Bioengineering breakthroughs:The impact of stem cell models on advanced therapy medicinal product development

The burgeoning field of bioengineering has witnessed significant strides due to the advent of stem cell models,particularly in their application in advanced therapy medicinal products(ATMPs).In this review,we examine the multifaceted impact of these developments,emphasizing the potential of stem cell models to enhance the sophistication of ATMPs and to offer alternatives to animal testing.Stem cell-derived tissues are particularly promising because they can reshape the preclinical landscape by providing more physiologically relevant and ethically sound platforms for drug screening and disease modelling.We also discuss the critical challenges of reproducibility and accuracy in measurements to ensure the integrity and utility of stem cell models in research and application.Moreover,this review highlights the imperative of stem cell models to align with regulatory standards,ensuring using stem cells in ATMPs translates into safe and effective clinical therapies.With regulatory approval serving as a gateway to clinical adoption,the collaborative efforts between scientists and regulators are vital for the progression of stem cell applications from bench to bedside.We advocate for a balanced approach that nurtures innovation within the framework of rigorous validation and regulatory compliance,ensuring that stem cell-base solutions are maximized to promote public trust and patient health in ATMPs.

Insights gained from single-cell analysis of chimeric antigen receptor T-cell immunotherapy in cancer

Advances in chimeric antigen receptor(CAR)-T cell therapy have significantly improved clinical outcomes of patients with relapsed or refractory hematologic malignancies.However,progress is still hindered as clinical benefit is only available for a fraction of patients.A lack of understanding of CAR-T cell behaviors in vivo at the single-cell level impedes their more extensive application in clinical practice.Mounting evidence suggests that single-cell sequencing techniques can help perfect the receptor design,guide gene-based T cell modification,and optimize the CAR-T manufacturing conditions,and all of them are essential for long-term immunosurveillance and more favorable clinical outcomes.The information generated by employing these methods also potentially informs our understanding of the numerous complex factors that dictate therapeutic efficacy and toxicities.In this review,we discuss the reasons why CAR-T immunotherapy fails in clinical practice and what this field has learned since the milestone of single-cell sequencing technologies.We further outline recent advances in the application of single-cell analyses in CAR-T immunotherapy.Specifically,we provide an overview of single-cell studies focusing on target antigens,CAR-transgene integration,and preclinical research and clinical applications,and then discuss how it will affect the future of CAR-T cell therapy.

Research Status of CAR-T Cell Immunotherapy in Tumor Treatment

In recent years, chimeric antigen receptor T-cell (CAR-T) therapy has made breakthroughs in the treatment of hematological tumors. However, due to the different characteristics of solid tumors from hematological tumors, CAR-T has not achieved good efficacy in the treatment of solid tumors. The key factors limiting the efficacy of CAR-T mainly include the solid tumor cells themselves and their special tumor microenvironment (TME), which damage CAR-T function in multiple processes such as CAR-T infiltration to tumor tissue sites, CAR-T maintaining anti-tumor activity in TME, and target recognition and killing of tumor cells by CAR-T. To solve these problems, more and more preclinical studies have proposed potentially effective solutions, and corresponding clinical studies have been carried out one after another. In this article, the existing challenges and corresponding optimization strategies of CAR-T cell therapy for solid tumors will be reviewed, to provide a reference for the future exploration of CAR-T therapy.

New era of personalized medicine:Advanced therapy medicinal products in Europe

Advanced therapy medicinal products are human medical therapies based on genes,cells,or tissues,and due to their characteristics,they offer new innovative opportunities for the treatment of diseases and injuries,especially for diseases beyond the reach of traditional approaches.These therapies are at the forefront of innovation and have historically been very controversial,although in the last decade they have gained prominence while the number of new advanced therapies has increased every year.In this regard,despite the controversy they may generate,they are expected to dominate the market in the coming decades.Technologies based on advanced therapies are the present and future of medicine and bring us closer to the long-awaited precision medicine.Here we review the field as it stands today,with a focus on the molecular mechanisms that guided the different advanced therapies approved by the European Medicines Agency,their current status,and their legal approval.

