药学学报

Material	Bacteria species	Application	Ref.
PLGA	E. coli MG1655	Anti-tumor	[10]
β-Cyclodextrin-PEI600	S. typhimurium	Cancer immunotherapy	[11]
Bacterial biofilms	S. aureus, B. subtilis	Improve oral bioavailability	[13]
Red cell membrane	Listeria monocytogenes, E. coli	Anti-tumor	[14, 15]
Silk fibroin	EcN	Enhancing bioavailability and treatment efficacy	[16]
Dioleoylphosphatydic acid and cholesterol	EcN, S. aureus, E. faecalis	Enhancing bioavailability and treatment efficacy	[17]
Chitosan and alginate	B. coagulans, Bifidobacterium breve	Enhancing bioavailability and treatment efficacy	[18, 19]
Poloxham F-127	B. subtilis	Anti-epidermal fungal infection	[25]
Methacrylated gelatin, HAMA	L. reuteri	Anti-infection and promoting wound healing	[27]
Poly(vinyl alcohol), sodium alginate	Bdellovibrio bacteriovorus	Anti-infection and promoting wound healing	[28]
Gelatin, alginate	E. coli	Radiation detection	[29]
Engineered bacterial curli fibers	E. coli	Mucosal healing and immunomodulation	[30]
Chitosan/tannic acid coating and calcium alginate microspheres	L. rhamnosus	Radiation enteritis and ulcerative colitis	[32]
Inulin gel	C. butyricum, B. adolescentis, A. muciniphila	Radiation enteritis and acute radiological sickness	[33]
HA	Shewanella oneidensis and EcN	Improving bacterial survival	[44]
Aminated DNA aptamer	Attenuated Salmonella typhimurium	Targeting tumor tissues	[45]
Fe₃O₄ nanoparticles	E. coli	Anti-tumor	[46]
Imidoester and mucoprotein	EcN	Mucosal protection and anti-inflammation	[47]
Polydopamine	Salmonella strain VNP20009 and EcN	Anti-tumor and enhancing stomach acid tolerance	[50, 52]
Metal nanozyme	VNP20009	Tumor radioimmunotherapy	[53]
Selenium quantum dots	L. casei	Ulcerative colitis	[54]
Enteric polymer L100-55, HA	EcN, S. oneidensis	Anti-tumor	[44]
Biotin-conjugated polyclonal antibodies, streptavidin-coupled PLGA nanoparticles	Photosynthetic bacteria (Synechococcus 7942)	Anti-tumor	[57-59]
PEG and PDA	EcN	Mucosal protection and intestinal microecological regulation	[60]

Material	Bacteria species	Application	Ref.
PLGA	E. coli MG1655	Anti-tumor	[10]
β-Cyclodextrin-PEI600	S. typhimurium	Cancer immunotherapy	[11]
Bacterial biofilms	S. aureus, B. subtilis	Improve oral bioavailability	[13]
Red cell membrane	Listeria monocytogenes, E. coli	Anti-tumor	[14, 15]
Silk fibroin	EcN	Enhancing bioavailability and treatment efficacy	[16]
Dioleoylphosphatydic acid and cholesterol	EcN, S. aureus, E. faecalis	Enhancing bioavailability and treatment efficacy	[17]
Chitosan and alginate	B. coagulans, Bifidobacterium breve	Enhancing bioavailability and treatment efficacy	[18, 19]
Poloxham F-127	B. subtilis	Anti-epidermal fungal infection	[25]
Methacrylated gelatin, HAMA	L. reuteri	Anti-infection and promoting wound healing	[27]
Poly(vinyl alcohol), sodium alginate	Bdellovibrio bacteriovorus	Anti-infection and promoting wound healing	[28]
Gelatin, alginate	E. coli	Radiation detection	[29]
Engineered bacterial curli fibers	E. coli	Mucosal healing and immunomodulation	[30]
Chitosan/tannic acid coating and calcium alginate microspheres	L. rhamnosus	Radiation enteritis and ulcerative colitis	[32]
Inulin gel	C. butyricum, B. adolescentis, A. muciniphila	Radiation enteritis and acute radiological sickness	[33]
HA	Shewanella oneidensis and EcN	Improving bacterial survival	[44]
Aminated DNA aptamer	Attenuated Salmonella typhimurium	Targeting tumor tissues	[45]
Fe₃O₄ nanoparticles	E. coli	Anti-tumor	[46]
Imidoester and mucoprotein	EcN	Mucosal protection and anti-inflammation	[47]
Polydopamine	Salmonella strain VNP20009 and EcN	Anti-tumor and enhancing stomach acid tolerance	[50, 52]
Metal nanozyme	VNP20009	Tumor radioimmunotherapy	[53]
Selenium quantum dots	L. casei	Ulcerative colitis	[54]
Enteric polymer L100-55, HA	EcN, S. oneidensis	Anti-tumor	[44]
Biotin-conjugated polyclonal antibodies, streptavidin-coupled PLGA nanoparticles	Photosynthetic bacteria (Synechococcus 7942)	Anti-tumor	[57-59]
PEG and PDA	EcN	Mucosal protection and intestinal microecological regulation	[60]

Genetic modification	Modification locus	Chassis species	Application	Ref.
chuA, hrtR, luxCDABE	Plasmid	E. coli	Monitor intestinal bleeding and inflammation	[70]
narX/L, thsS/R, sfGFP	Plasmid	EcN	Monitor intestinal inflammation	[71]
ttrS/R, cro, o_L1-3, neo, cl, o_R3-1, lacZ	gDNA	E. coli, S. typhimurium	Long-term monitor intestinal inflammation	[72]
hok, sok, alp	Plasmid	EcN	Noninvasive diagnosis of early liver cancer	[73]
ZsGreen	Plasmid	S. typhimurium	Early diagnosis of cancer	[74, 75]
lasR, GFP, CoPy (a fusion protein), prgX/Q, AMPs	Plasmid	P. aeruginosa, E. faecalis	Antimicrobial agent	[77, 78]
Δalr, ΔdadX, lasR, pyoS5, colE7, dspB	Plasmid	EcN	Against gut infections	[79]
luxR, luxI, GFP	Plasmid	S. typhimurium	Anti-tumor and safety control	[80]
hlyE, sfGFP, luxI, φ174E	Plasmid	S. typhimurium	Anti-tumor	[81]
GLP-1, P_slpA	Plasmid	L. gasseri	Diabetes	[85]
Decarboxylase, IL-10	Plasmid	L. lactis	Diabetes	[87]
Tumor-associated antigen	Plasmid and gDNA	L. monocytogenes	Anti-tumor	[88]
Cytidine deaminase	Plasmid	S. typhimurium	Anti-tumor	[89]
IL-10	Plasmid	L. lactis	Ulcerative colitis	[90]
csg	Plasmid	EcN	Ulcerative colitis	[91]
ΔargR, ΔthyA, malEK: : P_fnrS-argA^fbr	gDNA	EcN	Hyperammonemia	[92]
hlyB/D, Sj16	Plasmid	EcN	Inflammatory bowel disease (IBD)	[93]
anti-PD-L1nb, anti-CTLA-4nb, luxI, φ174E	gDNA	EcN	Anti-tumor	[94]
ΔargR, OE: : argA^fbr	gDNA	EcN	Anti-tumor	[95]
thsS/R, sfGFP, hly-avCys, BE2, sgRNA, mCherry, hlyB/D, ACG-lacZ	gDNA and plasmid	EcN	IBD diagnosis, record, and treatment	[96]
ΔthyA, Δcsg, OE: : csgA-tff3, SOD, CAT	gDNA and plasmid	EcN	Radiation enteritis	[97]
bsh, IL-10	gDNA	E. coli	IBD	[98]
virB, T3SA, nanoanbibody, Δalr, ΔdadX	gDNA and plasmid	E. coli	IBD	[99]
ΔompT, Δlon, OE: : LLO, neo antigen	gDNA and plasmid	EcN	Tumor vaccine	[100]
GM-CSF, SIRPα	Plasmid	EcN	Anti-tumor	[101]

