Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and NK cells have shown remarkable clinical efficacy in treating hematological malignancies, however current methods mainly utilize viral vectors which are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof of principle TcB-M engineering of CAR-NK and CAR-T cells show low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

B-cell targeted therapies in autoimmune encephalitis: mechanisms, clinical applications, and therapeutic potential.

Autoimmune encephalitis (AE) broadly refers to inflammation of the brain parenchyma mediated by autoimmune mechanisms. In most patients with AE, autoantibodies against neuronal cell surface antigens are produced by B-cells and induce neuronal dysfunction through various mechanisms, ultimately leading to disease progression. In recent years, B-cell targeted therapies, including monoclonal antibody (mAb) therapy and chimeric antigen receptor T-cell (CAR-T) therapy, have been widely used in autoimmune diseases. These therapies decrease autoantibody levels in patients and have shown favorable results. This review summarizes the mechanisms underlying these two B-cell targeted therapies and discusses their clinical applications and therapeutic potential in AE. Our research provides clinicians with more treatment options for AE patients whose conventional treatments are not effective.

CAR products from novel sources: a new avenue for the breakthrough in cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has transformed cancer immunotherapy. However, significant challenges limit its application beyond B cell-driven malignancies, including limited clinical efficacy, high toxicity, and complex autologous cell product manufacturing. Despite efforts to improve CAR T cell therapy outcomes, there is a growing interest in utilizing alternative immune cells to develop CAR cells. These immune cells offer several advantages, such as major histocompatibility complex (MHC)-independent function, tumor microenvironment (TME) modulation, and increased tissue infiltration capabilities. Currently, CAR products from various T cell subtypes, innate immune cells, hematopoietic progenitor cells, and even exosomes are being explored. These CAR products often show enhanced antitumor efficacy, diminished toxicity, and superior tumor penetration. With these benefits in mind, numerous clinical trials are underway to access the potential of these innovative CAR cells. This review aims to thoroughly examine the advantages, challenges, and existing insights on these new CAR products in cancer treatment.

Biparatopic antibodies: therapeutic applications and prospects.

Biparatopic antibodies (bpAbs) bind distinct, non-overlapping epitopes on an antigen. This unique binding mode enables new mechanisms of action beyond monospecific and bispecific antibodies (bsAbs) that can make bpAbs effective therapeutics for various indications, including oncology and infectious diseases. Biparatopic binding can lead to superior affinity and specificity, promote antagonism, lock target conformation, and result in higher-order target clustering. Such antibody-target complexes can elicit strong agonism, increase immune effector function, or result in rapid target downregulation and lysosomal trafficking. These are not only attractive properties for therapeutic antibodies but are increasingly being explored for other modalities such as antibody-drug conjugates, T-cell engagers and chimeric antigen receptors. Recent advances in bpAb engineering have enabled the construction of ever more sophisticated formats that are starting to show promise in the clinic.

Chimeric Antigen Receptor (CAR)-T Cell Therapy for Non-Hodgkin's Lymphoma.

This review focuses on the use of chimeric antigen receptor (CAR)-T cell therapy to treat non-Hodgkin's lymphoma (NHL), a classification of heterogeneous malignant neoplasms of the lymphoid tissue. Despite various conventional and multidrug chemotherapies, the poor prognosis for NHL patients remains and has prompted the utilization of groundbreaking personalized therapies such as CAR-T cells. CAR-T cells are T cells engineered to express a CAR that enables T cells to specifically lyse tumor cells with extracellular expression of a tumor antigen of choice. A CAR is composed of an extracellular antibody fragment or target protein binding domain that is conjugated to activating intracellular signaling motifs common to T cells. In general, CAR-T cell therapies for NHL are designed to recognize cellular markers ubiquitously expressed on B cells such as CD19+, CD20+, and CD22+. Clinical trials using CAR-T cells such as ZUMA-7 and TRANSFORM demonstrated promising results compared to standard of care and ultimately led to FDA approval for the treatment of relapsed/refractory NHL. Despite the success of CAR-T therapy for NHL, challenges include adverse side effects as well as extrinsic and intrinsic mechanisms of tumor resistance that lead to suboptimal outcomes. Overall, CAR-T cell therapies have improved clinical outcomes in NHL patients and generated optimism around their future applications.

SOHO State of the Art Updates and Next Questions: Will CAR-T Replace ASCT in NDMM.

