Cellular Therapeutics Research Articles

Framework Nucleic Acids: Innovative Tools for Cellular Sensing and Therapeutics.

As emerging biomaterials, framework nucleic acids (FNAs) have recently demonstrated great potential in the biomedical field due to their high programmability, biocompatibility, unique structural diversity, and precise molecular design capabilities. This review focuses on the applications of FNAs in cellular sensing and disease treatment. First, we systematically introduce the applications of FNAs in cellular sensing, including their precise recognition and response to the extracellular tumor microenvironment, cell membrane proteins, and intracellular biomarkers. Subsequently, we review the potential of FNAs in disease treatment, covering their applications and development in drug delivery, regulation of cell behavior, and immunomodulation. We also discuss the limitations and potential role of FNAs in personalized medicine, precision diagnostics, and advanced therapies. The broad application of FNAs is expected to drive significant breakthroughs in future biomedical technological innovations and clinical translation.

Living Cell-Mediated Catalyst-Free Spontaneous Polymerization of Zwitterionic Methacrylates for Preparation of Probiotic-Loaded Hydrogels.

Living cell-mediated polymerization offers promising applications in biomaterials, yet its further biological utilization is hindered by the need for metal ions or radical initiators with available methods. In this study, we introduce a living cell-mediated polymerization that leverages the intrinsic metabolic activities of living cells to initiate and sustain free radical polymerization of zwitterionic methacrylates. The polymerization proceeded in the absence of transition metal catalysts, radical initiators, or light sources. The conversion of zwitterionic methacrylate strongly correlated with cellular activities and achieved a maximum conversion of 98 % within 48 hours. Living cells efflux redox power across membranes through metabolism and that terminal electron fluxes are captured by zwitterionic methacrylates pre-assembled on the living cell surface to initiate radical polymerization reactions. The polymerization caused significant changes to the cell membrane surface and synthesized hydrogels with tailored mechanical properties. The polymer hydrogel obtained via probiotic E. coli Nissle 1917 was able to release the in situ encapsulated molecules, which demonstrated living cell-mediated polymer hydrogel as a vehicle for the delivery of both cellular and molecular therapeutic agents. This research offered a green and efficient method for synthesizing bioactive materials and advancing the field of cellular therapeutics and drug delivery.

Living Cell‐Mediated Catalyst‐Free Spontaneous Polymerization of Zwitterionic Methacrylates for Preparation of Probiotic‐Loaded Hydrogels

AbstractLiving cell‐mediated polymerization offers promising applications in biomaterials, yet its further biological utilization is hindered by the need for metal ions or radical initiators with available methods. In this study, we introduce a living cell‐mediated polymerization that leverages the intrinsic metabolic activities of living cells to initiate and sustain free radical polymerization of zwitterionic methacrylates. The polymerization proceeded in the absence of transition metal catalysts, radical initiators, or light sources. The conversion of zwitterionic methacrylate strongly correlated with cellular activities and achieved a maximum conversion of 98 % within 48 hours. Living cells efflux redox power across membranes through metabolism and that terminal electron fluxes are captured by zwitterionic methacrylates pre‐assembled on the living cell surface to initiate radical polymerization reactions. The polymerization caused significant changes to the cell membrane surface and synthesized hydrogels with tailored mechanical properties. The polymer hydrogel obtained via probiotic E. coli Nissle 1917 was able to release the in situ encapsulated molecules, which demonstrated living cell‐mediated polymer hydrogel as a vehicle for the delivery of both cellular and molecular therapeutic agents. This research offered a green and efficient method for synthesizing bioactive materials and advancing the field of cellular therapeutics and drug delivery.

Cytological Effects of Cadmium Poisoning and the Protective Effect of Quercetin: A Mechanism Exploration based on the Testicular Lamina Propria.

This comprehensive study delved into the detrimental effects of cadmium (Cd), a toxic heavy metal, on the testicular lamina propria (LP), a key player in spermatogenesis, and the maintenance of testicular stem cell niches. Utilizing transmission electron microscopy, immunohistochemistry, and double-labeling immunofluorescence, the research characterized the structural and cellular components of mouse testicular LP under Cd exposure and investigated the protective effects of quercetin. The findings illustrated that Cd exposure results in significant morphological and cellular modifications within the LP, including the apoptosis of peritubular myoid cells, an upsurge in CD34+ stromal cells displaying anti-apoptotic behaviors, and an excessive production of collagen Type I fibers and extracellular matrix. Remarkably, quercetin effectively counteracted these adverse changes by reversing apoptosis, reducing the proliferation of CD34+ stromal cells, and addressing fibrosis markers, thereby mitigating the cellular damage induced by Cd. This study not only highlighted the critical impact of apoptosis and fibrosis in Cd-related testicular damage but also elucidated the protective mechanism of quercetin, laying the groundwork for future clinical applications in addressing testicular damage from heavy metal poisoning through cellular therapeutics and pharmacological interventions.