Genetic modification	Modification locus	Chassis species	Application	Ref.
chuA, hrtR, luxCDABE	Plasmid	E. coli	Monitor intestinal bleeding and inflammation	[70]
narX/L, thsS/R, sfGFP	Plasmid	EcN	Monitor intestinal inflammation	[71]
ttrS/R, cro, o_L1-3, neo, cl, o_R3-1, lacZ	gDNA	E. coli, S. typhimurium	Long-term monitor intestinal inflammation	[72]
hok, sok, alp	Plasmid	EcN	Noninvasive diagnosis of early liver cancer	[73]
ZsGreen	Plasmid	S. typhimurium	Early diagnosis of cancer	[74, 75]
lasR, GFP, CoPy (a fusion protein), prgX/Q, AMPs	Plasmid	P. aeruginosa, E. faecalis	Antimicrobial agent	[77, 78]
Δalr, ΔdadX, lasR, pyoS5, colE7, dspB	Plasmid	EcN	Against gut infections	[79]
luxR, luxI, GFP	Plasmid	S. typhimurium	Anti-tumor and safety control	[80]
hlyE, sfGFP, luxI, φ174E	Plasmid	S. typhimurium	Anti-tumor	[81]
GLP-1, P_slpA	Plasmid	L. gasseri	Diabetes	[85]
Decarboxylase, IL-10	Plasmid	L. lactis	Diabetes	[87]
Tumor-associated antigen	Plasmid and gDNA	L. monocytogenes	Anti-tumor	[88]
Cytidine deaminase	Plasmid	S. typhimurium	Anti-tumor	[89]
IL-10	Plasmid	L. lactis	Ulcerative colitis	[90]
csg	Plasmid	EcN	Ulcerative colitis	[91]
ΔargR, ΔthyA, malEK: : P_fnrS-argA^fbr	gDNA	EcN	Hyperammonemia	[92]
hlyB/D, Sj16	Plasmid	EcN	Inflammatory bowel disease (IBD)	[93]
anti-PD-L1nb, anti-CTLA-4nb, luxI, φ174E	gDNA	EcN	Anti-tumor	[94]
ΔargR, OE: : argA^fbr	gDNA	EcN	Anti-tumor	[95]
thsS/R, sfGFP, hly-avCys, BE2, sgRNA, mCherry, hlyB/D, ACG-lacZ	gDNA and plasmid	EcN	IBD diagnosis, record, and treatment	[96]
ΔthyA, Δcsg, OE: : csgA-tff3, SOD, CAT	gDNA and plasmid	EcN	Radiation enteritis	[97]
bsh, IL-10	gDNA	E. coli	IBD	[98]
virB, T3SA, nanoanbibody, Δalr, ΔdadX	gDNA and plasmid	E. coli	IBD	[99]
ΔompT, Δlon, OE: : LLO, neo antigen	gDNA and plasmid	EcN	Tumor vaccine	[100]
GM-CSF, SIRPα	Plasmid	EcN	Anti-tumor	[101]

Phase	Disease	Treatment	Route	Significance	Identifier/Refs.
Ⅰ (completed)	Cancer (advanced or metastatic)	VNP20009 (engineered S. typhimurium)	i.v.	S. typhimurium is genetically engineered to delete purI, msbB and xyl	NCT00004988
Ⅰ (ongoing)	Glioblastoma multiforme	EGFR(Ⅴ)-EDV-Dox (engineered bacterial minicell)	i.v.	Bacterial minicell derived from S. typhimurium minCDE-strain is engineered to target EGFR and carry doxorubicin	NCT02766699
Ⅰ/Ⅱ (suspended)	Solid tumours (advanced and/or metastatic)	APS001F (engineered B. longum) in combination with flucytosine and maltose	i.v.	B. longum is genetically engineered to produce cytosine deaminase	NCT01562626
Ⅰ/Ⅱ (discontinued)	Familial adenomatous polyposis	CEQ508 (engineered E. coli)	Oral	An attenuated strain (undisclosed) of E. coli is genetically engineered to deliver β-catenin short-hairpin RNA	[118]
Ⅱ (recruiting)	Metastatic pancreatic cancer	Saltikva (engineered S. Typhimurium) in combination with either FOLFIRINOX or gemcitabine/paclitaxel	Oral	An attenuated strain (undisclosed) of S. typhimurium is genetically engineered to express IL-2	NCT04589234
Ⅲ (recruiting)	Phenylketonuria	SYNB1934 (engineered EcN)	Oral	EcN is genetically engineered to metabolize L-phenylalanine	NCT05764239

Phase	Disease	Treatment	Route	Significance	Identifier/Refs.
Ⅰ (completed)	Cancer (advanced or metastatic)	VNP20009 (engineered S. typhimurium)	i.v.	S. typhimurium is genetically engineered to delete purI, msbB and xyl	NCT00004988
Ⅰ (ongoing)	Glioblastoma multiforme	EGFR(Ⅴ)-EDV-Dox (engineered bacterial minicell)	i.v.	Bacterial minicell derived from S. typhimurium minCDE-strain is engineered to target EGFR and carry doxorubicin	NCT02766699
Ⅰ/Ⅱ (suspended)	Solid tumours (advanced and/or metastatic)	APS001F (engineered B. longum) in combination with flucytosine and maltose	i.v.	B. longum is genetically engineered to produce cytosine deaminase	NCT01562626
Ⅰ/Ⅱ (discontinued)	Familial adenomatous polyposis	CEQ508 (engineered E. coli)	Oral	An attenuated strain (undisclosed) of E. coli is genetically engineered to deliver β-catenin short-hairpin RNA	[118]
Ⅱ (recruiting)	Metastatic pancreatic cancer	Saltikva (engineered S. Typhimurium) in combination with either FOLFIRINOX or gemcitabine/paclitaxel	Oral	An attenuated strain (undisclosed) of S. typhimurium is genetically engineered to express IL-2	NCT04589234
Ⅲ (recruiting)	Phenylketonuria	SYNB1934 (engineered EcN)	Oral	EcN is genetically engineered to metabolize L-phenylalanine	NCT05764239

基于工程化细菌的活体生物药: 现状与未来

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袁伯川 , 金义光 ^*

药学学报 | 专题报道: 基于工程化细菌的活体生物药 2025,60(5): 1183-1196

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药学学报 | 专题报道: 基于工程化细菌的活体生物药 2025, 60(5): 1183-1196

基于工程化细菌的活体生物药: 现状与未来

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袁伯川, 金义光^*

作者信息

军事科学院军事医学研究院, 北京 100850

通讯作者:

^*金义光, E-mail: jinyg@sina.com

Living biotherapeutic products based on engineered bacteria: current status and future prospects

Bo-chuan YUAN, Yi-guang JIN^*

Affiliations

Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China

出版时间: 2025-05-12 doi: 10.16438/j.0513-4870.2025-0139

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摘要

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活体生物药(live biotherapeutic product, LBP) 是一类含有活性生物体(如细菌) 的用于预防或治疗人类疾病的生物制品(不包括疫苗)。目前LBP的研发主要聚焦于活细菌。LBP与传统药物相比具有可复制性、靶向性、响应性等特点, 成为多种重大疾病药物研发的热点, 适应症涉及恶性肿瘤、代谢性疾病、炎症性肠病、基因缺陷病等。由于天然细菌存在活性低、不稳定和安全性等问题, 对其进行工程化改造是改良细菌药物学特性、促进细菌向LBP应用转化的关键。本文详细调研了近年来基于工程化细菌的LBP研究进展, 总结了基于化学作用、物理作用、遗传改造的工程化策略, 发现单一途径的工程化细菌疗效、稳定性和安全性问题仍不能完全解决, 因此提出“多工程化细菌”(multi-engineered bacteria) 理念, 即通过物理、化学、生物的组合工程化提高细菌成药性。本文通过对基于工程化细菌的LBP研究进行综述, 为LBP研发提供前瞻性思考和展望。

关键词

活体生物药 / 工程化细菌 / 细菌疗法 / 合成生物学 / 理化修饰

Abstract

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Live biotherapeutic products (LBPs) represent a distinct category of biological products containing viable organisms, such as bacteria, utilized for the prevention and treatment of human diseases (excluding vaccines). Presently, research and development efforts in LBPs are predominantly centered on live bacteria. Compared to traditional drugs, the LBPs demonstrate unique characteristics, including replicability, target specificity, and responsiveness. Owing to these properties, LBPs have emerged as hotspots in the development of specialized treatments for various major diseases, with applications spanning malignant tumors, metabolic disorders, inflammatory bowel diseases, genetic defects, and more. Nevertheless, natural bacteria face inherent limitations—such as low activity, instability, and safety concerns—that hinder their pharmacological potential. As a result, engineering strategies have become essential for enhancing the properties of bacteria and facilitating their clinical applications. This article delves into recent advancements in LBPs derived from engineered bacteria, offering a systematic review of reported engineering strategies, which are broadly categorized into chemical, physical, and genetic modifications. The findings indicate that no single engineering approach can comprehensively address all the challenges associated with converting viable bacteria into effective LBPs. To overcome this limitation, a concept of "multi-engineered bacteria" is introduced. This framework advocates for the integration of physical, chemical, and biological engineering strategies to develop next-generation LBPs with enhanced functionality and clinical potential. This article provides a concise review of current research on LBPs based on engineered bacteria and outlines forward-looking perspectives for advancing their development through innovative engineering approaches.

