Engineered Probiotic Bacteria Colonize Tumors, Attract CAR-T Cells
来源：Science | 发布时间：2023-10-16
Researchers at Columbia University have developed a probiotic-guided chimeric antigen receptor (CAR)-T platform that uses engineered bacteria to infiltrate and produce synthetic antigen targets, enabling CAR-T cells to find, identify, and destroy tumor cells in situ. The results of in vivo preclinical tests suggest that the combined ProCAR cell therapy platform could expand the scope of CAR-T cell therapy to include difficult-to-target solid tumors.
Tal Danino, PhD, and Rosa L. Vincent, PhD, at Columbia University’s department of biomedical engineering, and colleagues, reported on their developments in Science, in a paper titled “Probiotic-guided CAR-T cells for solid tumor targeting,” in which they concluded, “These findings highlight the potential of the ProCAR platform to address the roadblock of identifying suitable CAR targets by providing an antigen that is orthogonal to both healthy tissue and tumor genetics … Overall, combining the advantages of tumor-homing bacteria and CAR-T cells provides a new strategy for tumor recognition and, in turn, builds the foundation for engineered communities of living therapies.”
Immunotherapies using CAR-T cells have proven successful in treating some types of blood cancers, but their efficacy against solid tumors remains elusive. A key challenge facing tumor-antigen targeting immunotherapies like CAR-T is the identification of suitable targets that are specifically and uniformly expressed on solid tumors, the authors noted. “A key challenge of antigen-targeted cell therapies relates to the expression patterns of the antigen itself, which makes the identification of optimal targets for solid tumor cell therapies an obstacle for the development of new CARs.” Solid tumors express heterogeneous and nonspecific antigens and are poorly infiltrated by T cells. As a result, the approach carries a high risk of fatal on-target, off-tumor toxicity, wherein CAR-T cells attack the targeted antigen on healthy vital tissues with potentially fatal effects. “Few tumor-associated antigens (TAAs) identified on solid tumors are tumor restricted, and thus, they carry a high risk of fatal on-target, off-tumor toxicity because of cross-reactivity against proteins found in vital tissues,” the team continued.
Previous studies have shown that, unlike CAR-T cells, which require “considerable engineering to target and infiltrate solid tumors,” some species of bacteria can selectively colonize and preferentially grow within the hostile tumor microenvironments (TMEs) of immune-privileged tumor cores, and can be engineered as antigen-independent platforms for therapeutic delivery.
In this study, Vincent, Candice Gurbatri, and colleagues combine probiotic therapy with CAR-T cell therapy to create a two-stage probiotic-guided CAR-T cell (ProCAR) platform, whereby T cells are engineered to sense and respond to synthetic CAR targets that are delivered by solid tumor-colonizing probiotic bacteria. “This approach leverages the antigen independence of tumor-seeking microbes to create a combined cell therapy platform that broadens the scope of CAR-T cell therapy to include difficult-to-target tumors,” the investigators explained.
Using synthetic gene circuit engineering on a well-characterized non-pathogenic strain of E. coli, Vincent et al. created a probiotic that could infiltrate and cyclically release synthetic CAR targets directly to the tumor core, effectively “tagging” the tumor tissue. “With this system, bacterial growth reaches a critical population density exclusively within the niche of the solid TME and subsequently triggers lysis events that cyclically release genetically encoded payloads in situ,” they further explained.
Then, CAR-T cells that were programmed to recognize the probiotic-delivered synthetic antigen tags could be generated that homed in on the tagged solid tumors, killing the tumor cells in situ. The scientists also engineered probiotics that co-released chemokines in addition to synthetic targets to further enhance CAR-T cell recruitment to the tumor, further boosting therapeutic response.