Infrared Light Activated Highly Efficient Cell Therapy Using Flower‐Shaped Microstructure Device

AbstractIn this pioneering study, an infrared light‐activated highly efficient and uniform, small to large biomolecular delivery into various cell types is developed using a flower‐shaped microstructure device (FMD). Featuring a unique structural design, this FMD consists of 8 µm in length, with edges of ≈3 and 20 µm gaps between FMD microstructure. When subjected to IR laser exposure at 1050 nm, the FMD triggers the generation of photothermal cavitation bubbles, exerting jet fluid flow on the cell's plasma membrane surface, and facilitating biomolecule delivery into cells. The platform achieves efficient intracellular delivery spanning various biomolecules — from low‐molecular‐weight propidium iodide dye to higher molecular weight siRNA, plasmid, and enzymes — across human cervical (SiHa), mouse fibroblast (L929), and neural crest‐derived (N2a) cancer cells, ensuring consistently high efficiency without compromising cell viability. 95% delivery efficacy and 96% cell viability are achieved for smaller molecules like PI dye in L929 cells. For larger biomolecules such as enzymes, transfection efficiency reached 82%, and cell viability is nearly 90% in SiHa cells. This is confirmed via confocal microscopy and flow cytometry, the FMD‐based delivery system holds broad potential for cellular diagnostics and therapeutics, promising significant advancements in cellular research and biomedical treatments.

Challenges in the Development of NK-92 Cells as an Effective Universal Off-the-Shelf Cellular Therapeutic.

The human-derived NK-92 cell-based CAR-NK therapy exhibits inconsistency with overall suboptimal efficacy and rapid invivo clearance of CAR-NK92 cells in cancer patients. Analysis indicates that although pre-existing IgM in healthy individuals (n = 10) strongly recognizes both NK-92 and CAR-NK92 cells, IgG and IgE do not. However, only a subset of cancer patients (3/8) exhibit strong IgM recognition of these cells, with some (2/8) showing pre-existing IgG recognition. These results suggest a natural immunoreactivity between NK-92 and CAR-NK92 cells and pre-existing human Abs. Furthermore, the therapy's immunogenicity is evidenced by enhanced IgG and IgM recognition postinfusion of CAR-NK92 cells. We also confirmed that healthy plasma's cytotoxicity toward these cells is reduced by complement inhibitors, suggesting that Abs may facilitate the rapid clearance of CAR-NK92 cells through complement-dependent cytotoxicity. Given that NK-92 cells lack known receptors for IgG and IgM, identifying and modifying the recognition targets for these Abs on NK-92 and CAR-NK92 cells may improve clinical outcomes. Moreover, we discovered that the 72nd amino acid of the NKG2D receptor on NK-92 cells is alanine. Previous studies have demonstrated polymorphism at the 72nd amino acid of the NKG2D on human NK cells, with NKG2D72Thr exhibiting a superior activation effect on NK cells compared with NKG2D72Ala. We confirmed this conclusion also applies to NK-92 cells by invitro cytotoxicity experiments. Therefore, reducing the immunoreactivity and immunogenicity of CAR-NK92 and directly switching NK-92 bearing NKG2D72Ala to NKG2D72Thr represent pressing challenges in realizing NK-92 cells as qualified universal off-the-shelf cellular therapeutics.

Harnessing cellular therapeutics for type 1 diabetes mellitus: progress, challenges, and the road ahead.

Type 1 diabetes mellitus (T1DM) is a growing global health concern that affects approximately 8.5 million individuals worldwide. T1DM is characterized by an autoimmune destruction of pancreatic β cells, leading to a disruption in glucose homeostasis. Therapeutic intervention for T1DM requires a complex regimen of glycaemic monitoring and the administration of exogenous insulin to regulate blood glucose levels. Advances in continuous glucose monitoring and algorithm-driven insulin delivery devices have improved the quality of life of patients. Despite this, mimicking islet function and complex physiological feedback remains challenging. Pancreatic islet transplantation represents a potential functional cure for T1DM but is hindered by donor scarcity, variability in harvested cells, aggressive immunosuppressive regimens and suboptimal clinical outcomes. Current research is directed towards generating alternative cell sources, improving transplantation methods, and enhancing cell survival without chronic immunosuppression. This Review maps the progress in cell replacement therapies for T1DM and outlines the remaining challenges and future directions. We explore the state-of-the-art strategies for generating replenishable β cells, cell delivery technologies and local targeted immune modulation. Finally, we highlight relevant animal models and the regulatory aspects for advancing these technologies towards clinical deployment.