Key words

living biotherapeutic product / engineered bacteria / bacteriotherapy / synthetic biology / physicochemical modification

引用本文

袁伯川, 金义光. 基于工程化细菌的活体生物药: 现状与未来. 药学学报, 2025 , 60 (5) : 1183 -1196 . DOI: 10.16438/j.0513-4870.2025-0139

Bo-chuan YUAN, Yi-guang JIN. Living biotherapeutic products based on engineered bacteria: current status and future prospects[J]. Acta Pharmaceutica Sinica, 2025 , 60 (5) : 1183 -1196 . DOI: 10.16438/j.0513-4870.2025-0139

正文

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活体生物药(live biotherapeutic product, LBP) 的广义定义是指用于诊断、治愈、缓解、治疗或预防人类或其他动物疾病的生命体^[1]。人类利用活菌进行食品制作、改善营养供应以及预防疾病的历史可以追溯至距今8 000年前, 现在还在服务于人类, 如各种含活菌的发酵食品。早在19世纪末, 纽约癌症医院的William Coley医生就尝试使用化脓性链球菌(Streptococcus pyogenes) 来对抗肿瘤, 成功在部分病例体内消除实体瘤^[2]。进入21世纪以来, 随着生命科学和医学的进步, 人们逐渐认识到LBP可作为一种新的药物形式用于治疗特定疾病^[3]。尤其是进入合成生物学时代以来, 合成生物学以“自上而下”的全新理念, 实现了对活体生物药的全面革新, 可对炎症性疾病、免疫性疾病、代谢性疾病、肿瘤等重大疾病进行精准诊断与治疗^[4]。然而这一阶段活体生物药的发展一直面临法规不健全、审批流程不完善的困境。直到2016年, 美国食品药品监督管理局(Food and Drug Administration, FDA) 颁布了新的指南——“Early clinical trials with live biotherapeutic products: chemistry, manufacturing, and control information”, 才产生第一部系统性、规范性的活体生物药技术指导原则。在新的指导原则中, 主要对微生物来源的LBP进行了法规规定^[5], 因此, 微生物是LBP概念中的主要研究内容。可用作活体生物药活性单元的微生物主要包括酵母菌和细菌, 而哺乳动物肠道中酵母菌丰度较低, 且酵母菌在肿瘤领域应用较少, 目前LBP的研究与开发重点集中在不同种属细菌^[6]。

现代社会也大量应用活菌制剂。在我国上市的药品包括枯草杆菌二联活菌颗粒、双歧杆菌四联活菌片、地衣芽孢杆菌活菌胶囊等, 但此类药物功能较单一、疗效不确切, 适应症主要是腹泻等常规疾病, 并未在药物领域引起颠覆性创新与进步。天然细菌虽然表现出不同于传统化学药与生物药的独特之处, 但受限于天然的特性, 不能很好地适用于重大疾病的治疗, 因此人为改造的细菌, 即工程化细菌, 成为解决问题的关键^[6]。细菌的工程化主要围绕其表面功能、有效载荷、可控性与安全性等方面展开, 具体可分为物理工程化、化学工程化和基因工程化, 改造后的细菌相比于天然细菌一般具有更强的定植能力、更精准的疾病靶向与响应能力, 以及更有效的治疗能力, 且随着合成生物学理念的引入, 工程化细菌的功能可以被定制, 以满足治疗人类重大疾病的需求^[7], 包括恶性肿瘤、基因缺陷疾病、炎症性肠病、代谢性疾病等(图 1)。本文将对目前用于医学领域的工程化细菌前沿进展进行综述, 以期为LBP的发展提供有价值的见解。

1 物理工程化细菌

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物理工程化细菌是指化合物或生物材料通过静电吸附、氢键、范德华力等作用力修饰的细菌, 同时也包括某些为细菌量身定制的药物递送载体, 如水凝胶、微球等^[8]。物理工程化细菌制备形式包括共挤出、包裹、表面沉积、物理混合等, 可达到改善细菌抗逆性、提高生物利用度、增加肠道滞留时间的目的。

1.1 生物材料物理包覆的工程化细菌

由于细菌外膜含有大量羟基肽聚糖和磷壁酸, 大多数细菌的外膜带负电, 因此可利用静电作用进行表面修饰^[9]。通过静电相互作用将纳米颗粒PR848装载到Escherichia coli上, 可以将药物靶向输送到缺氧的肿瘤组织, 并将M2巨噬细胞极化为M1巨噬细胞, 从而增强抗肿瘤免疫反应^[10]。用交联的β-环糊精-PEI600 (CP) 纳米颗粒包覆减毒沙门氏菌有助于沙门氏菌有效逃避体内吞噬作用并增强其环境耐受性, 通过杂化纳米载体的自组装VEGFR2 DNA疫苗的口服递送可显著刺激T细胞活化和细胞因子产生^[11]。

高聚物的物理包裹与自组装同样能够在细菌表面形成修饰层, 天然的细胞膜或生物膜是一种重要的修饰物, 用于改善活体生物药的生物相容性和免疫调节能力^[12]。利用枯草芽孢杆菌(Bacillus subtilis) 生物膜包裹金黄葡萄球菌, 以提高其胃肠道环境耐受性和定植能力, 与裸菌相比, 包膜益生菌的口服生物利用度提高了125倍, 肠道定植率提高了17倍^[13]。红细胞膜也是常用的物理改性材料, 可以将益生型大肠杆菌(Escherichia coli Nissle 1917, EcN) 或单核细胞增生李斯特菌等细菌与鼠红细胞膜混合, 然后通过微孔膜共挤出的方式生产工程化细菌, 该工程菌可减弱全身炎症反应, 增加肿瘤内细菌蓄积, 并增强治疗效果^{[14, 15]}。自组装是单个实体自发聚集形成紧密有序整体的过程, 利用丝素蛋白可以从随机卷曲转变为β片层构象的自组装特性, 将其与EcN混合在悬浮液中能够在细菌表面形成多层涂层, 显著增强EcN在模拟胃液(simulated gastric fluid, SGF, pH 2) 中的耐受性^[16]。二油酰磷脂酸(dioleoylphosphatydic acid, DOPA) 和胆固醇能够在磷酸钙缓冲液自组装形成脂质涂层覆盖在EcN表面, 由此产生的脂质膜包被细菌在暴露于各种恶劣环境条件(包括强酸、强碱、抗生素和乙醇) 时表现出显著增强的活力^[17]。通过静电相互作用将阳离子壳聚糖和阴离子海藻酸盐依次沉积在凝结芽孢杆菌(Bacillus coagulans) 上, 包被的益生菌在模拟肠液(simulated id intestinal fluid, SIF, pH 7) 和SGF中表现出更好的存活率^[18]; 使用该方法包被的双歧杆菌同样显示出对胃肠环境的抵抗力和增强的口服递送效率; 值得注意的是, 涂层层数与益生菌的存活率并不成正比, 一般涂有3层阳离子壳聚糖和阴离子海藻酸盐的细菌表现出了最平衡的存活率和功效关系^[19]。

1.2 新型纳米材料物理装载的工程化细菌

活体生物药以细菌细胞为最小有效单元, 针对细菌量身定制的递送载体有助于提高生物利用度、疾病靶向性、生物安全性, 从而扩展活体生物药应用范围, 因此基于材料科学和纳米技术的新型载菌药物制剂被不断开发, 促进活体生物药的临床转化。

水凝胶(hydrogels) 是一种具有三维网状结构的柔软凝胶, 含水量很高。一般地, 水凝胶可以通过聚合物的共价交联(即大分子水凝胶) 或非共价相互作用(例如静电相互作用和聚合物之间的氢键) 形成(即超分子水凝胶)^[20]。根据配方和设计的不同, 水凝胶具有各种特性和功能, 包括可调节的流动性和刚度、刺激响应性降解、自修复特性等, 因此常在伤口愈合、组织工程、药物递送中用作细菌的载体^[21-23]。更重要的是, 水凝胶可以支持细胞生长并模拟细胞外基质(extracellular matrix, ECM), 聚合物水凝胶壳可保护细菌免受环境侵害, 并且易于添加供细菌生长的营养成分^[24]。例如, 一种特殊的温敏型凝胶被制备, 由于配方中的泊洛沙姆F-127的最低临界溶解温度接近体温, 水凝胶会在1 min内通过溶胶-凝胶转变形成, 用它装载益生菌后形成的活菌凝胶可以有效地在表皮中积累, 在皮肤白色念珠菌感染模型中表现出良好的抗真菌功效^[25]。含有B. subtilis的水凝胶可以充当高效生产抗真菌药物的“微型工厂”^[26]。罗伊氏乳杆菌(Lactobacillus reuteri) 是一种常见的益生菌, 可通过乳液聚合包封在水凝胶微球中, 并通过甲基丙烯酸酯修饰透明质酸的共价交联进一步固定在水凝胶网络中, 这种载菌水凝胶不仅可以保护益生菌免受免疫系统攻击, 还可以防止益生菌逃逸, 能够抑制有害菌生长并促进伤口闭合^[27]。一种聚乙烯醇—海藻酸盐双网络水凝胶被制备, 用于装载掠食性细菌, 这种载菌凝胶表现出对耐药创伤弧菌感染伤口的强效抗菌和促愈合能力^[28]。

除了抗感染外, 活菌水凝胶还被用于生物传感、胃肠道疾病的治疗或可穿戴设备。基于细菌在电离辐射作用下的SOS响应, 一种在γ射线照射下剂量依赖性产生荧光的E. coli被制备出来以装载到明胶—海藻酸钙凝胶中, 结合生物3D打印, 这种活菌凝胶可被制备成任意形状的辐射生物传感器, 体现了载菌水凝胶的多用途性^[29]。通过基因工程可设计大肠杆菌高表达定制的卷曲纤维, 这种工程菌通过发酵可以自发形成含菌水凝胶并通过简单过滤获得, 其可以更牢固地黏附在胃肠道的特定组织上并自我更新实现功能持久性^[30]。一些特定的水凝胶可以进一步用作坚硬的外壳保护细菌并进行环境感知和信号交换, 同时避免细菌代谢物对环境的污染, 如以海藻酸盐为主的水凝胶可形成多层硬壳, 位于其中的基因工程菌在环境中泄漏的风险大幅降低, 并使细菌能够发挥所需的功能, 在重金属等典型环境污染物检测方面表现出巨大潜力^[31]。为了开发在肠道滞留时间更长的载菌制剂, 鼠李糖乳杆菌(Lactobacillus rhamnosus) 首先被壳聚糖包裹, 随后单宁酸(多酚) 被修饰形成双涂层细菌, 再被封装于海藻酸盐微球中, 这种水凝胶载涂层菌制剂相比于未修饰细菌的结肠定植数量提高3~4个数量级^[32]。水凝胶用于装载复合菌同样具有优势, 将丁酸梭菌(Clostridium butyricum)、婴儿双歧杆菌(Bifidobacterium adolescentis)、嗜黏蛋白阿克曼菌(Akkermansia muciniphila) 装载于菊粉凝胶中, 提高了口服递送效率并显示出良好的预防放射性肠炎的作用^[33]。将活菌装载于水凝胶后, 可进一步与可穿戴柔性电子设备集成用于体内实时炎症监测, 这进一步扩展了活体生物药的应用范围^[34]。