自体嵌合抗原受体T细胞(CAR-T)治疗产品药学变更实践与思考

近年来我国在嵌合抗原受体T细胞(CAR-T cell)治疗领域的研究非常活跃。由于该领域技术迭代快,生产工艺复杂,药学研究和变更方面的经验相对有限,CAR-T产品药学变更的评估、研究以及申报、审评对企业和监管机构提出了多方面的挑战。本课题组聚焦于自体CAR-T细胞治疗产品药学变更案例及其共性问题与挑战,进行了行业调查、法规与案例收集和研讨以及变更管理工具评估,并提出了多项建议,期望为企业和监管机构管理CAR-T药学变更提供参考。相关法规收集与分析结果显示,我国和美国在法规方面均走在前沿,总体原则均为基于科学和风险;在细节和实践上,监管机构均处于持续研究、探讨和完善的阶段。行业调研结果显示,企业药学变更主要目的之一是降低成本以提高产品可及性,主要挑战包括企业和监管机构对变更理解的差异、沟通机制以及审评审批时长。收集的药学变更案例包括多种变更情形,其中针对扩大生产产能,建议基于适当产品与工艺知识经验、风险管控和无菌验证,可使用同步验证,适当时使用模拟验证辅助。就上市后变更管理工具,建议试行ICH Q12批准后变更管理方案(PACMP)帮助管理自体CAR-T细胞治疗产品上市后药学变更。

Unlocking T cell exhaustion:Insights and implications for CAR-T cell therapy

Chimeric antigen receptor T(CAR-T)cell therapy as a form of adoptive cell therapy(ACT)has shown significant promise in cancer treatment,demonstrated by the FDA-approved CAR-T cell therapies targeting CD19 or B cell maturation antigen(BCMA)for hematological malignancies,albeit with moderate outcomes in solid tumors.However,despite these advancements,the efficacy of CAR-T therapy is often compromised by T cell exhaustion,a phenomenon that impedes the persistence and effector function of CAR-T cells,leading to a relapse rate of up to 75%in patients treated with CD19 or CD22 CAR-T cells for hematological malignancies.Strategies to overcome CAR-T exhaustion employ state-of-the-art genomic engineering tools and single-cell sequencing technologies.In this review,we provide a comprehensive understanding of the latest mechanistic insights into T cell exhaustion and their implications for the current efforts to optimize CAR-T cell therapy.These insights,combined with lessons learned from benchmarking CAR-T based products in recent clinical trials,aim to address the challenges posed by T cell exhaustion,potentially setting the stage for the development of tailored next-generation approaches to cancer treatment.

CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors

In the past decade,chimeric antigen receptor(CAR)-T cell therapy has emerged as a promising immunotherapeutic approach for combating cancers,demonstrating remarkable efficacy in relapsed/refractory hematological malignancies in both pediatric and adult patients.CAR-natural killer(CAR-NK)cell complements CAR-T cell therapy by offering several distinct advantages.CAR-NK cells do not require HLA compatibility and exhibit low safety concerns.Moreover,CAR-NK cells are conducive to“off-the-shelf”therapeutics,providing significant logistic advantages over CAR-T cells.Both CAR-T and CAR-NK cells have shown consistent and promising results in hematological malignancies.However,their efficacy against solid tumors remains limited due to various obstacles including limited tumor trafficking and infiltration,as well as an immuno-suppressive tumor microenvironment.In this review,we discuss the recent advances and current challenges of CAR-T and CAR-NK cell immunotherapies,with a specific focus on the obstacles to their application in solid tumors.We also analyze in depth the advantages and drawbacks of CAR-NK cells compared to CAR-T cells and highlight CAR-NK CAR optimization.Finally,we explore future perspectives of these adoptive immunotherapies,highlighting the increasing contribution of cutting-edge biotechnological tools in shaping the next generation of cellular immunotherapy.

