Novelty in improvement of CAR T cell-based immunotherapy with the aid of CRISPR system

Abstract Introduction Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. Method In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. Results The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. Conclusion: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

γδ T cells: Major advances in basic and clinical research in tumor immunotherapy / 中华医学杂志(英文版)

γδ T cells are a kind of innate immune T cell. They have not attracted sufficient attention because they account for only a small proportion of all immune cells, and many basic factors related to these cells remain unclear. However, in recent years, with the rapid development of tumor immunotherapy, γδ T cells have attracted increasing attention because of their ability to exert cytotoxic effects on most tumor cells without major histocompatibility complex (MHC) restriction. An increasing number of basic studies have focused on the development, antigen recognition, activation, and antitumor immune response of γδ T cells. Additionally, γδ T cell-based immunotherapeutic strategies are being developed, and the number of clinical trials investigating such strategies is increasing. This review mainly summarizes the progress of basic research and the clinical application of γδ T cells in tumor immunotherapy to provide a theoretical basis for further the development of γδ T cell-based strategies in the future.

Progress in Chimeric Antigen Receptor-Modified Natural Killer Cells for Multiple Myeloma / 中国医学科学院学报

Although the development of novel drugs has significantly improved the survival of patients with multiple myeloma (MM) over the past decades,the lack of effective therapeutic options for relapsed and refractory MM results in poor prognosis.The chimeric antigen receptor (CAR) T-cell therapy has achieved considerable progress in relapsed and refractory MM.Nevertheless,this therapy still has limitations such as cytokine release syndrome,neurotoxicity,and off-target effects.Natural killer (NK) cells,as a critical component of the innate immune system,play an essential role in tumor immunosurveillance.Therefore,CAR-modified NK (CAR-NK) cells are put forward as a therapeutic option for MM.The available studies have suggested that multiple targets can be used as specific therapeutic targets for CAR-NK cell therapy and confirmed their antitumor effects in MM cell lines and animal models.This review summarizes the anti-tumor mechanisms,biological characteristics,and dysfunction of NK cells in the MM tumor microenvironment,as well as the basic and clinical research progress of CAR-NK cells in treating MM.

Generating universal chimeric antigen receptor expressing cell products from induced pluripotent stem cells: beyond the autologous CAR-T cells / 中华医学杂志(英文版)

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.

Challenges and optimal strategies of CAR T therapy for hematological malignancies / 中华医学杂志(英文版)

Remarkable improvement relative to traditional approaches in the treatment of hematological malignancies by chimeric antigen receptor (CAR) T-cell therapy has promoted sequential approvals of eight commercial CAR T products within last 5 years. Although CAR T cells' productization is now rapidly boosting their extensive clinical application in real-world patients, the limitation of their clinical efficacy and related toxicities inspire further optimization of CAR structure and substantial development of innovative trials in various scenarios. Herein, we first summarized the current status and major progress in CAR T therapy for hematological malignancies, then described crucial factors which possibly compromise the clinical efficacies of CAR T cells, such as CAR T cell exhaustion and loss of antigen, and finally, we discussed the potential optimization strategies to tackle the challenges in the field of CAR T therapy.

Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma / 中华肿瘤杂志

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Long-term follow-up of humanized and murine CD19 CAR-T-cell therapy for B-cell acute lymphoblastic leukemia / 中华血液学杂志