活菌水凝胶用途广泛, 具有广阔的重大疾病临床应用前景。例如, 在皮肤感染中, 壳聚糖水凝胶伤口敷料可以将伤口部位与外界环境隔离, 以防止病原体相互作用, 并作为“药物工厂”用于经皮药物递送^[35]。活菌水凝胶还可以作为可注射制剂, 用于皮下植入细菌制剂, 促进再生医学的应用转化^[36]。还可以利用水凝胶可调节的黏附和降解特性开发载菌口服给药系统, 用于治疗胃肠道疾病^[37]。具有环境响应性的智能活菌水凝胶也已被广泛用于药物、蛋白质和基因输送^[38], 但水凝胶的成分会影响所装载细菌的活力和功能, 因此在将其应用于生物医学之前应进行彻底研究, 根据活菌的行为调整和优化水凝胶的配方和性能, 以最大限度地提高治疗效果。

2 化学工程化细菌

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化学工程化细菌是指利用化合物(无机物或有机物) 对细菌进行改性, 使其具备更良好的性能以满足特定用途的工程化细菌。这种化学工程化的前提是仍保证细菌的活力, 否则其产物不属于活体生物药范畴。化学工程化细菌的技术难点在于温和条件下进行的细菌与化合物偶联, 因此修饰物大部分为生物材料或常见无毒/低毒高聚物, 如聚多巴胺、多糖、多肽、多酚等^[39], 修饰后的细菌往往具有更高的鲁棒性、更强的靶向性, 从而实现更有效的治疗。细菌的化学修饰往往聚焦于细菌表面, 主要通过共价键等作用力与细菌偶联^[40]。

细菌表面主要由细胞膜、细胞壁及细菌表面附属物组成, 根据细菌细胞壁结构和成分, 可将其分为革兰阳性菌和革兰阴性菌^[41]。除了共有的、但厚度差异很大的肽聚糖层细胞壁外, 二者还有各自独特的外膜结构, 如革兰阴性菌的脂多糖(lipopolysaccharide, LPS) 和革兰阳性菌的脂磷壁酸^{[41, 42]}。细菌的表面附属物种类更加多样, 包括鞭毛、菌毛、分泌系统、脂蛋白、外膜蛋白等^[43]。细菌表面的化学本质是多糖、蛋白质和脂质的有机组合物。丰富的表面成分为化学修饰细菌提供了丰富的反应基团, 如硫醇、胺基、羟基和羧基等, 细菌表面的负电荷也为通过高分子静电作用进行修饰提供了机会^[39]。因此, 基于细菌的这些结构特征, 已经开发出多种细菌表面的化学工程化方法, 用于改良LBP的特性, 包括但不限于: ①降低或消除细菌的毒性; ②增强细菌在体内的生存力和增殖能力; ③赋予细菌额外的功能以增强治疗效果。

2.1 化合物共价连接的工程化细菌

许多高聚物或纳米粒子可以直接共价连接到细菌表面。透明质酸(hyaluronic acid, HA) 可以通过羟基与细菌细胞壁N-乙酰胞壁酸的羧基发生缩合反应沉积到E. coli表面, 可大幅提高口服E. coli在胃肠道的存活率^[44]。胺化的核酸适配体可通过酰胺缩合反应连接到N-乙酰胞壁酸的羧基上, 制备出化学工程化细菌, 增加细菌在肿瘤部位的定植^[45]。磁性Fe₃O₄纳米粒子也可通过酰胺缩合反应连接到细菌上, 制备成Fenton反应的催化剂, 该生物反应器将细菌不断合成的H₂O₂转化为有毒的羟基自由基(•OH) 杀死肿瘤细胞^[46]。在温和条件下, EcN表面的一级氨基残基可通过一步酰亚胺化反应转化为游离硫醇, 通过动态的硫醇-二硫键交换反应, 形成新的二硫键, 使改造后的细菌能自发地与富含多硫化物的黏膜层连接, 在小肠内表现出趋化和定植的优势^[47]。由于儿茶酚基团具有很强的黏附性, 聚多巴胺(polydopamine, PDA) 涂层在细菌修饰中得到了广泛的应用^{[48, 49]}, 通过多巴胺的氧化和自聚合可沉积于VNP20009活菌表面, 在808 nm的光照下, 工程菌诱导机体产生TNF-α和IL-4抑制肿瘤的生长^{[50, 51]}; 在添加胃蛋白酶的SGF中, PDA涂层可提高EcN的存活率, 在涂层中添加壳聚糖, 进一步增强了其对SGF的抵抗力^[52]。此外, 金、铂等金属离子可以通过还原剂还原形成纳米颗粒, 然后沉积在细菌表面形成稳定的涂层^[53]; 通过限制营养可诱导干酪乳杆菌(Lactobacillus casei) 表面形成菌膜, 该富含羟基的多糖结构可高效结合硒量子点, 显著提高了益生菌清除氧自由基的能力^[54]。

2.2 多级偶联的工程化细菌

多级偶联的策略也被用于细菌表面修饰。为了使EcN包裹于肠溶聚合物L100-55中, 钙离子首先通过静电作用吸附到EcN上, 通过形成离子键为L100-55的自组装提供可交联位点; 通过将pH降低至5.0左右, L100-55可以在细菌表面形成稳定的涂层, 实现靶向肠道的益生菌高口服生物利用度递送^{[44, 55, 56]}。聚乳酸-乙醇酸共聚物纳米粒[poly(lactic-co-glycolic acid), PLGA] 不具备与细菌表面反应的位点, 但可将链霉亲和素与PLGA纳米粒子偶联, 再将与生物素偶联的多克隆鼠伤寒沙门氏菌(Salmonella typhimurium) 抗体与细菌表面结合, 随后通过链霉亲和素-生物素结合组装形成PLGA修饰的细菌, 该平台可作为各种有效载荷肿瘤靶向的输送载体^[57-59]。为了将聚乙二醇(polyethylene glycol, PEG) 修饰于细菌表面以改善其在肠黏膜的定植生态位, 首先将多巴胺沉积于细菌表面提供反应位点, 再将末端氨基化的PEG修饰在聚多巴胺上, 实现PEG与细菌的偶联^[60]。上述理化修饰的工程菌相关信息在表 1中总结。

3 基因工程化细菌

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基因工程化细菌是指遗传物质经过改造的细菌, 修饰位点既可位于基因组DNA也可位于质粒DNA, 其可赋予细菌可遗传的特殊性状, 实现理化修饰难以达到的目的, 如持续合成治疗性分子、药物的自主分泌、改变的细菌趋向性等。合成生物学和微生物学的快速发展为工程菌在生物医学领域的应用提供了机遇, 上述化学修饰技术可以人为地丰富细菌的表面成分、包裹细菌或充当细菌的递送载体, 而基因工程化细菌则是利用生物合成、基因工程等手段改变细菌的固有性状, 赋予它们更多功能和更好的生物相容性, 甚至构建出能够自动感知疾病并释放特效药物的“智能细菌”^{[3, 40]}。相比于化学工程化细菌, 基因工程化细菌的优势在于功能和性状的可遗传性以及更多的工程化位点, 这有助于活体生物药药效的稳定以及更大的治疗潜力, 但同时也带来安全性挑战。

基因工程化细菌的实现策略一般分为两种: 通过质粒改造细菌和通过基因组编辑改造细菌。前者的优势在于操作简单、基因线路设计多样, 但存在稳定性不足、多质粒不兼容的缺点; 后者具有稳定性高、不易造成基因泄漏的优点, 但操作繁琐且受到基因调控影响^[7]。以有效性和安全性为目标, 综合利用各类遗传改造技术, 创造全新功能的基因工程菌是当前活体生物药菌株研发的热点与前沿。与传统的非活体疗法(即化学药物或生物制剂) 和天然菌相比, 基因工程菌还具如下特点: ①基因工程菌和天然菌都能将药物输送到患病组织的局部, 从而提高疗效, 减少不良反应; ②基因工程菌可原位生产药物, 省去了药物合成或发酵制备生物大分子生产过程中昂贵的纯化步骤; ③由于使用的是特性良好的工程基因调控网络, 而不是特性不佳的自然进化细菌, 因此与天然菌相比, 用基因工程菌激活体内的治疗途径更为可靠; ④在基因工程菌中, 产生治疗性分子的遗传途径是明确的, 而天然菌可能会产生不确定的生物活性化合物, 其作用难以区分; ⑤基因工程菌与天然菌形成鲜明对比, 天然菌只是“按原样”从大自然中提取, 而基因工程菌则是通过反复试验开发出来的。

