋白作为药物负载锚，以便进一步结合。为了确保基因工程功能蛋白在细胞膜上沉积，应在基因序列中加入信号肽和跨膜区域的额外基因序列（例如，pDisplay 载体中使用的血小板源生长因子受体的 Igκ信号肽（METDTLLLWVLLLWVPGSTGD）和跨膜区域（氨基酸 Ala513–Arg561））。此外，来自 CD3、CD4、CD8 和 CD28 的信号肽和跨膜区域已被用于生成嵌合抗原受体。基因工程可能导致特定蛋白质的瞬时或长期表达，具体取决于治疗的需求；例如，蛋白质的持续表达可以显著提高蛋白质药物的剂量，并延长蛋白质在细胞表面的持续时间。

Many diseases and pathological conditions are characterized by metabolic, functional and structural changes¹⁷⁹. Cellular carriers can recognize and respond to specific signals accordingly⁷², allowing the CDCs to function as both cells and drugs to match therapeutic requirements. In this section, we describe examples of CDCs developed for various disease settings, in particular cancers, autoimmune diseases, inflammatory diseases, cerebral diseases and thrombosis (Tables 2 and 3).
许多疾病和病理状态的特征是代谢、功能和结构的变化 179。细胞载体能够识别并相应地响应特定信号 72，使得 CDC 既可以作为细胞又可以作为药物来满足治疗需求。在本节中，我们描述了为各种疾病环境开发的 CDC 示例，特别是癌症、自身免疫疾病、炎症性疾病、脑部疾病和血栓形成（表 2 和表 3）。

CDCs for cancer therapy 癌症治疗的 CDC

A considerable number of tumour-targeting medications have been developed to improve therapeutic efficacy. Cell-based therapies, especially regulatorily approved chimaeric antigen receptor T cell therapies for B cell lymphomas and relapsed or refractory multiple myeloma, have shown impressive treatment outcomes^180,181. However, the heterogenous microenvironment of solid tumours limits the use of these types of cytopharmaceutical, and chemotherapeutic drugs and immune checkpoint inhibitors have shown poor efficacy and severe off-target toxicity. To overcome these bottlenecks, CDCs have been explored as cancer therapies, either loaded with adjuvant drugs to regulate the function of the carrier cell or incorporating drugs that exert therapeutic function towards cancer cells or the tumour microenvironment (Fig. 4).
已经开发出大量肿瘤靶向药物以提高治疗效果。基于细胞的疗法，特别是针对 B 细胞淋巴瘤和复发或难治性多发性骨髓瘤的监管批准的嵌合抗原受体 T 细胞疗法，已显示出令人印象深刻的治疗结果。然而，实体肿瘤的异质微环境限制了这些类型的细胞药物的使用，而化疗药物和免疫检查点抑制剂的疗效较差且具有严重的脱靶毒性。为了克服这些瓶颈，CDC 被探索作为癌症疗法，或加载辅助药物以调节载体细胞的功能，或结合对癌细胞或肿瘤微环境具有治疗作用的药物（图 4）。

a–g, Schematics of CDCs used in cancer treatment, including T cell-based CDCs (a), macrophage-based CDCs (b), dendritic cell (DC)-based CDCs (c), erythrocyte-based CDCs (d), platelet-based CDCs (e), stem cell-based CDCs (f) and natural killer cell-based CDCs (g). BTB, blood–tumour barrier; Fas, a type 1 transmembrane protein; FasL, Fas ligand (a type 2 transmembrane protein); IFNγ, interferon γ; HIF-1α, hypoxia-inducible factor 1α; iNOS, inducible nitric oxide synthase; MHC II, major histocompatibility complex class II; TRAIL, TNF-related apoptosis-inducing ligand; TRAILR, TRAIL receptor; VEGF, vascular endothelial growth factor.
a–g，癌症治疗中使用的 CDC 示意图，包括基于 T 细胞的 CDC（a）、基于巨噬细胞的 CDC（b）、基于树突状细胞（DC）的 CDC（c）、基于红细胞的 CDC（d）、基于血小板的 CDC（e）、基于干细胞的 CDC（f）和基于自然杀伤细胞的 CDC（g）。BTB，血液-肿瘤屏障；Fas，一种 1 型跨膜蛋白；FasL，Fas 配体（2 型跨膜蛋白）；IFNγ，干扰素γ；HIF-1α，缺氧诱导因子 1α；iNOS，诱导型一氧化氮合酶；MHC II，主要组织相容性复合体 II 类；TRAIL，肿瘤坏死因子相关的凋亡诱导配体；TRAILR，TRAIL 受体；VEGF，血管内皮生长因子。