Vincent et al. tested the resulting probiotic-guided CAR-T cell platform in humanized and immunocompetent mouse models of leukemia, colorectal cancer, and breast cancer and showed that it resulted in the safe reduction of tumor volume. “Collectively, these mouse model data demonstrate the use of engineered probiotics to selectively grow within the TME niche and safely release combinations of CAR-T cell enhancing payloads in situ,” they wrote. The team acknowledged that further development of the system will be needed before it can be considered for clinical application. Nevertheless, they stated, “We have demonstrated an approach to engineering interactions between living therapies, in which tumor-colonizing probiotics have been repurposed to guide the cytotoxicity of engineered T cells.”
In a related Perspective, Eric Bressler, PhD, and Wilson Wong, PhD, at Boston University Biomedical Engineering and Biological Design Center, also noted, “Translation of the ProCAR system to the clinic will depend on scalability to larger tumors and attenuation of bacterial strains for safety.” However, they concluded, “The study of Vincent et al. is an important proof-of-concept for a potential approach to treating heterogeneous, immunologically cold, and poorly infiltrated solid tumors.”
益生菌激发出CAR-T巨大潜力
嵌合抗原受体T细胞（CAR-T）疗法的出现，让部分血液肿瘤患者迎来了治愈的希望。通过收集患者自身的T细胞，然后工程改造带上识别肿瘤抗原的受体，科学家就能使T细胞更精准和高效地寻找肿瘤。
在应对血液肿瘤时，CAR-T会更加得心应手，因为血液循环中会充斥大量抗原靶标，T细胞也容易接触到肿瘤。但对藏在组织中的实体瘤，CAR-T会有点力不从心，首先它们很难浸润到肿瘤区域，此外实体瘤的抗原往往不够特异性，改造的T细胞还可能会攻击健康组织，造成严重的副作用。如何利用CAR-T细胞安全且高效地治疗实体瘤，是科学家努力攻克的新方向。
在最新一期的《科学》期刊上，哥伦比亚大学的研究者创新地提出了利用益生菌来引导CAR-T的方式，此举能帮助T细胞找到实体瘤，并且提升T细胞清除肿瘤的效率，他们将其称作益生菌引导的CAR疗法（probiotic-guided CAR-T cell，ProCAR）。
其实拆分来看，细菌和CAR-T都是治疗肿瘤的有效帮手，比如肿瘤定植细菌早就用于癌症研究。得益于这类细菌的肿瘤归巢特性，它们在进入体内后会朝着肿瘤区域前进，最终在肿瘤组织中定植和生长。
后续，细菌会吸引免疫细胞前往来攻击肿瘤，这一过程仍然比较依赖自身的T细胞水平和活性。但如果让它们吸引一个本来就强力的CAR-T细胞，结果会不会更好呢？这就是新研究尝试解决的问题。
研究团队分两步开展了实验，首先是选取一批原本就喜爱肿瘤微环境的细菌，测试中使用了改造后的大肠杆菌菌株，这些细菌会定向朝肿瘤移动，并且在肿瘤微环境中定植，当细菌种群密度达到一定程度后，细菌会引发后续的溶解事件，释放出可靶向的抗原，其中包括提前设计的特异性抗原。
▲研究示意图
这一次，CAR-T细胞上携带的不再是识别肿瘤抗原的受体，而是用于专门靶向肿瘤定植细菌的抗原标签。作者在小鼠体内移植了肿瘤，然后使用了这种全新的ProCAR技术，结果他们看到与对照组相比，经过ProCAR治疗的小鼠肿瘤生长速度要更慢，小鼠的生存率也更高。
他们同样在结直肠癌模型小鼠中观察到了相似的结果，ProCAR对小鼠没有明显毒副作用，不会影响小鼠的活动行为、体重等，但能够显著抑制肿瘤的生长和远端转移。
除了T细胞本身的作用，工程细菌还会额外释放趋化因子，增强肿瘤微环境中的免疫敏感程度。该研究的作者之一Rosa Vincent博士指出，“新研究结合了两类疗法的优势，并弥补了它们的局限性，即用细菌来放置靶标，用T细胞来破坏肿瘤。”研究团队未来计划开展临床试验，以验证ProCAR方式在人体中的安全性和有效性。