3.1 疾病检测和诊断型基因工程菌

针对特定疾病的独特标志物, 利用合成生物学技术构建传感线路, 创建能感知一种或多种疾病生物标志物的“智能工程菌”, 用于诊断疾病。细菌用于感知待测物的基因回路通常是单组分或双组分系统, 并且可以对体内相关的分子做出反应, 包括IL-1β、TNF、IFN-γ等细胞因子^[61-63]、肾上腺素和γ-氨基丁酸等激素^{[64, 65]}、温度以及代谢物等^[66-69]。改造后的细菌可以直接在体内或在离体样本中进行检测, 其中利用工程菌来感知在离开肠道之前被降解、修饰或吸收的瞬时分子是它在非侵入检测领域的一项重要用途。

基于血红素特异性转录调节因子HrtR的单组分工程菌, 能够封装在可吞服微电子胶囊中从而检测肠内出血, 可以不依赖肠镜实时监测黏膜损伤^[70]。基于细菌NarX/L和ThsS/R的双组分系统构建的基因工程菌, 可在AND逻辑门基因线路下同时检测肠道中NO₃^-和S₂O₃^2-, 用于准确判断肠道炎症^[71]。基于细菌TtrR/S的单组分系统, 同时结合Cro记忆元件构建的工程菌, 能够在哺乳动物体内长期滞留监测炎症的发生, 同时可将炎症记录于转录谱实现检测和记录的一体化诊断, 连续监测时间长达6个月^[72]。除了肠道炎症, 工程菌用于肿瘤的检测和报告的线路设计也被报道。研究发现口服的EcN可穿过小鼠肠道并优先在转移性肝肿瘤中生长^[73], 将该菌株遗传改造使其表达一种酶, 该酶可以切割系统给药的化合物, 从而导致尿液中检测到的颜色变化, 以无创诊断早期肝癌^[73]。不过虽然已在人类肠道中检测到EcN, 但其穿过肠道达到肝脏仅在一小部分健康个体中发生, 这限制了该技术的应用^[74]。另一项研究改造了减毒S. typhimurium, 使其在肿瘤内生长期间分泌荧光蛋白ZsGreen, 检测灵敏度比断层摄影技术高2 500倍, 可以改善癌症的早期诊断^{[74, 75]}。

群体感应是细菌一种独特的群体行为调控机制, 即细菌通过感应自身分泌的自诱导剂来判断菌群密度和周围环境变化, 如生物膜的形成是一种典型的群体感应效应。目前已有若干研究通过基因工程改造细菌群体感应系统, 检测病原体感染或定植调控^[76], 其中用于检测铜绿假单胞菌Pseudomonas aeruginosa和肠球菌Enterococcus faecalis的系统已用于体外检测^{[77, 78]}。对群体信号响应性工程菌株最常用的底盘是EcN, 该菌株通过感知P. aeruginosa的群体感应信号N-乙酰高丝氨酸内酯来做出反应, 产生抗生物膜分子, 该工程菌在小鼠和秀丽隐杆线虫模型中能有效减少P. aeruginosa的肠道感染^[79]。群体感应还被用来控制细菌特定功能的表达并控制体内分布。例如, 使用群体感应来诱导体内注射给药的S. typhimurium仅在肿瘤组织维持高密度, 而其他部位群体感应诱导较弱而导致生长受阻, 这种策略脱靶率很低^[80]; 该策略还可以用于抗肿瘤分子、溶菌素等效应物的诱导表达, 如体内注射的S. typhimurium因在肿瘤组织中特异性定植导致菌密度增加触发lysin E7的表达^[81], 同时释放胞内的抗肿瘤分子, 还有助于维持体内相对较低的细菌总体定植水平, 触发细菌的条件包括瘤内的乏氧环境^[82]、外部给予的化合物或信号(超声、近红外光等)^{[66, 83]}。该基因工程化策略在输送治疗性分子时减少脱靶效应, 是限制活体生物药不良反应的重要方法。

3.2 治疗型基因工程菌

细菌已被用于向人体递送治疗剂, 尤其是利用其原位生产、原位递送的能力, 可将一些在血液或上消化道迅速降解的药物递送至靶标, 还可以将治疗剂递送到细菌可以生存但难以通过口服或注射递送到的身体部位, 如结肠或肿瘤中心^[84]。通过简单的重组表达, 已使多种细菌成为向人体特定部位输送治疗剂的工具。例如, 表达胰高血糖素样肽1 (GLP-1) 的乳杆菌能够重编程肠上皮细胞以响应葡萄糖并产生胰岛素, 实现糖尿病的治疗^[85]; 口服表达胰岛素原和GAD65的乳球菌联合注射IL-10可预防甚至逆转胰岛β细胞破坏^{[86, 87]}; 基因改造减毒李斯特菌Listeria monocytogenes以表达肿瘤相关抗原可激活针对肿瘤的先天性和适应性免疫^[88]; 工程化S. typhimurium以表达胞嘧啶脱氨酶, 将无毒的前药5-氟胞嘧啶转化为抗癌剂5-氟尿嘧啶实现肿瘤的靶向治疗^[89]; 口服重组表达IL-10的L. lactis能够向肠道局部输送高剂量IL-10, 有效治疗结肠炎^[90]。

随着合成生物学和微生物学的发展, 定制和创造具有显著药效的活体生物药成为可能, 工程学原理的引入让科学家在细菌这个微生物平台可以充分发挥想象力和创造力, 让“智能药物”成为可能。利用合成生物技术能实现细菌表面的可遗传改造, 最近的一项研究开发了细菌表面curli纤维改造平台, 将肠三叶因子肽与菌毛蛋白CsgA融合用于缓解炎症性肠病^[91]; 针对EcN的氨-精氨酸代谢通路的基因组重编程赋予了其高效的氨转化能力, 口服能够显著降低肠道有害代谢物氨的累积, 缓解高氨血症^[92]; 经基因改造的EcN能够表达免疫调节蛋白Sj16, 并通过异源表达专属蛋白质分泌系统HlyB/D实现蛋白药物的分泌, 在小鼠溃疡性结肠炎模型中取得显著疗效^[93]; 同样, 基于EcN底盘研究者设计了一种群体感应裂解释放免疫检查点阻断纳米抗体的基因线路, 利用细菌的肿瘤趋向性工程菌在肿瘤内定植, 当密度增加达到诱导浓度时裂解蛋白φX174E表达导致菌膜破裂, 释放PD-L1和CTLA-4的纳米抗体, 实现肿瘤的免疫治疗^[94]; 经代谢重编程可将氨转化为精氨酸的工程菌, 通过瘤内定植可将肿瘤细胞高产的氨转化为L-精氨酸, 这种氨基酸能够提高免疫检查点抑制剂的有效性, 实现联合抗癌^[95]; 一种基于质粒和基因组双重基因工程的EcN, 可实现炎症性肠病的诊断、治疗、记录的一体化解决方案^[96]; 同样基于质粒和基因组双重基因工程的EcN, 可以同时过表达肠黏附菌毛和抗氧化酶, 实现高效防治放射性疾病^[97], 扩展了活体生物药的适应症范围; 鉴于外源益生菌在哺乳动物肠道的定植抵抗, 研究者采用类似CAR-T疗法的策略, 分离宿主自身肠道大肠杆菌作为底盘, 通过基因工程化使其表达胆盐水解酶, 该同源工程菌可实现6个月以上的肠道定植, 实现一次使用长期治疗的目的^[98]; 工程菌表达的大分子治疗剂的分泌是一个棘手的问题, 基于胞内注射理念发展的工程化Ⅲ型细菌分泌系统可实现蛋白质从细菌内向细胞内注射的目的, 改造后的工程菌可高效向肠上皮细胞输送TNF-α纳米抗体, 缓解肠炎^[99]; 微生物系统已经被合成工程化以在体内部署治疗性有效载荷, 近期一项最新研究显示, 工程化EcN可作为优化的抗肿瘤疫苗接种平台, 用于增强新表位的产生和胞质递送, 并增加血液清除和吞噬作用的敏感性, 这些特征增强了安全性和免疫原性, 实现一个系统驱动有效和特异性的T细胞介导的抗癌免疫^[100]; 此外, 将光遗传工程菌分泌刺激因子与工程菌胞内连续生产释放载RNA药物的外膜囊泡(OMVs) 相结合, 实现了良好的肿瘤抑制作用^[101]。总之, 在合成生物学的加持下, 基因工程化细菌在重大疾病治疗领域展示出无限潜力, 其适应症可能囊括炎症性疾病、代谢性疾病、肿瘤和基因缺陷病等人类重大疾病^[102]。上述以基因工程化为主的活体生物药构建案例被总结于表 2。