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CD8⁺ T cells are the primary mediators of cellular antitumour immunotherapy. One approach to realizing T cell-mediated immunotherapy is engineering and expanding tumour antigen-specific CD8⁺ T cells ex vivo and infusing them back into the patient¹⁸². However, the proportion of responders to this therapy is suboptimal, particularly for therapies against solid tumours. Hence, specific cytokines or immunomodulators have been conjugated to T cells to prolong the lifetime of the infused cells, to increase their proliferation and boost the immune responses. Lipid nanoparticles loaded with interleukin-15 super-agonist complex (IL-15SA) and IL-21 (ref. ³⁹), nanogels composed of IL-2/Fc or IL-15SA (ref. ³⁷), nanoparticles encapsulating small inhibitors such as adenosine A2A receptor-specific antagonists and a dual inhibitor of tyrosine phosphatase 1 and 2 (refs. ^183,184) and liposomes loaded with avasimibe¹⁸⁵ have all been evaluated for the levels of sustained proliferation of CD8⁺ T cells that they elicit. For example, IL-15SA and IL-21 can work synergistically to stimulate the expansion and effector function of CD8⁺ T cells³⁹. Multilamellar lipid particles encapsulating both IL-15SA and IL-21 were tethered onto CD8⁺ Pmel-1 effector T cells. Remarkably amplified proliferation, long-term persistence and enhanced levels of memory T cells were observed, with the tumour cell targeting of the T cells and their cytolytic capability remaining intact. Furthermore, the conjugated cytokines were primarily localized in the cellular carrier and showed limited effects on bystander cells. In another study, T cells carrying avasimibe (a drug that can enrich T cell membranes with cholesterol) could reach a glioblastoma and completely eradicate tumours in an animal model¹⁸⁵.
CD8+ T 细胞是细胞抗肿瘤免疫疗法的主要介导者。实现 T 细胞介导的免疫疗法的一种方法是体外工程化和扩增肿瘤抗原特异性的 CD8+ T 细胞，并将其输注回患者体内。然而，对这种疗法的响应者比例并不理想，特别是在针对实体肿瘤的疗法中。因此，特定的细胞因子或免疫调节剂已与 T 细胞结合，以延长输注细胞的生命周期，增加其增殖并增强免疫反应。加载有白细胞介素-15 超激动剂复合物（IL-15SA）和 IL-21 的脂质纳米颗粒（参考文献 39）、由 IL-2/Fc 或 IL-15SA 组成的纳米凝胶（参考文献 37）、包裹小型抑制剂如腺苷 A2A 受体特异性拮抗剂和酪氨酸磷酸酶 1 和 2 的双重抑制剂的纳米颗粒（参考文献 183,184）以及加载有阿伐西普的脂质体（参考文献 185）均已被评估其引发的 CD8+ T 细胞持续增殖水平。例如，IL-15SA 和 IL-21 可以协同作用，刺激 CD8+ T 细胞的扩增和效应功能（参考文献 39）。 多层脂质颗粒包裹了 IL-15SA 和 IL-21，并与 CD8+ Pmel-1 效应 T 细胞结合。观察到显著增强的增殖、长期持久性和提高的记忆 T 细胞水平，同时 T 细胞的肿瘤细胞靶向能力和细胞毒性保持完整。此外，结合的细胞因子主要定位于细胞载体，并对旁观细胞的影响有限。在另一项研究中，携带阿伐西普（可以使 T 细胞膜富含胆固醇的药物）的 T 细胞能够到达胶质母细胞瘤，并在动物模型中完全消灭肿瘤。

Similarly, IL-15SA-loaded nanogels have been conjugated onto T cells via electrostatic attraction and the specific recognition between CD45 and its antibody. The on-demand release of IL-15SA was triggered by reductive signals when T cells encountered and recognized tumour cells^37,186. This approach is attractive in that it targets IL-15SA release to tumours, maximizing the intratumoural IL-15SA concentration and the associated treatment efficacy to enhance T cell expansion. This method may also prevent the premature release of IL-15SA and minimize systemic off-target effects.
同样，IL-15SA 负载的纳米凝胶通过静电吸引和 CD45 与其抗体之间的特异性识别结合到 T 细胞上。当 T 细胞遇到并识别肿瘤细胞时，IL-15SA 的按需释放会被还原信号触发。这种方法的吸引之处在于它将 IL-15SA 的释放靶向于肿瘤，最大化肿瘤内 IL-15SA 的浓度及相关的治疗效果，以增强 T 细胞扩增。这种方法还可以防止 IL-15SA 的过早释放，并最小化系统性非靶向效应。