Dawn of CAR-T cell therapy in autoimmune diseases

Chimeric antigen receptor(CAR)-T cell therapy has achieved remarkable success in the treatment of hematological malignancies.Based on the immunomodulatory capability of CAR-T cells,efforts have turned toward exploring their potential in treating autoimmune diseases.Bibliometric analysis of 210 records from 128 academic journals published by 372 institutions in 40 countries/regions indicates a growing number of publications on CAR-T therapy for autoimmune diseases,covering a range of subtypes such as systemic lupus erythematosus,multiple sclerosis,among others.CAR-T therapy holds promise in mitigating several shortcomings,including the indiscriminate suppression of the immune system by traditional immunosuppressants,and non-sustaining therapeutic levels of monoclonal antibodies due to inherent pharmacokinetic constraints.By persisting and proliferating in vivo,CAR-T cells can offer a tailored and precise therapeutics.This paper reviewed preclinical experiments and clinical trials involving CAR-T and CAR-related therapies in various autoimmune diseases,incorporating innovations well-studied in the field of hematological tumors,aiming to explore a safe and effective therapeutic option for relapsed/refractory autoimmune diseases.

Therapeutic potential of thymoquinone in combination therapy against cancer and cancer stem cells

The long-term success of standard anticancer monotherapeutic strategies has been hampered by intolerable side effects,resistance to treatment and cancer relapse.These monotherapeutic strategies shrink the tumor bulk but do not effectively eliminate the population of self-renewing cancer stem cells(CSCs)that are normally present within the tumor.These surviving CSCs develop mechanisms of resistance to treatment and refuel the tumor,thus causing cancer relapse.To ensure durable tumor control,research has moved away from adopting the monotreatment paradigm towards developing and using combination therapy.Combining different therapeutic modalities has demonstrated significant therapeutic outcomes by strengthening the anti-tumor potential of monotreatment against cancer and cancer stem cells,mitigating their toxic adverse effects,and ultimately overcoming resistance.Recently,there has been growing interest in combining natural products from different sources or with clinically used chemotherapeutics to further improve treatment efficacy and tolerability.Thymoquinone(TQ),the main bioactive constituent of Nigella sativa,has gained great attention in combination therapy research after demonstrating its low toxicity to normal cells and remarkable anticancer efficacy in extensive preclinical studies in addition to its ability to target chemoresistant CSCs.Here,we provide an overview of the therapeutic responses resulting from combining TQ with conventional therapeutic agents such as alkylating agents,antimetabolites and antimicrotubules as well as with topoisomerase inhibitors and non-coding RNA.We also review data on anticancer effects of TQ when combined with ionizing radiation and several natural products such as vitamin D3,melatonin and other compounds derived from Chinese medicinal plants.The focus of this review is on two outcomes of TQ combination therapy,namely eradicating CSCs and treating various types of cancers.In conclusion,the ability of TQ to potentiate the anticancer activity of many chemotherapeutic agents and sensitize cancer cells to radiotherapy makes it a promising molecule that could be used in combination therapy to overcome resistance to standard chemotherapeutic agents and reduce their associated toxicities.

Formulation strategies in immunotherapeutic pharmaceutical products

Development of immunologic-based biopharmaceutical products have strikingly increased in recent years and have made evident contributions to human health.Antibodies are the leading entity in immunotherapy,while chimeric antigen receptor T cells therapies are the advent of a novel strategy in this area.In order to enable antibody candidates or cells available as products,formulation is critical in terms of stabilize molecules or cells to achieve practical shelf life,storage and handling conditions.Here we provide a concise and contemporary review of ongoing formulation strategies and excipients used in approved antibodies and cellular therapeutic products.Excipients are categorized,and their function in formulations are discussed.