4 多工程化细菌

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多工程化细菌(multi-engineered bacteria, MEB) 是作者在总结上述工程化手段的基础上提出的新概念, 它是指至少利用物理、化学、基因工程化策略中的2种修饰的细菌, 旨在用最简单有效的方式获得活性强、稳定性高、安全性好的活体生物药。目前活体生物药的基础研究已经由最初功能菌种的发现与培育转变为菌株、工程化、制剂相结合的研发模式, 因为过去20年间的诸多临床试验表明, 任何单一途径的工程化细菌在后期临床转化过程中风险较大, 难以在药物稳定性、有效性、安全性方面全面符合高效活体生物药的预期, 因此多种工程化策略相结合的活体生物药逐渐被科学家重视^{[7, 40]}。

基因工程修饰与化学修饰相结合的活体生物药首先被报道。研究者设计并构建了一种过表达酪氨酸酶的基因工程EcN, 该菌株高产内在光热黑色素, 进一步将免疫检查点抑制剂αPD-1修饰在其表面, 大大增强了双重免疫激活效果^[103]。针对溃疡性结肠炎的高水平ROS, 设计了一种过表达抗氧化酶的基因工程菌, 能够高效清除肠道ROS抗炎, 但差的口服生物利用度导致该菌株效果不佳, 随后研究者为该基因工程菌修饰了壳聚糖/海藻酸钠涂层, 这种双重修饰的工程菌表现出更好的治疗效果^[104]。光遗传学可用于工程菌的可控药物释放^[105], 天津大学王汉杰教授团队在该方面取得较多进展, 通过基因工程重编程细菌, 再利用纳米技术与工程菌相结合实现肿瘤、炎症、感染等疾病的治疗。例如, 构建的蓝光诱导表达TGF-β1和IFN-γ的基因工程菌被表面修饰上转换纳米粒, 在体外近红外光的照射下特异性分泌两种细胞因子, 实现肿瘤的治疗^[106]; 同样转化纳米粒结合基因工程菌可控表达并分泌黏附蛋白, 实现细菌的肠道定植, 有效治疗溃疡性结肠炎^[107]; 这种蓝光响应性工程益生菌结合上转换纳米粒还能够通过调控肠脑轴缓解脑部疾病, 如焦虑行为、帕金森病等^[108]。虽然化学修饰可以使细菌具备在生物系统中难以实现的功能, 但基因工程也可以赋予细菌当前纳米技术和分子工程不可能实现的复杂行为, 因此化学修饰和基因工程具有互补的特性, 二者结合可以发挥彼此协同作用。不过值得注意的是, 多重工程化细菌在设计和构建中需要考虑未来工业规模生产的可行性, 过于复杂精巧的设计不易被开发为活体生物药。

通过文献调研不难发现, 物理、化学、基因工程化策略各有优势, 也各具不足(图 2)。例如, 用化学修饰使细菌递送特定蛋白质至体内可能很困难, 但用基因工程则很容易实现蛋白的表达和分泌; 用基因工程手段提高细菌的胃酸耐受能力可能涉及复杂的遗传操作, 但用材料包衣技术可能很容易提高细菌胃酸中存活率; 用化学修饰不易实现的肠道靶向细菌控释, 但生物材料物理装载的细菌很容易实现靶向控释。因此, 现阶段许多有前景的工程菌折戟于临床试验很可能由于没有采用合理的工程化策略。作者长期致力于活体生物药研究, 近期在多工程化细菌研究方面也取得不错进展, 例如在文献^[92]报道的基础上, 综合利用物理、化学、基因三者结合工程化策略, 显著提高了基因工程菌治疗高血氨症的效果, 相信未来细菌活体生物药的突破将很大程度上依赖多工程化细菌的策略。

5 细菌工程化的目的

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以天然细菌为主要成分的活菌药物或功能性食品已在全球多个国家和地区获批上市, 但此类产品的适应症很狭窄, 通常只用于普通腹泻, 或者仅作为改善胃肠道功能的营养品。被称为“下一代益生菌”的活体生物药理念出现后, 人们认识到细菌作为一种新的药物形式——活体药物, 有望在更广阔的范围发挥它们的优势, 但如何赋予细菌稳定、安全、有效治疗各类重大疾病的能力仍是目前科学家正在关注的问题。得益于微生物合成生物学和纳米技术的发展, 通过工程学原理重新设计细菌、改造细菌使其满足药物的基本要求, 将加快活体生物药的医学转化。

5.1 增加有效载荷

细菌工程化的最主要目的是提高疾病治疗有效性。利用细菌的靶向性、可复制性、定植性等特点, 可将有效载荷高效输送至所需部位, 实现靶向、高效的治疗。常见的有效载荷包括各类活性蛋白(酶、抗体、治疗性蛋白)、核酸、小分子药物、多肽等, 工程化方法根据实际需要既能采用化学、物理法, 也能采用基因工程法, 两种方法的区别主要在于可遗传性^[109]。增加有效载荷的细菌能够充当“药物工厂”, 在病灶部位原位生产治疗性分子, 避免了药物在输送过程中的损失和变化, 显著提高细菌疗法有效性^[110]。工程化赋予细菌更多的可能性, 极大拓展细菌疗法的应用边界。

5.2 提高细菌鲁棒性

尽管细菌能够作为“药物工厂”原位输送治疗剂, 但活菌药物在递送过程中同样面临各类失活挑战。例如, 口服递送的细菌面临胃肠道恶劣的环境, 包括胃酸、消化酶、胆汁酸等的侵蚀和免疫细胞摄取^[111], 哺乳动物肠道原有的生态对外源性益生菌天然具有定植抵抗作用^[98]; 体内注射或瘤内注射细菌面临血液或组织微环境各类淋巴细胞的吞噬和清除作用, 导致低剂量细菌的快速清除和高剂量细菌的不良反应存在难以调和的矛盾^[112]; 冻干细菌制剂在体内恢复活力的时效性差导致最终细菌制剂的无效性。因此, 通过工程化方法提高细菌鲁棒性, 使其能在体内的各类场景下耐受性增强、复愈迅速, 同时具有可接受的血清清除期具有重要意义。目前通过化学修饰和制剂技术能够应对口服细菌递送中的挑战, 而通过精巧的基因线路设计也实现了治疗性细菌在小鼠体内的可编程封装系统从而改善体内递送, 但是上述理论和技术还未实现工业化, 距离真正获得高鲁棒性、高效的活体生物药仍有差距。

5.3 提高细菌体内定植能力

细菌在肿瘤内的定植虽然已有广泛报道, 但外源性细菌, 尤其是外源性益生菌, 在哺乳动物肠道内定植抵抗是显著的, 大部分益生菌在肠道内保留时间较短导致差的疗效。虽然通过扩大给药剂量和给药频次可以提高肠道活菌的保有量, 但过短的保留时间可能导致细菌还未恢复活力或发挥功能就被排出体外, 实际有效活菌量仍无法提高, 因此提高细菌体内定植能力有助于活性的发挥。提高细菌体内定植能力的工程化方法较多, 主要聚焦于细菌表面修饰或递送载体的设计, 包括基因工程改造细菌表面组成、细菌表面化学修饰特定基团增加肠道黏附以及黏附载体的包裹等^[39]。

5.4 提高细菌疾病响应性和靶向性

围绕细菌有效性, 通过工程学手段提高其对疾病的响应性和靶向性也是提高活体生物药疗效的有效途径。因为细菌具有细胞结构, 能够对感知外界环境并做出相应反应, 其遗传基础是各类基因操纵子, 如乳糖操纵子等^[113], 所以基于合成生物学设计的基因线路能够赋予细菌感知特定物质并做出响应的能力, 从而实现疾病响应性的药物控释, 制备真正意义上的“智能药物”, 因此, 可设计疾病响应性是活体生物药区别于其他传统药物的一大特点^[3]。目前该功能的实现主要依赖于合成生物学技术, 现有的化学、物理修饰难以实现类似功能。

5.5 生物安全控制

活体生物药的使用不可避免地将其所含有的活菌释放到环境中, 因此生物安全控制是批准临床试验和使用活体生物药的关键因素, 防止个体间转移、控制生长和治疗性分子表达、防止基因转入和转出工程菌株等因素都很重要。底盘菌株对于生物安全控制起到基础性作用, 到目前为止, 临床试验中很少发生严重不良事件, 使用长期定植细菌将变得越来越有吸引力和可行性, 但是通过工程化手段进行生物安全控制显得尤为重要。

目前有几种工程化策略可用于生物安全控制。①设计“死亡开关”基因线路: 通过基因工程使细菌只在特定环境下生长, 否则将表达致死蛋白(如DNA解旋酶、温敏型基因回路), 但任何编码生物体死亡的基因线路都存在强烈的选择压力^[67]; ②营养缺陷型是十分有效的控制方法: 最近的两项研究建立在大肠杆菌的基础上, 通过基因编辑使其密码子被重新编码, 以匹配与外源提供的非天然氨基酸相对应的非天然tRNA合成酶^[114]; 通过工程改造几种必需蛋白质, 使其在结构上依赖于非天然氨基酸, 细菌仅在非天然氨基酸存在的情况下被迫生长^{[115, 116]}, 这种策略防止细菌在无非天然氨基酸的情况下生长; ③大片段基因编辑同样能实现有效控制: 用重新编码的细菌替换200 kb (相当于鼠伤寒沙门氏菌基因组的4%以上) 合成的DNA片段^[117], 创造“人造细菌”实现生物安全控制, 但这种方法也带来未知的生物泄露风险。总之, 通过基因工程化方法进行细菌生物安全控制更为有效。