In addition to biomacromolecules, lipid nanoparticles encapsulating immunosuppressive signal-targeting small molecules have been conjugated to cells through the maleimide–thiol Michael addition¹⁸⁴. This conjugation method can induce lipid nanoparticles to selectively localize within immunological synapses, efficiently shielding T cells from multiple tumour-derived inhibitory signals. Macrophages can also substantially affect tumour immunotherapeutic responses^187,188, as M1 macrophages generally cause a positive immune response and enhance antitumour immunity by secreting pro-inflammatory biological molecules such as IL-1 and TNFα^187,189,190. However, the tumour immunosuppressive microenvironment can induce the switching of M1 macrophages towards the M2 phenotype. Yet, an interferon γ-loaded polyelectrolyte multilayer device that adhered to the macrophage surface maintained the pro-inflammatory phenotype of macrophages. These CDCs polarized tumour-associated macrophages to the M1 phenotype and led to a lower burden of metastatic colonies and inhibited tumour growth⁴².
除了生物大分子外，包裹免疫抑制信号靶向小分子的脂质纳米颗粒通过马来酰亚胺-硫醇迈克尔加成反应与细胞结合。这种结合方法可以诱导脂质纳米颗粒选择性地定位于免疫突触内，有效地保护 T 细胞免受多种肿瘤来源的抑制信号。巨噬细胞也可以显著影响肿瘤免疫治疗反应，因为 M1 巨噬细胞通常会通过分泌促炎生物分子如 IL-1 和 TNFα引发积极的免疫反应并增强抗肿瘤免疫。然而，肿瘤免疫抑制微环境可以诱导 M1 巨噬细胞向 M2 表型转变。然而，粘附在巨噬细胞表面的干扰素γ负载聚电解质多层装置维持了巨噬细胞的促炎表型。这些 CDC 将肿瘤相关巨噬细胞极化为 M1 表型，导致转移性克隆负担降低并抑制肿瘤生长。

Cells carrying chemomodulating and immunomodulating therapeutics can selectively deliver drugs to tumours. In these systems, the drugs themselves exert the therapeutic effect rather than the cellular moiety. For instance, injured or senescent erythrocytes are mainly cleared by the spleen¹⁹¹. Thus, ovalbumin, a model antigen, was coupled to 200 nm polystyrene carboxylate particles, which were then adsorbed on erythrocytes⁷³. A nanoparticle-to-erythrocyte ratio of 300:1 was optimal for efficient spleen targeting and a comprehensive immune response was observed. Because nanoparticle–erythrocyte conjugates are not stable under high shear stress, erythrocytes carrying DOX-loaded nanoparticles have been used for the treatment of pulmonary metastases of melanoma¹⁴². The same strategy has also been applied to other chemotherapeutic agents, including camptothecin, paclitaxel, gemcitabine and methotrexate, for lung-targeted cell-assisted chemotherapy¹⁹². Poly(lactic-co-glycolic acid) nanoparticles loaded with the immunotherapeutic CXCL10 have been conjugated to erythrocytes for lung-targeted transportation, establishing a local chemokine gradient to attract the migration of immune effector cells towards the lungs and to activate adaptive immunity¹⁹³. Platelets can capture circulating tumour cells and target metastases via the transmembrane protein P-selectin and CD44, and cytotoxic complexes loaded with granzyme B and perforin have been conjugated to platelets to eliminate circulating tumour cells and suppress lung metastasis¹⁹⁴. Inspired by the tumour-targeting ability of bacteria, DOX³⁸, rhenium¹⁹⁵ and gold nanoparticles¹⁹⁶ have also been coupled to bacterial surfaces for tumour-targeted delivery. Furthermore, bacteria carrying SN-38-loaded liposomes have been employed to deliver drugs into tumours via an external weak magnetic field¹⁹⁷. The bacteria can overexpress the respiratory chain enzyme II (via genetic engineering) and carry Fe₃O₄ (via covalent modification), thus generating reactive oxygen species in tumours and thereby anticancer effects¹⁹⁸.
携带化学调节和免疫调节治疗药物的细胞可以选择性地将药物输送到肿瘤。在这些系统中，药物本身发挥治疗效果，而不是细胞部分。例如，受损或衰老的红细胞主要由脾脏清除。因此，模型抗原卵白蛋白被偶联到 200 纳米的聚苯乙烯羧酸盐颗粒上，然后被吸附在红细胞上。纳米颗粒与红细胞的比例为 300:1 时，能够实现有效的脾脏靶向，并观察到全面的免疫反应。由于纳米颗粒-红细胞结合物在高剪切应力下不稳定，因此携带 DOX 负载纳米颗粒的红细胞已被用于治疗黑色素瘤的肺转移。同样的策略也已应用于其他化疗药物，包括喜树碱、紫杉醇、吉西他滨和甲氨蝶呤，以实现肺靶向细胞辅助化疗。 负载免疫治疗剂 CXCL10 的聚乳酸-聚乙烯醇酸（PLGA）纳米颗粒已与红细胞结合，以实现肺部靶向运输，建立局部趋化因子梯度以吸引免疫效应细胞向肺部迁移并激活适应性免疫。血小板可以通过跨膜蛋白 P 选择素和 CD44 捕获循环肿瘤细胞并靶向转移灶，负载有颗粒酶 B 和穿孔素的细胞毒性复合物已与血小板结合，以消除循环肿瘤细胞并抑制肺部转移。受到细菌肿瘤靶向能力的启发，DOX、铼和金纳米颗粒也已与细菌表面结合以实现肿瘤靶向递送。此外，携带 SN-38 负载脂质体的细菌已被用于通过外部弱磁场将药物递送到肿瘤中。这些细菌可以通过基因工程过表达呼吸链酶 II，并通过共价修饰携带 Fe3O4，从而在肿瘤中产生活性氧物质，从而产生抗癌效果。