The treatment landscape for multiple myeloma (MM) has rapidly evolved over the last 2 decades. The development of triplet and quadruplet regimens including proteasome inhibitors (PI), immunomodulatory agents (IMiDs), and anti-CD38 monoclonal antibodies has dramatically extended overall survival. In addition to effective multidrug regimens, autologous stem cell transplant (ASCT) is a cornerstone of management in newly diagnosed multiple myeloma (NDMM). However, despite these combined treatment modalities, curative therapy for MM remains elusive. Recent, novel immunotherapies including chimeric antigen T-cell (CAR-T) therapy have demonstrated deep and durable responses in relapsed and refractory multiple myeloma (RRMM). Currently 2 CAR-T products, ciltacabtagene autoleucel (cilta-cel) and idecabtagene vicleucel (ide-cel), are approved by the FDA for the treatment of RRMM. The success of CAR-T therapy revolutionized the management of RRMM prompting clinical trials studying CAR-T therapy in the first line setting. The ongoing KarMMa-4, CARTITUDE-5, and CARTITUDE-6 clinical trials may establish CAR-T therapy as a first line option potentially supplanting ASCT in the initial treatment of NDMM. In this review, we discuss the current standard of care management of NDMM, trace the evolution of CAR-T clinical trials in RRMM, and survey ongoing clinical trials studying CAR-T therapy in NDMM.

Melatonin as an immunomodulator in CD19-targeting CAR-T cell therapy: managing cytokine release syndrome.

BACKGROUND: Chimeric antigen receptor CAR-T cell therapies have ushered in a new era of treatment for specific blood cancers, offering unparalleled efficacy in cases of treatment resistance or relapse. However, the emergence of cytokine release syndrome (CRS) as a side effect poses a challenge to the widespread application of CAR-T cell therapies. Melatonin, a natural hormone produced by the pineal gland known for its antioxidant and anti-inflammatory properties, has been explored for its potential immunomodulatory effects. Despite this, its specific role in mitigating CAR-T cell-induced CRS remains poorly understood. METHODS: In this study, our aim was to investigate the potential of melatonin as an immunomodulatory agent in the context of CD19-targeting CAR-T cell therapy and its impact on associated side effects. Using a mouse model, we evaluated the effects of melatonin on CAR-T cell-induced CRS and overall survival. Additionally, we assessed whether melatonin administration had any detrimental effects on the antitumor efficacy and persistence of CD19 CAR-T cells. RESULTS: Our findings demonstrate that melatonin effectively mitigated the severity of CAR-T cell-induced CRS in the mouse model, leading to improved overall survival outcomes. Remarkably, melatonin administration did not compromise the antitumor effectiveness or persistence of CD19 CAR-T cells, indicating its compatibility with therapeutic goals. These results suggest melatonin's potential as an immunomodulatory compound to alleviate CRS without compromising the therapeutic benefits of CAR-T cell therapy. CONCLUSION: The study's outcomes shed light on melatonin's promise as a valuable addition to the existing treatment protocols for CAR-T cell therapies. By attenuating CAR-T cell-induced CRS while preserving the therapeutic impact of CAR-T cells, melatonin offers a potential strategy for optimizing and refining the safety and efficacy profile of CAR-T cell therapy. This research contributes to the evolving understanding of how to harness immunomodulatory agents to enhance the clinical application of innovative cancer treatments.

Advanced Strategies of CAR-T Cell Therapy in Solid Tumors and Hematological Malignancies.

Chimeric antigen receptor T-cells, known as CAR-T cells, represent a promising breakthrough in the realm of adoptive cell therapy. These T-cells are genetically engineered to carry chimeric antigen receptors that specifically target tumors. They have achieved notable success in the treatment of blood-related cancers, breathing new life into this field of medical research. However, numerous obstacles limit chimeric antigen receptors T-cell therapy's efficacy, such as it cannot survive in the body long. It is prone to fatigue and exhaustion, leading to difficult tumor elimination and repeated recurrence, affecting solid tumors and hematological malignancies. The challenges posed by solid tumors, especially in the context of the complex solid-tumor microenvironment, require specific strategies. This review outlines recent advancements in improving chimeric antigen receptors T-cell therapy by focusing on the chimeric antigen receptors protein, modifying T-cells, and optimizing the interaction between T-cells and other components within the tumor microenvironment. This article aims to provide an extensive summary of the latest discoveries regarding CAR-T cell therapy, encompassing its application across various types of human cancers. Moreover, it will delve into the obstacles that have emerged in recent times, offering insights into the challenges faced by this innovative approach. Finally, it highlights novel therapeutic options in treating hematological and solid malignancies with chimeric antigen receptors T-cell therapies.

Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors.

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.

Abnormal bone marrow findings in patients following treatment with chimeric antigen receptor-T cell therapy.