Objective: Murine CD19 chimeric antigen receptor T-cell (CAR-T) products have been approved for the treatment of refractory/relapsed (R/R) B-cell acute lymphocytic leukemia (B-ALL) ; moreover, humanized products are also undergoing clinical trials. This study aimed to explore the differences in safety and short- and long-term follow-up efficacy between humanized and murine CD19 CAR-T-cells for treating relapsed and refractory B-ALL. Methods: Clinical data of 80 patients with R/R B-ALL treated with CD19-targeted CAR-T-cells at the Union Hospital of Tongji Medical College of Huazhong University of Science and Technology between May 2016 and March 2023 were analyzed, which included 31 patients with murine CAR-T and 49 with humanized products. Results: The proportion of patients with cytokine-release syndrome (CRS) in the murine and humanized groups was 63.1% and 65.3%, respectively. Moreover, a higher proportion of patients suffered from severe CRS in the murine group than in the humanized CAR-T group (19.4% vs 8.2%, P=0.174). Furthermore, one patient per group died of grade 5 CRS. The incidence of grade 1-2 immune effector cell-associated neurotoxicity syndrome (ICANS) was 12.9% and 6.1%, respectively; severe ICANS were not observed. Among patients receiving murine CAR-T-cells, an overall response (OR) was observed in 74.2%. Conversely, the OR rate of patients receiving humanized CAR-T-cells was 87.8%. During the median follow-up time of 10.5 months, the median recurrence-free survival (RFS) of patients with murine CAR-T-cells was 12 months, which was as long as that of patients with humanized CAR-T-cells. The median overall survival (OS) were not reached in both groups. Of the 45 patients with a bone marrow burden over 20% at baseline, humanized CAR-T therapy was associated with a significantly improved RFS (43.25% vs 33.33%, P=0.027). Bridging transplantation was an independent factor in prolonging OS (χ(2)=8.017, P=0.005) and PFS (χ(2)=6.584, P=0.010). Common risk factors, such as age, high proportion of bone marrow blasts, and BCR-ABL fusion gene expression, had no significant effect on patients' long-term follow-up outcomes. Three patients reached complete remission after reinfusion of humanized CAR-T-cells. However, one patient relapsed one month after his second infusion of murine CAR-T-cells. Conclusions: The results indicate that humanized CAR-T therapy showed durable efficacy in patients with a higher tumor burden in the bone marrow without any influence on safety. Moreover, it could overcome immunogenicity-induced CAR-T resistance, providing treatment options for patients who were not treated successfully with CAR-T therapies.

Clinical analysis of long-term survival and influencing factors of chimeric antigen receptor T-cell therapy in relapsed/refractory acute B-cell lymphoblastic leukemia / 中华血液学杂志

Objective: To analyze the survival and influencing factors of chimeric antigen receptor (CAR) T-cell therapy in relapsed/refractory acute B-cell lymphoblastic leukemia (R/R B-ALL) . Methods: Clinical information of patients who received CAR-T-cell therapy and achieved complete remission of R/R B-ALL between May 2015 and June 2018 at the Shaanxi Provincial People's Hospital was obtained. Kaplan-Meier analysis was used to evaluate the overall survival (OS) and leukemia-free survival (LFS) times of patients, and Cox regression analysis was performed to analyze the prognostic factors that affect patient survival after CAR-T therapy. Results: Among the 38 patients with R/R B-ALL, 21 were men, with a median age of 25 (6-59) years and a median OS time of 18 (95% CI 3-33) months. Multivariate Cox regression analysis showed that positive MLL-AF4 fusion gene expression was an independent risk factor for OS and LFS (OS: HR=4.888, 95% CI 1.375-17.374, P=0.014; LFS: HR=6.683, 95% CI 1.815-24.608, P=0.004). Maintenance therapy was a protective factor for OS and LFS (OS: HR=0.153, 95% CI 0.054-0.432, P<0.001; LFS: HR=0.138, 95% CI 0.050-0.382, P<0.001). In patients with MRD negative conversion, LFS benefit (HR=0.209, 95% CI 0.055-0.797, P=0.022) and OS difference was statistically insignificant (P=0.111). Moreover, patients with high tumor burden were risk factors for OS and LFS at the level of 0.1 (OS: HR=2.662, 95% CI 0.987-7.184, P=0.053; LFS: HR=2.452, 95% CI 0.949-6.339, P=0.064) . Conclusion: High tumor burden and high-risk genetics may affect the long-term survival rate of patients with R/R B-ALL receiving CAR-T, and lenalidomide-based maintenance therapy may improve their prognosis.

Efficacy and safety analysis of the zanubrutinib-based bridging regimen in chimeric antigen receptor T-cell therapy for relapsed/refractory diffuse large B-cell lymphoma / 中华血液学杂志