Blocking programmed cell death protein 1 (PD-1) on T cells from binding PD-L1 on cancer cells and antigen-presenting cells to reactivate antitumour responses has resulted in impressive clinical outcomes^{199,200,201,202}. However, the suboptimal response rates and undesirable side effects of this type of therapy require new treatment strategies associated with delivery techniques^203,204. One option is to use platelets, which target wounds as well as circulating tumour cells^205,206,207. Conjugates of anti-PD-L1 antibody and platelets can deliver anti-PD-L1 to residual cancer cells post-surgery to inhibit tumour recurrence^54,208 and integrating blood vessel-disrupting agents with anti-PD-L1 antibody–platelet conjugates led to a tenfold increase in accumulated antibody in lung metastasis²⁰⁹. Notably, platelets can be activated by Toll-like receptor 4-binding mediators and generate platelet-derived microparticles from their plasma membrane, thereby increasing the exposure of antibody to residual tumour cells²⁰⁵. Additionally, PD-1-presenting platelets can be obtained by genetically engineering megakaryocyte progenitor cells. Such therapeutic platelets were loaded with cyclophosphamide as a combination for the inhibition of tumour relapse after surgery²¹⁰. Another approach for targeted delivery was developed for the treatment of acute myeloid leukaemia—a malignant disease characterized by abnormal myeloblast expansion in the bone marrow. Conjugation of anti-PD-1 antibody to platelets that are then attached to the surface of haematopoietic stem cells via a click reaction yielded a conjugate¹³¹ that, on intravenous injection, migrated to bone marrow and led to a 25-fold increase in antibody signal (with anti-PD-1 antibody exerting its checkpoint-blocking function).
阻断程序性细胞死亡蛋白 1（PD-1）在 T 细胞上与癌细胞和抗原呈递细胞上的 PD-L1 结合，以重新激活抗肿瘤反应，已取得显著的临床效果。然而，这种疗法的亚最佳反应率和不良副作用需要与给药技术相关的新治疗策略。一个选择是使用靶向伤口以及循环肿瘤细胞的血小板。抗 PD-L1 抗体与血小板的结合物可以在手术后将抗 PD-L1 输送到残余癌细胞，以抑制肿瘤复发，并且将破坏血管的药物与抗 PD-L1 抗体-血小板结合物结合，导致肺转移中抗体的积累增加十倍。值得注意的是，血小板可以通过结合 Toll 样受体 4 的介质被激活，并从其质膜生成血小板衍生微粒，从而增加抗体对残余肿瘤细胞的暴露。此外，可以通过基因工程改造巨核细胞祖细胞获得呈现 PD-1 的血小板。 这种治疗性血小板负载了环磷酰胺，作为手术后抑制肿瘤复发的组合方案 210。另一种针对急性髓性白血病的靶向递送方法被开发出来，这是一种以骨髓中异常的髓母细胞扩增为特征的恶性疾病。将抗 PD-1 抗体与血小板结合，然后通过点击反应附着在造血干细胞表面，产生了一种结合物 131，该结合物在静脉注射后迁移到骨髓，并导致抗体信号增加 25 倍（抗 PD-1 抗体发挥其检查点阻断功能）。

CDCs consisting of cells and conjugated drugs that both have therapeutic functions may result in synergetic effects. For example, autologous polyclonal T cells expressing lymph node-homing receptors were engineered to carry SN-38-loaded nanocapsules to transport them to neoplastic lymphoid organs²¹¹. This CDC led to a 90-fold greater SN-38 accumulation at the tumour compared with SN-38 injected intravenously at a dose of tenfold, substantially reducing tumour burden and prolonging animal survival²¹¹. Similar to T cells, natural killer cells participate in innate immunity against tumours; also, they natively possess the capability to home to lymph nodes. Natural killer cells can also express or conjugate to tumour cell-targeting moieties to acquire a tumour-targeting capability. Natural killer cells have therefore been loaded with nanoparticulate carriers encapsulating DOX²¹², paclitaxel²¹³ and TNFα-related apoptosis-inducing ligands²¹⁴.
由具有治疗功能的细胞和结合药物组成的 CDC 可能会产生协同效应。例如，工程化的自体多克隆 T 细胞表达淋巴结归巢受体，携带装载 SN-38 的纳米胶囊，将其运输到肿瘤淋巴器官。这种 CDC 使得肿瘤处的 SN-38 积累量比以十倍剂量静脉注射的 SN-38 高出 90 倍，显著减少了肿瘤负担并延长了动物的生存期。与 T 细胞类似，自然杀伤细胞参与对肿瘤的先天免疫；此外，它们本身具备归巢到淋巴结的能力。自然杀伤细胞还可以表达或结合肿瘤细胞靶向部分，以获得肿瘤靶向能力。因此，自然杀伤细胞被加载了包裹 DOX、紫杉醇和 TNFα相关诱导凋亡配体的纳米颗粒载体。