Generating universal chimeric antigen receptor expressing cell products from induced pluripotent stem cells:beyond the autologous CAR-T cells

Adoptive therapeutic immune cells, such as chimeric antigen receptor (CAR)-T cells and natural killer cells, have established a new generation of precision medicine based on which dramatic breakthroughs have been achieved in intractable lymphoma treatments. Currently, well-explored approaches focus on autologous cells due to their low immunogenicity, but they are highly restricted by the high costs, time consumption of processing, and the insufficiency of primary cells in some patients. Induced pluripotent stem cells (iPSCs) are cell sources that can theoretically produce indefinite well-differentiated immune cells. Based on the above facts, it may be reasonable to combine the iPSC technology and the CAR design to produce a series of highly controllable and economical "live" drugs. Manufacturing hypoimmunogenic iPSCs by inactivation or over-expression at the genetic level and then arming the derived cells with CAR have emerged as a form of "off-the-shelf" strategy to eliminate tumor cells efficiently and safely in a broader range of patients. This review describes the reasonability, feasibility, superiority, and drawbacks of such approaches, summarizes the current practices and relevant research progress, and provides insights into the possible new paths for personalized cell-based therapies.

Nanotherapeutics approaches to improve the efficacy of CAR-T cells in solid tumors

Adoptive cell therapy and Immune Checkpoint Blockade Inhibitors have recently revolutionized the field of oncology.However,these types of immunotherapeutic approaches have limited success in treating solid tumors.In particular,chimeric antigen receptor(CAR)-T cells efficacy is hampered by immunosuppressive signals in the tumor microenvironment(TME)and by a limited infiltration of re-infused T cells to the tumor site.The field of nanobiotechnology applied to oncology is also rapidly expanding.Nanoparticles-based delivery systems can be employed to modulate the activity of immune cells present in the TME enhancing the efficacy of CAR-T cells.Interestingly,nano-backpacks can be attached to CAR-T cells prior to re-infusion to support their homing to the tumor site and to slowly release immunopotentiators directly in the TME.Furthermore,nanovaccines can also be employed to support the in vivo expansion of CAR-T cells with consequent enhancement of their therapeutic potential.In this viewpoint,recent advancement in the field of nanobiotechnology to support CAR-T cell therapy will be discussed.The development of novel therapeutic CAR-T cells protocols together with nanotherapies is warranted in order to take full advantage of the high therapeutic potential of CAR-T cell therapy.

Direct anti-atherosclerotic therapy preventing intracellular cholesterol retention

The key initiating process in atherogenesis is the subendothelial cholesterol retention, which is both necessary and sufficient to provoke lesion initiation. Retention of cholesterol transported by low density lipoprotien (LDL) in subendothelial cells of arterial wall, is an absolute requirement for lesion development. This allows us to consider intracellular cholesterol retention as a novel target for anti-atherosclerotic therapy. In this case, the target is not the level of blood cholesterol but the level of cholesterol in vascular cells. This review summarizes the results of our basic studies shedding light on the mechanisms of intracellular cholesterol retention. We describe our cellular models to search for anti-atherosclerotic agents and demonstrate the use of these models for the development of anti-atherosclerotic drugs. We use natural products as the basis of anti-atherosclerotic drugs because anti-atherosclerotic therapy should be long-term or even lifelong. Using cellular models and natural products, we have developed an approach to prevent intracellular cholesterol retention in cultured subendothelial aortic cells. We have developed drugs that reduce intracellular cholesterol retention, namely Allicor on the basis of garlic powder, anti-inflammatory drug Inflaminat (calendula, elder, and violet) possessing anti-cytokine activity and phytoestrogen-rich drug Karinat (garlic powder, extract of grape seeds, green tea leaves, hop cones, β-carotene, α-tocopherol, and ascorbic acid). Treatment with Allicor or Inflaminat caused regression of carotid atherosclerosis in asymptomatic men. Karinat prevented the development of new atherosclerotic plaques in postmenopausal women. Thus, the main findings of our basic research have been successfully translated into clinics. As a result, this translation, a novel approach to the development of anti-atherosclerotic therapy, has been established. Our clinical trials have confirmed the suitability of innovative approach and the efficacy of novel drugs developed on the basis our methodology.