BACKGROUND: Bone marrow (BM) assessment after CAR-T cell immunotherapy infusion is not routinely performed to monitor adverse events such as cytopenias, hemophagocytic lymphohistiocytosis, or infections. Our institution has performed BM biopsies as part of CAR-T cell treatment protocols, encompassing pre- and post-treatment time points and during long-term follow-up. METHODS: We conducted a systematic retrospective review of BM abnormalities observed in samples from 259 patients following CAR-T cell immunotherapy. We correlated BM pathology findings with mortality, relapse/residual disease, and laboratory values. RESULTS: At a median of 35.5 days post-CAR-T infusion, 25.5% showed severe marrow hypocellularity, and 6.2% showed serous atrophy, and peripheral blood cytopenias corroborated these observations. Marrow features associated with reduced disease burden post-CAR-T infusion include increased lymphocytes seen in 16 patients and an increase of macrophages or granulomatous response seen in 25 patients. However, a 100-day landmark analysis also showed increased marrow histiocytes were associated with lower survival (median OS 6.0 vs. 21.4 months, p = .026), as was grade 2-3 marrow reticulin (18 patients) (median OS 12.5 vs. 24.2 months, p = .034). CONCLUSIONS: These data represent the first systematic observations of BM changes in patients receiving CAR-T cell immunotherapy.

Genetic Modification of Tumor-Infiltrating Lymphocytes, Peripheral T Cells, and T-Cell Model Cell Lines.

Genetic modification of tumor-infiltrating lymphocytes (TILs) or circulating T cells has become an important avenue in cancer therapy. Here we describe a comprehensive method for establishing and expanding TIL cultures and genetically modifying them with a gene of interest (GOI) via retroviral transduction or mRNA transfection. The method includes all the important steps starting with TIL extraction from tumors through to the maintenance of the genetically modified TILs. The protocol includes instructions for retroviral transduction and mRNA transfection of circulating T cells or T-cell lines. The GOIs most commonly introduced into the target cells are chimeric antigen receptors (CARs); genetic adjuvants, such as membrane-bound interleukins; and antitumor T-cell receptors (TCRs).

Chimeric antigen receptor Treg therapy in transplantation.

In the quest for more precise and effective organ transplantation therapies, chimeric antigen receptor (CAR) regulatory T cell (Treg) therapies represent a potential cutting-edge advance. This review comprehensively analyses CAR Tregs and how they may address important drawbacks of polyclonal Tregs and conventional immunosuppressants. We examine a growing body of preclinical findings of CAR Treg therapy in transplantation, discuss CAR Treg design specifics, and explore established and attractive new targets in transplantation. In addition, we explore present impediments where future studies will be necessary to determine the efficacy of CAR Tregs in reshaping alloimmune responses and transplant microenvironments to reduce reliance on chemical immunosuppressants. Overall, ongoing studies and trials are crucial for understanding the full scope of CAR Treg therapy in transplantation.

ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

Development of CAR-NK Cells Targeting cSCC-Specific Antigens for Precision Immunotherapy.

Cutaneous squamous cell carcinoma (cSCC), a non-melanoma skin cancer that is frequently diagnosed, is distinguished by its propensity for aggressive behavior, frequent poor response to standard therapy, and capacity to metastasize to distant areas. Utilizing the body's natural immune defense mechanisms, particularly through the use of chimeric antigen receptor (CAR) technology, is receiving increasing attention in the dynamic field of oncological therapies. Although T cells have received most of the attention, this strategy has proven to be highly effective in battling some blood-related malignancies. However, there are considerable challenges when using this method in the context of solid tumors. The innate immune system's natural killer (NK) cells are essential parts because they have the ability to detect and destroy cancer cells. CAR-NK cells are a very promising approach because they combine the natural cytotoxic properties of natural killer (NK) cells with the precise targeting skills of chimeric antigen receptor (CAR) technology. With the use of this integrated strategy, the intrinsic diversity of cutaneous squamous cell carcinoma (cSCC) tumors may be successfully targeted, increasing treatment effectiveness and lowering the risk of tumor recurrence. This tactic is improved by the development of dual-specificity chimeric antigen receptors (CARs), which fully resolve the antigen presentation heterogeneity among tumor cells. In conclusion, the use of CAR-NK cells that precisely target cSCC-specific antigens has the potential to drastically transform therapy approaches for cSCC as well as other difficult solid tumors as oncological research advances. In order to create chimeric antigen receptor (CAR)-natural killer (NK) cells that particularly target antigens linked to cutaneous squamous cell carcinoma (cSCC), the goal of this protocol is to present a detailed method. The ultimate objective is to lay the groundwork for the development of precision immunotherapy.

Adoptive cell therapy for cancer treatment.