6 工程化细菌的医学转化前景与挑战

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尽管在过去20余年的时间里, 工程化细菌在向活体生物药转化的过程中产生很多里程碑式的研究和药物研发进展, 但时至今日, 按照活体生物药指导原则研发的新一代工程菌药物仍未有一款获批上市, 说明在基础研究取得突破的同时需要着重考虑其在临床转化、工业制造面临的挑战, 围绕药物稳定性、有效性、安全性的讨论将持续存在。

微生物合成生物学的进步使设计和编程具有复杂功能的细菌变得简单, 治疗性细菌, 特别是那些在临床试验中测试的工程菌, 在所使用的工程复杂性方面才刚刚开始发展, 例如细菌“药物工厂”、逻辑门和响应性治疗等工程设计均未在临床中试验, 此类药物未来的潜力值得挖掘。尽管目前已经进行了几项基于工程菌的临床试验, 但只有少数进展到高级临床阶段和FDA批准(表 3^{[7, 118]})。一般来说, 许多药物由于临床前模型和人类患者之间的差异而在临床试验中失败^[119]。此外, 细菌的体内成像对于获得实时生物分布数据是必要的。虽然光学成像对于小动物模型是有用的^[120], 但是在人类患者中建立药代动力学将需要具有深层组织分子成像技术^[121]。同时, 对基因工程菌进入体内后的鉴定, 以核实遗传元件和转录组的稳定性也是至关重要的^{[122, 123]}。

基于化学和物理原理的修饰同样极大地促进了活菌药物的发展, 使它们更容易地获得增强的药效和安全性, 同时避免遗传改造带来的潜在风险。当细菌在向靶组织递送途中遇到不同的生物屏障时, 细菌行为的动态调节是确保治疗安全性和有效性的关键。纳米技术的化学多样性导致更复杂的表面工程策略, 能够使细菌满足特定疾病的治疗需求。非基因工程的修饰技术与细菌结合的应该面向未来的工业生产而设计, 并增加构建多功能平台的组合技术路径, 通过将细菌和化学/生物材料结合, 实现二者特性的互补, 以改善其预期功能, 甚至创造单独不可能实现的新功能^[124]。本文提出的MEB是总结现有文献后发展而来的概念, 即充分发挥物理、化学、基因工程化手段的优势去解决活体生物药的研发难题, 促进基于工程化细菌的活体生物药转化。

近年来LBP的临床转化已引起全球药物研发与监管机构重视, 例如美国已出台相关指导原则, 包括中国在内的一些国家虽无专用指南, 但对LBP的临床转化保有支持态度。在满足药物安全性、有效性的基础上, 监管部门接受天然细菌、工程化细菌、复合细菌作为药物使用。由于不同于传统小分子、大分子药物(分子药物), LBP的有效成分为活细菌, 可能传统药物开发流程将不再适用, 例如LBP体内过程研究挑战很大, 制剂工艺显著不同等, 因此探索一条新的适用于LBP的药物研发路径仍然难度较大。然而, LBP药物可通过大规模发酵制备, 且省去了分子纯化的烦琐流程, 因此相比于十分昂贵的细胞疗法与纯化成本高的抗体药物, 该疗法可能具有明显的经济优势。

由于缺乏同类产品获批的先例, 基于工程化细菌的活体生物药在临床开发和监管审批方面将面临相当大的挑战, 有必要通过推广和持续的技术进步来建立患者接受度, 以满足推广要求。虽然这类药物未来很可能被首先用于治疗危及生命的疾病, 但必须警惕最初不良的人体实验报道, 否则可能成为阻碍该领域发展的不良先例。未来随着各类细菌工程化理念和技术的不断进步, 合成生物学和纳米医学的融合将有助于定制更好的工程化细菌, 以确保稳定性、安全性和有效性, 并推动细菌疗法的临床转化。

作者贡献: 袁伯川负责文献调研、整理归纳、系统分析和文章撰写; 金义光负责项目管理、技术指导和文章修改。

利益冲突: 所有作者声明不存在利益冲突。

基金

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国家自然科学基金资助项目(82404486)

参考文献

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文献

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参考文献引证文献

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2025年第60卷第5期

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doi: 10.16438/j.0513-4870.2025-0139

接收时间：2025-02-17
首发时间：2025-10-29
出版时间：2025-05-12

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收稿日期：2025-02-17
修回日期：2025-03-19

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国家自然科学基金资助项目(82404486)

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军事科学院军事医学研究院, 北京 100850

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^*金义光, E-mail: jinyg@sina.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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EndNote
RefWorks
TxT

Material	Bacteria species	Application	Ref.
PLGA	E. coli MG1655	Anti-tumor	[10]
β-Cyclodextrin-PEI600	S. typhimurium	Cancer immunotherapy	[11]
Bacterial biofilms	S. aureus, B. subtilis	Improve oral bioavailability	[13]
Red cell membrane	Listeria monocytogenes, E. coli	Anti-tumor	[14, 15]
Silk fibroin	EcN	Enhancing bioavailability and treatment efficacy	[16]
Dioleoylphosphatydic acid and cholesterol	EcN, S. aureus, E. faecalis	Enhancing bioavailability and treatment efficacy	[17]
Chitosan and alginate	B. coagulans, Bifidobacterium breve	Enhancing bioavailability and treatment efficacy	[18, 19]
Poloxham F-127	B. subtilis	Anti-epidermal fungal infection	[25]
Methacrylated gelatin, HAMA	L. reuteri	Anti-infection and promoting wound healing	[27]
Poly(vinyl alcohol), sodium alginate	Bdellovibrio bacteriovorus	Anti-infection and promoting wound healing	[28]
Gelatin, alginate	E. coli	Radiation detection	[29]
Engineered bacterial curli fibers	E. coli	Mucosal healing and immunomodulation	[30]
Chitosan/tannic acid coating and calcium alginate microspheres	L. rhamnosus	Radiation enteritis and ulcerative colitis	[32]
Inulin gel	C. butyricum, B. adolescentis, A. muciniphila	Radiation enteritis and acute radiological sickness	[33]
HA	Shewanella oneidensis and EcN	Improving bacterial survival	[44]
Aminated DNA aptamer	Attenuated Salmonella typhimurium	Targeting tumor tissues	[45]
Fe₃O₄ nanoparticles	E. coli	Anti-tumor	[46]
Imidoester and mucoprotein	EcN	Mucosal protection and anti-inflammation	[47]
Polydopamine	Salmonella strain VNP20009 and EcN	Anti-tumor and enhancing stomach acid tolerance	[50, 52]
Metal nanozyme	VNP20009	Tumor radioimmunotherapy	[53]
Selenium quantum dots	L. casei	Ulcerative colitis	[54]
Enteric polymer L100-55, HA	EcN, S. oneidensis	Anti-tumor	[44]
Biotin-conjugated polyclonal antibodies, streptavidin-coupled PLGA nanoparticles	Photosynthetic bacteria (Synechococcus 7942)	Anti-tumor	[57-59]
PEG and PDA	EcN	Mucosal protection and intestinal microecological regulation	[60]

Material

Bacteria species

Application

Ref.

PLGA

E. coli MG1655

Anti-tumor

[10]

β-Cyclodextrin-PEI600

S. typhimurium

Cancer immunotherapy

[11]

Bacterial biofilms

S. aureus, B. subtilis

Improve oral bioavailability

[13]

Red cell membrane

Listeria monocytogenes, E. coli

Anti-tumor

[14, 15]

Silk fibroin

EcN

Enhancing bioavailability and treatment efficacy

[16]

Dioleoylphosphatydic acid and cholesterol

EcN, S. aureus, E. faecalis

Enhancing bioavailability and treatment efficacy

[17]

Chitosan and alginate

B. coagulans, Bifidobacterium breve

Enhancing bioavailability and treatment efficacy

[18, 19]

Poloxham F-127

B. subtilis

Anti-epidermal fungal infection

[25]

Methacrylated gelatin, HAMA

L. reuteri

Anti-infection and promoting wound healing

[27]

Poly(vinyl alcohol), sodium alginate

Bdellovibrio bacteriovorus

Anti-infection and promoting wound healing

[28]

Gelatin, alginate

E. coli

Radiation detection

[29]

Engineered bacterial curli fibers

E. coli

Mucosal healing and immunomodulation

[30]

Chitosan/tannic acid coating and calcium alginate microspheres

L. rhamnosus

Radiation enteritis and ulcerative colitis

[32]

Inulin gel

C. butyricum, B. adolescentis, A. muciniphila

Radiation enteritis and acute radiological sickness

[33]

Shewanella oneidensis and EcN

Improving bacterial survival

[44]

Aminated DNA aptamer

Attenuated Salmonella typhimurium

Targeting tumor tissues

[45]

Fe₃O₄ nanoparticles

E. coli

Anti-tumor

[46]

Imidoester and mucoprotein

EcN

Mucosal protection and anti-inflammation

[47]

Polydopamine

Salmonella strain VNP20009 and EcN

Anti-tumor and enhancing stomach acid tolerance

[50, 52]

Metal nanozyme

VNP20009

Tumor radioimmunotherapy

[53]

Selenium quantum dots

L. casei

Ulcerative colitis

[54]

Enteric polymer L100-55, HA

EcN, S. oneidensis

Anti-tumor

[44]