CDCs for the treatment of autoimmune diseases
自体免疫疾病的 CDC 治疗

Immunosuppressive drugs for the treatment of autoimmune disorders can lead to persistent immunosuppression and increase the risk of infection and cancer^215,216. Pathology-specific CDCs as immunosuppressive therapy may increase treatment efficacy and reduce systemic side effects²¹⁷ (Fig. 5).
用于治疗自身免疫性疾病的免疫抑制药物可能导致持续的免疫抑制，并增加感染和癌症的风险 215,216。特定病理的 CDC 作为免疫抑制治疗可能提高治疗效果并减少全身副作用 217（图 5）。

a,b, Schematics of CDC-mediated immunotherapy (a) and CDC-mediated tolerance induction (b). APC, antigen-presenting cell; HSPC, haematopoietic stem and progenitor cell.
CDC 介导的免疫治疗示意图(a)和 CDC 介导的耐受诱导示意图(b)。APC，抗原呈递细胞；HSPC，造血干细胞和祖细胞。

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T_reg cells help to maintain the body’s internal homeostasis^218,219. T_reg cells suppress natural killer cells and T cells by secreting inhibitor cytokines, inducing cytolysis, interfering with cell metabolism and impairing dendritic cell maturation²²⁰. T_reg cells have been used to treat type 1 diabetes^221,222, yet maintaining their function required exogenous IL-2, which is also a stimulator for other effector T cells. Thus, covalently linking T_reg cells to IL-2/Fc-loaded nanogels may lead to enhanced function¹⁰¹. Another method for inhibiting the function of CD8⁺ T cells involves inhibitor proteins such as PD-L1, which has been genetically introduced on the surface of stem cells¹⁷³ and platelets¹⁷⁴ to reverse type 1 diabetes via PD-L1-induced T cell exhaustion²²³. Also, the inflammation-targeting capability of the two cell types promoted PD-L1 accumulation in the diseased pancreas of non-obese diabetic mice.
Treg 细胞有助于维持身体的内部稳态 218,219。Treg 细胞通过分泌抑制性细胞因子、诱导细胞溶解、干扰细胞代谢和损害树突状细胞成熟来抑制自然杀伤细胞和 T 细胞 220。Treg 细胞已被用于治疗 1 型糖尿病 221,222，但维持其功能需要外源性 IL-2，而 IL-2 也是其他效应 T 细胞的刺激因子。因此，将 Treg 细胞与 IL-2/Fc 负载的纳米凝胶共价连接可能会增强其功能 101。抑制 CD8+ T 细胞功能的另一种方法涉及抑制蛋白，如 PD-L1，已在干细胞 173 和血小板 174 的表面进行基因引入，以通过 PD-L1 诱导的 T 细胞耗竭逆转 1 型糖尿病 223。此外，这两种细胞类型的炎症靶向能力促进了 PD-L1 在非肥胖糖尿病小鼠病变胰腺中的积累。

Thyroglobulin- or insulin-coupled spleen cells can induce tolerance via the PD-1 and PD-L1 axis^224,225. Because erythrocytes are cleared daily by phagocytes and the reticuloendothelial system in the spleen, ovalbumin attached to erythrocytes has also resulted in the induction of tolerance to CD4⁺ and CD8⁺ T cells²²⁶. Immune tolerance was also achieved via Escherichia coli l-asparaginase type II–erythrocyte conjugates²²⁷. Moreover, immunogenic peptides such as the immunodominant peptide of myelin oligodendrocyte glycoprotein_35–55 and insulin B-chain peptide 9–23 have been introduced on the surface of erythrocytes to induce immune tolerance in mice for multiple sclerosis and type 1 diabetes⁵⁰ and myelin peptide-conjugated mononuclear cells have been transfused back to patients with multiple sclerosis to induce antigen-specific tolerance²²⁸ (NCT01414634).
甲状腺球蛋白或胰岛素耦合的脾细胞可以通过 PD-1 和 PD-L1 轴诱导耐受性 224,225。由于红细胞每天被脾脏中的吞噬细胞和网状内皮系统清除，附着在红细胞上的卵白蛋白也导致了对 CD4+和 CD8+ T 细胞的耐受性诱导 226。通过大肠杆菌 L-天冬氨酸酶 II 型-红细胞结合物也实现了免疫耐受 227。此外，免疫原性肽如髓鞘少突胶质细胞糖蛋白的免疫优势肽 35–55 和胰岛素 B 链肽 9–23 已被引入红细胞表面，以诱导小鼠对多发性硬化症和 1 型糖尿病的免疫耐受 50，而髓鞘肽结合的单核细胞已被输回多发性硬化症患者体内，以诱导抗原特异性耐受 228（NCT01414634）。