Dopaminergic progenitors generated by small molecule approach survived, integrated, and promoted functional recovery in (6-OHDA) mouse model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder resulting from the loss of dopamine-producing neurons in the brain, causing motor symptoms like tremors and stiffness. Although current treatments like medication and deep brain stimulation can alleviate symptoms, they don't address the root cause of neuron loss. Therefore, cell replacement therapy emerges as a promising treatment strategy. However, the generation of engraftable dopaminergic (DA) cells in clinically relevant quantities is still a challenge. Recent advances in cell reprogramming technologies open up vast possibilities to produce patient-specific cells of a desired type in therapeutic quantities. The main cell reprogramming strategies involve the enforced expression of individual or sets of genes through viral transduction or transfection, or through small molecules, known as the chemical approach, which is a much easier and safer method. In our previous studies, using a small molecule approach (combinations of epigenetic modifiers and SMAD inhibitors such asDorsomorphin and SB431542), we have been able to generate DA progenitors from human mesenchymal stem cells (hMSCs). The aim of this study was to further improve the method for the generation of DA progenitors and to test their therapeutic effect in an animal model of Parkinson's. The results showed that the addition of an autophagy enhancer (AE) to our DA cell induction protocol further increased the yield of DA progenitor cells. The results also showed that DA progenitors transplanted into the mouse model of PD survived, integrated, and improved PD motor symptoms. These data suggest that chemically-produced DA cells can be very promising and safe cellular therapeutics for PD.

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

A brief chronicle of research on human pluripotent stem cells.

Today, human pluripotent stem cell technologies find widespread application across biomedical research, as models for early human development, as platforms for functional human genomics, as tools for the study of disease, drug screening and toxicology, and as a renewable source of cellular therapeutics for a range of intractable diseases. The foundations of this human pluripotent stem cell revolution rest on advances in a wide range of disciplines, including cancer biology, assisted reproduction, cell culture and organoid technology, somatic cell nuclear transfer, primate embryology, single-cell biology, and gene editing. This review surveys the slow emergence of the study of human pluripotency and the exponential growth of the field during the past several decades.

Criteria for developing active cellular targeting miRNA oligonucleotide therapeutics with a peptide nucleic acid backbone: Combating cardiometabolic pandemics

Oligonucleotide therapeutics (ONTs) represent a growing new class of therapeutic agents aimed at addressing chronic diseases that remain untreatable by small molecules and antibodies. Our goal was to establish a selection of several criteria to design and develop miRNA-based ONTs, focusing on improved chemistry, pharmacokinetics/pharmacodynamics (PK/PD) profiles, and safety characteristics to combat cardiometabolic pandemics. By leveraging our own experimental data obtained from experiments involving miR-22-3p antagomirs and a careful review of the literature, we established a set of seven criteria to optimize the design of miRNA ONTs. These criteria prioritize simplified drug synthesis, optimized PK/PD properties, and reduced potential toxicities. This proposed set of seven criteria represents a novel strategy for developing active cellular targeting miRNA ONTs for various therapeutic indications.

Upstream Process Protocol for MSCs Isolated from Different Human-Based Tissue Origins.

This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.

Image-based discrimination of the early stages of mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells, which can be used in cellular and tissue therapeutics. MSCs cell number can be expanded in vitro, but premature differentiation results in reduced cell number and compromised therapeutic efficacies. Current techniques fail to discriminate the "stem-like" population from early stages (12 h) of differentiated MSC population. Here, we imaged nuclear structure and actin architecture using immunofluorescence and used deep learning-based computer vision technology to discriminate the early stages (6-12 h) of MSC differentiation. Convolutional neural network models trained by nucleus and actin images have high accuracy in reporting MSC differentiation; nuclear images alone can identify early stages of differentiation. Concurrently, we show that chromatin fluidity and heterochromatin levels or localization change during early MSC differentiation. This study quantifies changes in cell architecture during early MSC differentiation and describes a novel image-based diagnostic tool that could be widely used in MSC culture, expansion and utilization.

Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications

BackgroundMacrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ.Aim of the studyTo scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype.MethodsDifferentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling.ResultsMreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased.ConclusionExposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

Vascular endothelial growth factor secretion and immunosuppression are distinct potency mechanisms of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are investigated as cellular therapeutics for inflammatory bowel diseases and associated perianal fistula, although consistent efficacy remains a concern. Determining host factors that modulate MSCs' potency including their secretion of angiogenic and wound-healing factors, immunosuppression, and anti-inflammatory properties are important determinants of their functionality. We investigated the mechanisms that regulate the secretion of angiogenic and wound-healing factors and immune suppression of human bone marrow MSCs. Secretory analysis of MSCs focusing on 18 angiogenic and wound-healing secretory molecules identified the most abundancy of vascular endothelial growth factor A (VEGF-A). MSC viability and secretion of other angiogenic factors are not dependent on VEGF-A secretion which exclude the autocrine role of VEGF-A on MSC's fitness. However, the combination of inflammatory cytokines IFNγ and TNFα reduces MSC's VEGF-A secretion. To identify the effect of intestinal microvasculature on MSCs' potency, coculture analysis was performed between human large intestine microvascular endothelial cells (HLMVECs) and human bone marrow-derived MSCs. HLMVECs do not attenuate MSCs' viability despite blocking their VEGF-A secretion. In addition, HLMVECs neither attenuate MSC's IFNγ mediated upregulation of immunosuppressive enzyme indoleamine 2,3-dioxygenase nor abrogate suppression of T-cell proliferation despite the attenuation of VEGF-A secretion. We found that HLMVECs express copious amounts of endothelial nitric oxide synthase and mechanistic analysis showed that pharmacological blocking reverses HLMVEC-mediated attenuation of MSC's VEGF-A secretion. Together these results suggest that secretion of VEGF-A and immunosuppression are separable functions of MSCs which are regulated by distinct mechanisms in the host.

Stealth transgenes enable CAR-T cells to evade host immune responses

BackgroundAdoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based...

Engineering immune-evasive allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold a great promise for cancer treatment owing to their potential cost-effectiveness, scalability and on-demand availability. However, immune rejection of adoptively transferred allogeneic T and natural killer (NK) cells is a substantial obstacle to achieving clinical responses that are comparable to responses obtained with current autologous chimeric antigen receptor T cell therapies. In this Perspective, we discuss strategies to confer cell-intrinsic, immune-evasive properties to allogeneic T cellsand NK cells in order to prevent or delay their immune rejection, thereby widening the therapeutic window. We discuss how common viral and cancer immune escape mechanisms can serve as a blueprint for improving the persistence of off-the-shelf allogeneic cell therapies. The prospects of harnessing genome editing and synthetic biology to design cell-based precision immunotherapies extend beyond programming target specificities and require careful consideration of innate and adaptive responses in the recipient that may curtail the biodistribution, in vivo expansion and persistence of cellular therapeutics.

Immune modulation in transplant medicine: a comprehensive review of cell therapy applications and future directions.

Balancing the immune response after solid organ transplantation (SOT) and vascularized composite allotransplantation (VCA) remains an ongoing clinical challenge. While immunosuppressants can effectively reduce acute rejection rates following transplant surgery, some patients still experience recurrent acute rejection episodes, which in turn may progress to chronic rejection. Furthermore, these immunosuppressive regimens are associated with an increased risk of malignancies and metabolic disorders. Despite significant advancements in the field, these IS related side effects persist as clinical hurdles, emphasizing the need for innovative therapeutic strategies to improve transplant survival and longevity. Cellular therapy, a novel therapeutic approach, has emerged as a potential pathway to promote immune tolerance while minimizing systemic side-effects of standard IS regiments. Various cell types, including chimeric antigen receptor T cells (CAR-T), mesenchymal stromal cells (MSCs), regulatory myeloid cells (RMCs) and regulatory T cells (Tregs), offer unique immunomodulatory properties that may help achieve improved outcomes in transplant patients. This review aims to elucidate the role of cellular therapies, particularly MSCs, T cells, Tregs, RMCs, macrophages, and dendritic cells in SOT and VCA. We explore the immunological features of each cell type, their capacity for immune regulation, and the prospective advantages and obstacles linked to their application in transplant patients. An in-depth outline of the current state of the technology may help SOT and VCA providers refine their perioperative treatment strategies while laying the foundation for further trials that investigate cellular therapeutics in transplantation surgery.