Biotin-conjugated polyclonal antibodies, streptavidin-coupled PLGA nanoparticles

Photosynthetic bacteria (Synechococcus 7942)

Anti-tumor

[57-59]

PEG and PDA

EcN

Mucosal protection and intestinal microecological regulation

[60]

Genetic modification	Modification locus	Chassis species	Application	Ref.
chuA, hrtR, luxCDABE	Plasmid	E. coli	Monitor intestinal bleeding and inflammation	[70]
narX/L, thsS/R, sfGFP	Plasmid	EcN	Monitor intestinal inflammation	[71]
ttrS/R, cro, o_L1-3, neo, cl, o_R3-1, lacZ	gDNA	E. coli, S. typhimurium	Long-term monitor intestinal inflammation	[72]
hok, sok, alp	Plasmid	EcN	Noninvasive diagnosis of early liver cancer	[73]
ZsGreen	Plasmid	S. typhimurium	Early diagnosis of cancer	[74, 75]
lasR, GFP, CoPy (a fusion protein), prgX/Q, AMPs	Plasmid	P. aeruginosa, E. faecalis	Antimicrobial agent	[77, 78]
Δalr, ΔdadX, lasR, pyoS5, colE7, dspB	Plasmid	EcN	Against gut infections	[79]
luxR, luxI, GFP	Plasmid	S. typhimurium	Anti-tumor and safety control	[80]
hlyE, sfGFP, luxI, φ174E	Plasmid	S. typhimurium	Anti-tumor	[81]
GLP-1, P_slpA	Plasmid	L. gasseri	Diabetes	[85]
Decarboxylase, IL-10	Plasmid	L. lactis	Diabetes	[87]
Tumor-associated antigen	Plasmid and gDNA	L. monocytogenes	Anti-tumor	[88]
Cytidine deaminase	Plasmid	S. typhimurium	Anti-tumor	[89]
IL-10	Plasmid	L. lactis	Ulcerative colitis	[90]
csg	Plasmid	EcN	Ulcerative colitis	[91]
ΔargR, ΔthyA, malEK: : P_fnrS-argA^fbr	gDNA	EcN	Hyperammonemia	[92]
hlyB/D, Sj16	Plasmid	EcN	Inflammatory bowel disease (IBD)	[93]
anti-PD-L1nb, anti-CTLA-4nb, luxI, φ174E	gDNA	EcN	Anti-tumor	[94]
ΔargR, OE: : argA^fbr	gDNA	EcN	Anti-tumor	[95]
thsS/R, sfGFP, hly-avCys, BE2, sgRNA, mCherry, hlyB/D, ACG-lacZ	gDNA and plasmid	EcN	IBD diagnosis, record, and treatment	[96]
ΔthyA, Δcsg, OE: : csgA-tff3, SOD, CAT	gDNA and plasmid	EcN	Radiation enteritis	[97]
bsh, IL-10	gDNA	E. coli	IBD	[98]
virB, T3SA, nanoanbibody, Δalr, ΔdadX	gDNA and plasmid	E. coli	IBD	[99]
ΔompT, Δlon, OE: : LLO, neo antigen	gDNA and plasmid	EcN	Tumor vaccine	[100]
GM-CSF, SIRPα	Plasmid	EcN	Anti-tumor	[101]

Genetic modification

Modification locus

Chassis species

Application

Ref.

chuA, hrtR, luxCDABE

Plasmid

E. coli

Monitor intestinal bleeding and inflammation

[70]

narX/L, thsS/R, sfGFP

Plasmid

EcN

Monitor intestinal inflammation

[71]

ttrS/R, cro, o_L1-3, neo, cl, o_R3-1, lacZ

gDNA

E. coli, S. typhimurium

Long-term monitor intestinal inflammation

[72]

hok, sok, alp

Plasmid

EcN

Noninvasive diagnosis of early liver cancer

[73]

ZsGreen

Plasmid

S. typhimurium

Early diagnosis of cancer

[74, 75]

lasR, GFP, CoPy (a fusion protein), prgX/Q, AMPs

Plasmid

P. aeruginosa, E. faecalis

Antimicrobial agent

[77, 78]

Δalr, ΔdadX, lasR, pyoS5, colE7, dspB

Plasmid

EcN

Against gut infections

[79]

luxR, luxI, GFP

Plasmid

S. typhimurium

Anti-tumor and safety control

[80]

hlyE, sfGFP, luxI, φ174E

Plasmid

S. typhimurium

Anti-tumor

[81]

GLP-1, P_slpA

Plasmid

L. gasseri

Diabetes

[85]

Decarboxylase, IL-10

Plasmid

L. lactis

Diabetes

[87]

Tumor-associated antigen

Plasmid and gDNA

L. monocytogenes

Anti-tumor

[88]

Cytidine deaminase

Plasmid

S. typhimurium

Anti-tumor

[89]

IL-10

Plasmid

L. lactis

Ulcerative colitis

[90]

csg

Plasmid

EcN

Ulcerative colitis

[91]

ΔargR, ΔthyA, malEK: : P_fnrS-argA^fbr

gDNA

EcN

Hyperammonemia

[92]

hlyB/D, Sj16

Plasmid

EcN

Inflammatory bowel disease (IBD)

[93]

anti-PD-L1nb, anti-CTLA-4nb, luxI, φ174E

gDNA

EcN

Anti-tumor

[94]

ΔargR, OE: : argA^fbr

gDNA

EcN

Anti-tumor

[95]

thsS/R, sfGFP, hly-avCys, BE2, sgRNA, mCherry, hlyB/D, ACG-lacZ

gDNA and plasmid

EcN

IBD diagnosis, record, and treatment

[96]

ΔthyA, Δcsg, OE: : csgA-tff3, SOD, CAT

gDNA and plasmid

EcN

Radiation enteritis

[97]

bsh, IL-10

gDNA

E. coli

IBD

[98]

virB, T3SA, nanoanbibody, Δalr, ΔdadX

gDNA and plasmid

E. coli

IBD

[99]

ΔompT, Δlon, OE: : LLO, neo antigen

gDNA and plasmid

EcN

Tumor vaccine

[100]

GM-CSF, SIRPα

Plasmid

EcN

Anti-tumor

[101]

Phase	Disease	Treatment	Route	Significance	Identifier/Refs.
Ⅰ (completed)	Cancer (advanced or metastatic)	VNP20009 (engineered S. typhimurium)	i.v.	S. typhimurium is genetically engineered to delete purI, msbB and xyl	NCT00004988
Ⅰ (ongoing)	Glioblastoma multiforme	EGFR(Ⅴ)-EDV-Dox (engineered bacterial minicell)	i.v.	Bacterial minicell derived from S. typhimurium minCDE-strain is engineered to target EGFR and carry doxorubicin	NCT02766699
Ⅰ/Ⅱ (suspended)	Solid tumours (advanced and/or metastatic)	APS001F (engineered B. longum) in combination with flucytosine and maltose	i.v.	B. longum is genetically engineered to produce cytosine deaminase	NCT01562626
Ⅰ/Ⅱ (discontinued)	Familial adenomatous polyposis	CEQ508 (engineered E. coli)	Oral	An attenuated strain (undisclosed) of E. coli is genetically engineered to deliver β-catenin short-hairpin RNA	[118]
Ⅱ (recruiting)	Metastatic pancreatic cancer	Saltikva (engineered S. Typhimurium) in combination with either FOLFIRINOX or gemcitabine/paclitaxel	Oral	An attenuated strain (undisclosed) of S. typhimurium is genetically engineered to express IL-2	NCT04589234
Ⅲ (recruiting)	Phenylketonuria	SYNB1934 (engineered EcN)	Oral	EcN is genetically engineered to metabolize L-phenylalanine	NCT05764239

Phase

Disease

Treatment

Route

Significance

Identifier/Refs.

Ⅰ (completed)

Cancer (advanced or metastatic)

VNP20009 (engineered S. typhimurium)

i.v.

S. typhimurium is genetically engineered to delete purI, msbB and xyl

NCT00004988

Ⅰ (ongoing)

Glioblastoma multiforme

EGFR(Ⅴ)-EDV-Dox (engineered bacterial minicell)

i.v.

Bacterial minicell derived from S. typhimurium minCDE-strain is engineered to target EGFR and carry doxorubicin

NCT02766699

Ⅰ/Ⅱ (suspended)

Solid tumours (advanced and/or metastatic)

APS001F (engineered B. longum) in combination with flucytosine and maltose

i.v.

B. longum is genetically engineered to produce cytosine deaminase

NCT01562626

Ⅰ/Ⅱ (discontinued)

Familial adenomatous polyposis

CEQ508 (engineered E. coli)

Oral

An attenuated strain (undisclosed) of E. coli is genetically engineered to deliver β-catenin short-hairpin RNA

[118]

Ⅱ (recruiting)

Metastatic pancreatic cancer

Saltikva (engineered S. Typhimurium) in combination with either FOLFIRINOX or gemcitabine/paclitaxel

Oral

An attenuated strain (undisclosed) of S. typhimurium is genetically engineered to express IL-2

NCT04589234

Ⅲ (recruiting)

Phenylketonuria

SYNB1934 (engineered EcN)

Oral

EcN is genetically engineered to metabolize L-phenylalanine

NCT05764239