CDCs for treating other diseases
用于治疗其他疾病的 CDC

Inflammation is a physiological response to infection, tissue damage and other external stimuli^229,230. Inflammatory cytokines, plasma enzyme mediators and lipid inflammatory mediators play key roles in driving pro-inflammatory processes^230,231. Activation of the pro-inflammatory pathway can trigger a local or systemic inflammatory response and lead to increases in the adhesion of endothelial cells and in chemokine levels. All of these events attract circulating leukocytes to the site of infection or tissue damage. These effector cells can be leveraged to transport anti-inflammatory drugs to strengthen endogenous defences against inflammation^232,233,234. For example, macrophage-carrying catalase-loaded cargo can accumulate in brain tissue with lipopolysaccharide-induced inflammation¹⁵⁰ and nanocarriers loaded on erythrocytes have been selectively delivered to the lungs to treat pulmonary inflammation²³⁵ (the released nanocarriers were internalized by endothelial cells, leukocytes and other intravascular resident cells).
炎症是对感染、组织损伤和其他外部刺激的生理反应 229,230。炎症细胞因子、血浆酶介质和脂质炎症介质在驱动促炎过程方面发挥关键作用 230,231。促炎通路的激活可以触发局部或全身的炎症反应，并导致内皮细胞的粘附性增加和趋化因子水平的升高。所有这些事件都吸引循环中的白细胞到感染或组织损伤的部位。这些效应细胞可以被利用来运输抗炎药物，以增强机体对抗炎症的内源性防御 232,233,234。例如，携带过氧化氢酶的巨噬细胞载荷可以在脂多糖诱导的炎症中积聚在脑组织中 150，而加载在红细胞上的纳米载体已被选择性地输送到肺部以治疗肺部炎症 235（释放的纳米载体被内皮细胞、白细胞和其他血管内常驻细胞内化）。

Cerebral pathologies are associated with high morbidity, severe sequelae and high mortality²³⁶. The complex cerebral environment, especially the BBB, often inhibits the efficacy of systemically administered drugs. Insufficient blood flow further hampers the use of systemically administered drugs to reach the core and penumbra following stroke. Better delivery systems are needed to increase drug concentrations at cerebral sites^150,237 (for instance, intravenously injected platelet–microglia hybrids carrying IL-4-containing liposomes homed to injured brain sites; under ultrasound stimulation, released IL-4 stimulated microglia to become anti-inflammatory and led to positive treatment effects²³⁸).
脑部病理与高发病率、严重后遗症和高死亡率相关。复杂的脑环境，尤其是血脑屏障，常常抑制系统性给药的疗效。血流不足进一步妨碍了系统性给药药物到达中风后的核心和边缘区。需要更好的给药系统以提高药物在脑部的浓度（例如，静脉注射的携带 IL-4 含量脂质体的血小板-小胶质细胞混合物定向到受损脑部；在超声刺激下，释放的 IL-4 刺激小胶质细胞转变为抗炎状态，并产生积极的治疗效果）。

Thrombosis, which can be caused by impairment of the vascular endothelium (by abnormalities in the coagulation system and by blood rheology alterations) is a central causative factor of many cerebral and cardiovascular diseases and is associated with high morbidity and mortality^239,240. Although anticoagulants and thrombolytics, such as warfarin and heparin, have been used clinically to prevent or inhibit the formation of blood clots²⁴¹, short lifespans, bleeding risk and extravasation toxicity have limited their use. To address the limitations of standard anticoagulation, erythrocyte-conjugated anti-thrombotic agents have been shown to effectively prolong the circulation time of drugs and to allow for thromboprophylaxis¹⁴⁸. Moreover, erythrocyte–fibrinolytic agent conjugates can absorb into neointimal clots and start to dissolve the neo-thrombus from within. These studies exemplify the potential of CDCs to treat thrombotic stroke, pulmonary embolism¹⁴⁸ and cerebral ischaemia²⁴², at least in animal models.
血栓形成可能由血管内皮功能障碍（由凝血系统异常和血液流变学改变引起）导致，是许多脑血管和心血管疾病的主要致病因素，并与高发病率和死亡率相关。尽管抗凝剂和溶栓剂，如华法林和肝素，已在临床上用于预防或抑制血栓形成，但其短暂的半衰期、出血风险和外渗毒性限制了它们的使用。为了解决标准抗凝治疗的局限性，红细胞结合的抗血栓药物已被证明能够有效延长药物的循环时间，并允许进行血栓预防。此外，红细胞-溶栓剂结合物可以吸附到新生内膜血栓中，并从内部开始溶解新血栓。这些研究展示了 CDC 在治疗血栓性中风、肺栓塞和脑缺血方面的潜力，至少在动物模型中是如此。

Substantial advances at the interfaces of material science, microtechnology and nanotechnology, cell biology, bioconjugate chemistry and medicine have led to new opportunities for introducing drugs onto cells with high efficiency and precision^25,26,243. The integration of cellular physiological function with a drug’s therapeutic effect can increase the precision of targeted delivery and ultimately treatment efficacy.
在材料科学、微技术和纳米技术、细胞生物学、生物偶联化学和医学的交叉领域取得了重大进展，这为以高效率和精确度将药物引入细胞提供了新的机会。将细胞生理功能与药物的治疗效果相结合，可以提高靶向递送的精确性，最终提高治疗效果。