Intracellular Cholesterol Retention—New Target for Direct Anti-Atherosclerotic Therapy

Accumulation of cholesterol in arterial cells, intracellular cholesterol retention, may be responsible for all major manifestations of atherosclerosis on a cellular level. Previously we have shown that intracellular cholesterol retention is the principal event in the genesis of atherosclerotic lesions. This allows us to consider cellular retention of cholesterol as a novel target for anti-atherosclerotic therapy. In this case the target is not the level of blood cholesterol but the level of cholesterol in vascular cells. This review describes our approach based on the use of cultured human arterial cells for the development of direct anti-atherosclerotic therapy. We use natural products as the basis of promising drugs for anti-atherosclerotic therapy. Using natural products, we have developed an approach to prevent intracellular cholesterol retention in cultured cells. Our knowledge of the mechanisms of atherosclerosis is the foundation on which we have developed drugs that have a direct anti-atherosclerotic effect, namely Allicor on the basis of garlic powder, anti-inflammatory drug Inflaminat (calendula, elder, and violet) possessing anti-cytokine activity and phytoestrogen-rich drug Karinat (garlic powder, extract of grape seeds, green tea leaves, hop cones, β-carotene, α-tocopherol, and ascorbic acid). Treatment with allicor or inflaminat has a direct anti-atherosclerotic effect on carotid atherosclerosis in asymptomatic men. Karinat prevents the development of carotid atherosclerosis in postmenopausal women. Thus, the main findings of our basic research have been successfully translated into clinical practice. As a result, this translation, a novel approach to the development of anti-atherosclerotic therapy, has been established. Our clinical trials have confirmed the suitability of innovative approach and the efficacy of novel drugs developed on the basis our methodology.

省级药监局视角下CAR-T治疗药品生产监管探讨

目的:为省级药监局建立和完善已上市CAR-T治疗药品生产监管制度提供参考。方法:分析CAR-T治疗药品的产品、药学研究、临床研究、生产、临床使用等方面特点,介绍WHO、PIC/S、美国、欧盟等监管机构的监管政策,梳理我国目前的监管体系。结果与结论:从省级药监局的职责出发,提出已上市CAR-T治疗药品生产监管应重点关注的内容,包括变更管理关注可比性研究、GMP符合性检查关注6个关注点和延伸检查、GVP符合性检查关注上市后监测、日常监督检查关注风险信号和企业对医疗机构的管理策略。

Research on the Regulatory Framework of Advanced Therapies in the European Union and the United States

Objective To study the regulatory framework of advanced therapies in the European Union and the United States,and to provide reference for the regulation of cell-and gene-based therapeutic products in China.Methods The legal and regulatory documents,annual reports,work information and related literature published on the websites of the FDA and European Medicines Agency(EMA)were reviewed to analyze the regulatory models of advanced therapies in the European Union and the United States.Results and Conclusion the United States and the European Union have carried out a lot of work in the classification standards of advanced therapies,policy formulation and accelerated listing procedures.Therefore,they have established a relatively mature regulatory system.China can learn from their experience and continuously improve the regulatory system to help the sustainable development of gene and cell therapy industry.