Handling the different requirements for commercial and investigational MNC collections by apheresis

BackgroundWith the success of chimeric antigen receptor T-cell (CAR-T) and similar cellular-based therapies, the demand for collection of autologous mononuclear cells by apheresis (MNC(A)) from blood by apheresis has increased. From an apheresis technical standpoint, the collection of MNC(A) is relatively straightforward, especially when compared with collection of hematopoietic progenitor cells (HPC(A)). Most of the collection for MNC(A) are performed for the commercial entities, who use the product for manufacturing cellular therapeutics. We have noticed discrepancies in the handling and apheresis processes required by different companies in obtaining essentially the same product (all companies in the study manufacture CAR-T-based products). We have analyzed the MNC collection requirements from all FDA-approved CAR-T cellular products and some investigational products collected at University of Nebraska Medical Center. We identified discrepancies in the process and suggested mitigation strategies. MethodsStep-by-step analysis of the collection requirements. Review of the current guidelines and recommendations on this issue. ResultsMultiple discrepancies in the collection process have been identified, even in the products collected for the same company. Practical approach of satisfying all the requirements based on University of Nebraska Medical Center experience has been suggested. ConclusionThe current recommendations from multiple sources were reviewed in discussion.

Abstract LB341: Clinical validation and mechanistic insights of ThisCART: A novel allogeneic platform enhancing CAR-T signaling via intracellular TCRαβ/CD3 retention

Abstract Background: Current allogeneic CAR-T cell therapies face challenges with post-infusion expansion and efficacy. In response, we developed ThisCART, a non-genetic editing, allogeneic platform that significantly enhances CAR-T cell expansion and efficacy. This is achieved through a novel approach of intracellular retention of membrane proteins, reducing surface expression of TCRαβ/CD3 complexes and HLA-I molecules. Our lead product, ThisCART19, has demonstrated superior efficacy compared to conventional AutoCART19 in preclinical and early clinical studies for B-ALL. Methods: In the clinical trial NCT04384393, we assessed the safety and efficacy of ThisCART19 in patients with B-cell malignancies. Post-lymphodepletion, participants received infusions of ThisCART19 and AutoCART19, both personalized from the patient's own T cells, in equal proportions. Safety and efficacy were the primary endpoints, evaluated on Day 28 post-infusion and throughout extended follow-up. Mechanistic insights were gained through comprehensive multi-omics studies, including RNAseq, whole-cell proteomics, and phosphoproteomics. Results: ThisCART19 showed enhanced expansion and an improved safety profile in vivo. As of 01 January 2024, Two patients diagnosed as relapse/refractory acute B cell leukemia (R/R B-ALL) were enrolled in the clinical trial. Pt1 is a 55yo male with Ph-positive B-ALL (36% blasts) who relapsed after multiagent chemotherapy and TKIs. Pt2 is a 16yo male with CNS leukemia (78.5% blasts) after multiagent chemotherapy. No DLTs or ICANS were observed. Pt1 experienced Grade 1 CRS that resolved without treatment, and Pt2 experienced Grade 2 CRS that resolved after tocilizumab and corticosteroids. Complete remission with MRD-negative status was achieved by Day 28. Pharmacodynamic analysis indicated a significant expansion of CAR-T cells, predominantly in the CAR+TCR- subset, highlighting autonomous CAR signaling enhancement. Multi-omics analysis identified upregulation in JAK-STAT, PI3K-Akt, and NFKB pathways, suggesting improved cell proliferation, survival, anti-apoptotic activity, and effector function. This molecular profiling provides a mechanistic understanding of ThisCART19A's therapeutic efficacy. Conclusion: The ThisCART platform marks a transformative advancement in allogeneic CAR-T cell therapy. By enhancing CAR signaling autonomously through a unique intracellular retention of TCRαβ/CD3 complexes, ThisCART has shown promising safety and efficacy in early clinical trials. Its potential in reshaping the treatment landscape for hematological and autoimmune disorders positions ThisCART as a crucial development in cellular therapeutics. Citation Format: Jun Li, Lei Xue, Sujun Li, Ling He, Kaichun Liu, Rong Jin, Tao Wang, Yinhang Zhang, Shanshan Chen, Yulian Gao, Dan Liu, Liyun Yang, Xingbing Wang. Clinical validation and mechanistic insights of ThisCART: A novel allogeneic platform enhancing CAR-T signaling via intracellular TCRαβ/CD3 retention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB341.