Designing effective CDCs requires an in-depth understanding of the biochemical properties of drugs, the physiological functions of cells and the pathological characteristics of disease. Unfortunately, insufficient knowledge of cellular physiology and disease pathology often hinders progress in the design of effective CDCs for the treatment of a specific disease. At present, only a few types of cell and drug have been used to generate CDCs, and expansion of the sources of cells and therapeutic indications is necessary. The most widely studied cells for preparing CDCs are erythrocytes, platelets, leucocytes, stem cells and bacteria. Yet, the use of tissue-resident cells, such as adipocytes, hepatic cells and neuron cells, should be explored. For example, tumour-associated adipocytes have been proven to facilitate angiogenesis²⁴⁴, energy uptake^245,246 and PD-L1 expression in tumour cells, and engineered adipocytes conjugated with immunomodulating drugs might be recruited by tumours, at which point the release of the drug would exert antitumour activity by reprogramming the tumour’s immunosuppressive microenvironment²⁴⁷.
设计有效的细胞药物复合体（CDCs）需要深入了解药物的生化特性、细胞的生理功能以及疾病的病理特征。不幸的是，对细胞生理学和疾病病理学的知识不足常常阻碍了针对特定疾病的有效 CDC 设计的进展。目前，仅有少数类型的细胞和药物被用于生成 CDC，因此有必要扩展细胞来源和治疗适应症。用于制备 CDC 的细胞中，红细胞、血小板、白细胞、干细胞和细菌是研究最广泛的。然而，组织驻留细胞，如脂肪细胞、肝细胞和神经细胞的使用应当被探索。例如，肿瘤相关脂肪细胞已被证明能够促进血管生成、能量摄取和肿瘤细胞中 PD-L1 的表达，工程化的脂肪细胞与免疫调节药物结合后可能被肿瘤招募，此时药物的释放将通过重编程肿瘤的免疫抑制微环境发挥抗肿瘤活性。

Another challenge for the translation of CDCs is the need to minimize the host’s immune response against allogeneic cells and hence the graft-versus-host reaction. Clinically, the transfusion of cells from a donor to a recipient requires a pre-transfusion test to ascertain the degree of matching of surface antigens. For example, the use of allogeneic stem cells and cells differentiated from stem cells requires matching human leukocyte antigens. Advanced technologies to block the antigens of allogenic cells may expand the use of CDCs^248,249,250. Moreover, using allogeneic cells bears a risk of infection. For autologous cells, the development of methods to expand the cells to the amounts required for therapeutic effect is crucial. Also, standard isolation and storage techniques, particularly for cell types that can be easily activated, require further optimization.
另一个关于 CDC 翻译的挑战是需要最小化宿主对异体细胞的免疫反应，从而减少移植物抗宿主反应。在临床上，从供体向受体输注细胞需要进行输注前测试，以确定表面抗原的匹配程度。例如，使用异体干细胞和从干细胞分化而来的细胞需要匹配人类白细胞抗原。阻断异体细胞抗原的先进技术可能会扩大 CDC 的使用。此外，使用异体细胞存在感染风险。对于自体细胞，开发扩增细胞至治疗效果所需数量的方法至关重要。此外，标准的分离和储存技术，特别是对于容易被激活的细胞类型，需要进一步优化。

Moreover, the inherent physiological function of the cells may not meet clinical needs. Engineering cells to enhance their relevance for treating disease may help to generate new approaches for the production of CDCs. For example, in the tumour microenvironment, myeloid-derived suppressor cells, fibroblasts, stromal cells and other cell types can influence anticancer outcomes; hence, CDCs could be engineered to target these bystander cells.
此外，细胞的固有生理功能可能无法满足临床需求。工程化细胞以增强其在治疗疾病中的相关性可能有助于产生新的 CDC 生产方法。例如，在肿瘤微环境中，髓源抑制细胞、成纤维细胞、基质细胞和其他细胞类型可以影响抗癌结果；因此，CDC 可以被工程化以靶向这些旁观细胞。

Cytotoxic drugs can enhance the target cell-killing effect directly, and the conjugated drug may also modulate the tumour microenvironment and enhance the proliferation of the carrier cells and their therapeutic activity. In cancer therapy, the drug in CDCs can further modulate the phenotype and metabolic pathways of the carrier cells, change the hypoxic or acidic microenvironment, or modify the immunogenicity of tumour cells in the tumour microenvironment to promote anticancer activity^{251,252,253,254,255,256,257}.
细胞毒性药物可以直接增强靶细胞的杀伤效果，结合药物也可能调节肿瘤微环境，增强载体细胞的增殖及其治疗活性。在癌症治疗中，CDC 中的药物可以进一步调节载体细胞的表型和代谢途径，改变缺氧或酸性微环境，或修饰肿瘤微环境中肿瘤细胞的免疫原性，以促进抗癌活性 251,252,253,254,255,256,257。