Adoptive cell therapy (ACT) is a rapidly growing anti-cancer strategy that has shown promise in treating various cancer types. The concept of ACT involves activating patients' own immune cells ex vivo and then transferring them back to the patients to recognize and eliminate cancer cells. Currently, the commonly used ACT includes tumor-infiltrating lymphocytes (TILs), genetically engineered immune cells, and dendritic cells (DCs) vaccines. With the advancement of cell culture and genetic engineering techniques, ACT has been used in clinics to treat malignant hematological diseases and many new ACT-based regimens are in different stages of clinical trials. Here, representative ACT approaches are introduced and the opportunities and challenges for clinical translation of ACT are discussed.

Characterization of CAR T Cells Manufactured Using Genetically Engineered Artificial Antigen Presenting Cells.

OBJECTIVE: Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for the treatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is an important concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansion is to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 for T cell activation. The aim of this study was to characterize CAR T cells generated using this aAPC-mediated approach in terms of expansion efficiency, immunophenotype, and cytotoxicity. MATERIALS AND METHODS: In this experimental study, we generated an aAPC line by engineering K562 cells to express a membrane-bound anti-CD3 (mOKT3). T cell activation was performed by co-culturing PBMCs with either mitomycin C-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CARtransduced T cells were characterized in terms of expansion, activation markers, interferon gamma (IFN-γ) secretion, CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCs and antibodies were also investigated using a bioluminescence-based co-culture assay. RESULTS: Our findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to that using the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memory T cells in the final product, we did not observe a significant difference in IFN-γ secretion, cytotoxic activity or exhaustion between CAR T cells generated with aAPC or antibodies. CONCLUSION: Our results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR T cells, both methods can be used to manufacture potent CAR T cells. These findings are instrumental for the improvement of the CAR T cell manufacturing process and future applications of aAPC-mediated expansion of CAR T cells.

Anti­BCMA CAR­T cell immunotherapy for relapsed or refractory multiple myeloma.

The present study aimed to study the efficacy and adverse effects of anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T (CAR-T) cell therapy in relapsed or refractory multiple myeloma. Patients were divided into three dose groups based on cell therapy concentration. After CAR-T cell therapy for 10 patients with recurrent or refractory multiple myeloma, the patients were monitored and evaluated regularly to observe the efficacy and adverse reactions of CAR-T cell therapy. At a median follow-up of 337 (253-504) days, one patient succumbed 24 days due to rapidly progressing disease. The overall response rate of nine patients was 88.9%, including 77.8% (7/9) with minimal residual disease negative complete remission (CR) and 11.1% (1/9) with partial remission. A total of three patients were maintained in remission state for more than a year and eight were maintained for more than six months. Among the three patients with extramedullary invasion, two extramedullary lesions disappeared and one was stable. The highest copy number of CAR-T cells in seven patients with CR was >1x105 copies/µl gDNA, and the best therapeutic effect can be achieved within 30 (7-30) days after the copy number of CAR-T cells reached 1x105 copies/µl genomic DNA. The median onset time in the nine patients was 43 (22-169) days, and the median progression-free survival was 337 (253-504). Among the 10 patients, nine (90%) had cytokine release syndrome, all of which were below grade II. There were nine (90%) patients with hematological adverse reactions, six (60%) patients with severe anemia, five (50%) patients with grade III and above leukopenia, five (50%) patients with granulocytopenia, four (40%) patients with grade III and above thrombocytopenia, and three (30%) patients with grade III and above pancytopenia. It was concluded that anti-BCMA CAR-T cell therapy is a promising treatment method for relapsed or refractory multiple myeloma and extramedullary invasion, with stable efficacy and controllable adverse effects.

Hallmarks of the Tumour Microenvironment of Gliomas and Its Interaction with Emerging Immunotherapy Modalities.

Gliomas are aggressive, primary central nervous system tumours arising from glial cells. Glioblastomas are the most malignant. They are known for their poor prognosis or median overall survival. The current standard of care is overwhelmed by the heterogeneous, immunosuppressive tumour microenvironment promoting immune evasion and tumour proliferation. The advent of immunotherapy with its various modalities-immune checkpoint inhibitors, cancer vaccines, oncolytic viruses and chimeric antigen receptor T cells and NK cells-has shown promise. Clinical trials incorporating combination immunotherapies have overcome the microenvironment resistance and yielded promising survival and prognostic benefits. Rolling these new therapies out in the real-world scenario in a low-cost, high-throughput manner is the unmet need of the hour. These will have practice-changing implications to the glioma treatment landscape. Here, we review the immunobiological hallmarks of the TME of gliomas, how the TME evades immunotherapies and the work that is being conducted to overcome this interplay.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.