我国先进治疗药品的范围及分类研究和建议

近年来先进治疗药品研发申报呈爆发式增长,引领生物医药的第三次产业革命。全球范围内嵌合抗原受体T细胞(CAR-T)治疗产品、腺相关病毒载体基因治疗产品等不同类型的细胞和基因治疗产品相继批准上市,为复发难治疾病提供了有效的治疗手段。先进治疗药品通常采用细胞或组织经基因修饰和(或)体外操作制备,种类包括基因修饰细胞、核酸、病毒载体和人工组织等。由于这类产品类型复杂、多样,明确其定义和分类,有利于优化产品注册申报路径,加速各类产品技术指南的发布,推动产品快速研发上市,助力我国药品监管与国际接轨。本文通过调研美国食品药品监督管理局(FDA)、欧洲药品管理局(EMA)、日本药品医疗器械综合机构(PMDA)等药品监管机构在法规层面对先进治疗药品的定义及监管分类情况,结合我国产品申报现状和审评积累,提出了对先进治疗药品分类和描述的建议,为我国相关监管政策制定提供参考。

欧盟先进疗法医药产品的医院豁免条款研究及对我国的启示

目的 研究欧盟先进疗法医药产品的医院豁免条款特点,为完善我国细胞和基因治疗产品的监管体系提供启示与建议。方法 通过文献研究及检索欧盟各成员国官网,从“非常规”定义、生产质量标准、药物警戒要求方面比较各成员国适用和实施医院豁免条款的差异,分析医院豁免条款在实践中可能存在的问题,并结合我国细胞和基因治疗产品的监管现状提出政策建议。结果与结论 欧盟医院豁免条款为罕见病、缺乏有效治疗或更好治疗方案的患者提供了获得新治疗方法的机会,有效提高了患者用药的可及性。但欧盟各成员国针对医院豁免条款的规定存在一定差异,譬如部分成员国未明确界定“非常规”情形,各个成员国在生产质量标准及药物警戒要求方面不同。医院豁免条款在实施过程中也存在信息透明度不高、缺乏一定的限制条件等问题。结合我国细胞和基因治疗产业的发展及监管现状,建议我国在“非常规”情形下探索低风险细胞和基因治疗方法的临床转化应用,并从生产质量标准、药物警戒要求等方面加强对临床转化应用疗法的管理;应当进一步规范研究者发起的临床研究,并注意平衡临床应用与药品注册上市的关系,引导我国细胞和基因治疗产业持续健康发展。

基于免疫细胞治疗产品生产技术探讨监管检查关注要点

目的:梳理免疫细胞治疗产品的开发以及监管检查文件依据,为此类产品的生产监管提供参考。方法:针对免疫细胞治疗产品的生产特点进行整理分析,介绍国内相关监管法规及指导意见,并对此类产品的生产现场检查面临的关键问题进行汇总分析,提出相关建议。结果与结论:细胞作为药品上市与传统药品相比具有多种独特的特性,因此在生产管理、质量控制、共线生产导致的系统污染风险等方面均存在挑战,本文从省级监管部门监管角度结合免疫细胞治疗产品的生产工艺,针对此类产品提出免疫细胞治疗产品现场检查关注重点及风险研判的建议,希望进一步完善免疫细胞治疗产品的上市后监管制度。

Targeting metabolism to improve CAR-T cells therapeutic efficacy

Chimeric antigen receptor T(CAR-T)cell therapy achieved advanced progress in the treatment of hematological tumors.However,the application of CAR-T cell therapy for solid tumors still faces many challenges.Competition with tumor cells for metabolic resources in an already nutrient-poor tumor microenvironment is a major contributing cause to CAR-T cell therapy’s low effectiveness.Abnormal metabolic processes are now acknowledged to shape the tumor microenvironment,which is characterized by increased interstitial fluid pressure,low pH level,hypoxia,accumulation of immunosuppressive metabolites,and mitochondrial dysfunction.These factors are important contributors to restriction of T cell proliferation,cytokine release,and suppression of tumor cell-killing ability.This review provides an overview of how different metabolites regulate T cell activity,analyzes the current dilemmas,and proposes key strategies to reestablish the CAR-T cell therapy’s effectiveness through targeting metabolism,with the aim of providing new strategies to surmount the obstacle in the way of solid tumor CAR-T cell treatment.