The therapeutic efficacy of CDCs can be hindered by insufficient drug-loading capacity, the degradation of the internalized drug, its uncontrolled release (typically associated with cell encapsulation strategies), the endocytosis of the drug, the detachment of the drug from the cell’s surface, and disruption of the natural function of the cell. Indeed, covering the cell’s surface with drugs may substantially alter the cell’s character and result in loss of function. These challenges can be overcome by tailoring the conjugation methods. For example, the use of maleimide–thiol linkers to conjugate ~153 particles per cell led to toxicity, whereas conjugating ~100 particles per cell did not change the proliferation and killing function of the cells³⁹. Also, introducing genes into effector cells to continuously express the therapeutic proteins on the cell’s surface may be advantageous in certain cases. Other strategies could involve using more potent drugs and leveraging methods that allow for the administrated drugs to bind carrier cells in vivo. Additionally, in future, microrobots²⁵⁸ may improve the tumour penetration and distribution of CDCs.
CDC 的治疗效果可能受到药物负载能力不足、内化药物的降解、药物的 uncontrolled release（通常与细胞包裹策略相关）、药物的内吞作用、药物从细胞表面的脱离以及细胞自然功能的破坏等因素的影响。实际上，药物覆盖细胞表面可能会显著改变细胞的特性并导致功能丧失。这些挑战可以通过定制连接方法来克服。例如，使用马来酰亚胺-硫醇连接子将每个细胞连接约 153 个颗粒会导致毒性，而将每个细胞连接约 100 个颗粒则不会改变细胞的增殖和杀伤功能。此外，在某些情况下，将基因引入效应细胞以持续在细胞表面表达治疗蛋白可能是有利的。其他策略可能涉及使用更强效的药物，并利用允许在体内将药物与载体细胞结合的方法。此外，未来微型机器人可能会改善 CDC 的肿瘤渗透和分布。

As for conjugation methods integral to the development of CDCs, the dynamics of the plasma membrane and phagocytosis hamper reliable cell-surface modification. Therefore, strategies to evade internalization of the drug are necessary. In most cases, conjugating a drug to the cell’s surface while minimizing its uptake by the cell requires the encapsulation of the drug in nanoparticles. Using carrier materials thus offers the opportunity to design drug carriers that respond to physiological signals. Such stimuli-responsive carriers and/or conjugation linkers may be leveraged to improve the spatiotemporal distribution of the drug to augment therapeutic efficacy and reduce any side effects²⁵⁹. Hence, ensuring that all conjugation materials are highly biocompatible is central for clinical translation. In addition, precise covalent conjugation to certain proteins or polysaccharides on the cell’s surface may be possible, which may allow for pre-designed drug distributions on the cell’s surface and for the minimization of interferences with cell viability and function. Furthermore, it is important to avoid compromising the integrity of the cell membrane through the drug-loading process. To this end, enzyme-catalysed reactions may be advantageous.

Genetic engineering techniques enable the introduction of therapeutic proteins on the cell’s surface without disturbing cell-membrane integrity and hence cell viability. However, genetic engineering methods are typically time consuming (whereas biochemical conjugation requires hours, genetic engineering usually requires weeks of work to construct the vectors, transfect the drug, and screen for successful conjugation). In addition, viral vectors tend to integrate the target gene into the genome and thus bear non-negligible risks of tumorigenesis and immunogenicity¹⁶². When designing cell-surface-anchoring proteins, multiple strategies can be combined skilfully on the basis of the requirements of the treatment. In Supplementary Table 2, we list the modification yield, drug stability, drug activity, and drug effects on cellular viability and cellular-function maintenance for the most relevant strategies.

Over the past few decades, several CDCs have been regulatorily approved for use in clinical trials. In particular, myelin peptide-conjugated mononuclear cells for treating multiple sclerosis were used in a phase 1 clinical trial (NCT01414634)²²⁸. IL-15-conjugated T cells for the treatment of solid tumours and lymphomas have advanced clinically (phase 1; NCT03815682). Also, IL-2-conjugated platelets for advanced malignant solid tumours have been in clinical research (NCT05829057). Erythrocyte-based CDCs have also been clinically tested: IL-15-expressing erythrocytes with a 4-1BB ligand (RTX-240; NCT04372706) for relapsed/refractory or locally advanced solid tumours and relapsed/refractory acute myeloid leukaemia; human papillomavirus peptide antigen, 4-1BB ligand and IL-12-expressing erythrocytes for advanced malignancies (RTX-321; NCT04672980); and 4-1BB ligand and IL-12-expressing erythrocytes for advanced solid tumours (RTX-224; NCT05219578). Yet, to accelerate the clinical testing of CDCs, it will be necessary to guarantee consistency in product quality and to work towards large-scale manufacturing at a low cost. Manufacturing CDCs involves many steps: typically, cell isolation, cell expansion and cell modification, all of which need to be standardized. Also, large-scale production of qualified CDCs relies on sterilization, purification and storage/transportation procedures that must ensure that the activity and stability of the CDCs are retained. Moreover, any risks associated with altered pharmacokinetics, abnormal thrombosis and uncontrolled release of the drug require thorough evaluation. In particular, undesired cell or tissue targeting or accumulation, as well as undesired immune responses, may all lead to severe side effects. Yet, when the disease is locally constrained (as in wounds and localized tumours), direct injection or scaffold-mediated transplantation of the CDCs may be the easiest strategy^208,260.