Objective: To further elucidate the clinical efficacy and safety of a combination regimen based on the BTK inhibitor zebutanil bridging CD19 Chimeric antigen receptor T cells (CAR-T cells) in the treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) . Methods: Twenty-one patients with high-risk r/r DLBCL were treated with a zanubrutinib-based regimen bridging CAR-T between June 2020 and June 2023 at the Department of Hematology, Tongji Hospital, Tongji University and the Second Affiliated Hospital of Zhejiang University, and the efficacy and safety were retrospectively analyzed. Results: All 21 patients were enrolled, and the median age was 57 years (range: 38-76). Fourteen patients (66.7%) had an eastern cooperative oncology group performance status score (ECOG score) of ≥2. Eighteen patients (85.7%) had an international prognostic index (IPI) score of ≥3. Three patients (14.3%) had an IPI score of 2 but had extranodal infiltration. Fourteen patients (66.7%) had double-expression of DLBCL and seven (33.3%) had TP53 mutations. With a median follow-up of 24.8 (95% CI 17.0-31.6) months, the objective response rate was 81.0%, and 11 patients (52.4%) achieved complete remission. The median progression-free survival (PFS) was 12.8 months, and the median overall survival (OS) was not reached. The 1-year PFS rate was 52.4% (95% CI 29.8% -74.3%), and the 1-year OS rate was 80.1% (95% CI 58.1% -94.6%). Moreover, 18 patients (85.7%) had grade 1-2 cytokine-release syndrome, and two patients (9.5%) had grade 1 immune effector cell-associated neurotoxicity syndrome. Conclusion: Zanubrutinib-based combination bridging regimen of CAR-T therapy for r/r DLBCL has high efficacy and demonstrated a good safety profile.

Efficacy and safety of fourth-generation CD19 CAR-T expressing IL7 and CCL19 along with PD-1 monoclonal antibody for relapsed or refractory large B-cell lymphoma / 中华血液学杂志

Objective: This study systematically explore the efficacy and safety of fourth-generation chimeric antigen receptor T-cells (CAR-T), which express interleukin 7 (IL7) and chemokine C-C motif ligand 19 (CCL19) and target CD19, in relapsed or refractory large B-cell lymphoma. Methods: Our center applied autologous 7×19 CAR-T combined with tirelizumab to treat 11 patients with relapsed or refractory large B-cell lymphoma. The efficacy and adverse effects were explored. Results: All 11 enrolled patients completed autologous 7×19 CAR-T preparation and infusion. Nine patients completed the scheduled six sessions of tirolizumab treatment, one completed four sessions, and one completed one session. Furthermore, five cases (45.5%) achieved complete remission, and three cases (27.3%) achieved partial remission with an objective remission rate of 72.7%. Two cases were evaluated for disease progression, and one died two months after reinfusion because of uncontrollable disease. The median follow-up time was 31 (2-34) months, with a median overall survival not achieved and a median progression-free survival of 28 (1-34) months. Two patients with partial remission achieved complete remission at the 9th and 12th months of follow-up. Therefore, the best complete remission rate was 63.6%. Cytokine-release syndrome and immune effector cell-associated neurotoxicity syndrome were controllable, and no immune-related adverse reactions occurred. Conclusion: Autologous 7×19 CAR-T combined with tirelizumab for treating relapsed or refractory large B-cell lymphoma achieved good efficacy with controllable adverse reactions.

Observation of liver indexes in patients with relapsed/refractory multiple myeloma treated with CAR-T-cells based on BCMA / 中华血液学杂志

Objective: To observe the characteristics of the evolution of liver indexes in patients with relapsed/refractory multiple myeloma (RRMM) treated with CAR-T-cells based on BCMA. Methods: Retrospective analysis was performed of patients with RRMM who received an infusion of anti-BCMA CAR-T-cells and anti-BCMA combined with anti-CD19 CAR-T-cells at our center between June 1, 2019, and February 28, 2023. Clinical data were collected to observe the characteristics of changes in liver indexes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL) in patients, and its relationship with cytokine-release syndrome (CRS) . Results: Ninety-two patients were included in the analysis, including 41 patients (44.6%) in the group receiving a single infusion of anti-BCMA CAR-T-cells, and 51 patients (55.4%) in the group receiving an infusion of anti-BCMA combined with anti-CD19 CAR-T-cells. After infusing CAR-T-cells, 31 patients (33.7%) experienced changes in liver indexes at or above grade 2, which included 20 patients (21.7%) with changes in one index, five patients (5.4%) with changes in two indexes, and six patients (6.5%) with changes in three or more indexes. The median time of peak values of ALT and AST were d17 and d14, respectively, and the median duration of exceeding grade 2 was 5.0 and 3.5 days, respectively. The median time of peak values of TBIL and DBIL was on d19 and d21, respectively, and the median duration of exceeding grade 2 was 4.0 days, respectively. The median time of onset of CRS was d8, and the peak time of fever was d9. The ALT, AST, and TBIL of patients with CRS were higher than those of patients without CRS (P=0.011, 0.002, and 0.015, respectively). CRS is an independent factor that affects ALT and TBIL levels (OR=19.668, 95% CI 18.959-20.173, P=0.001). The evolution of liver indexes can be reversed through anti-CRS and liver-protection treatments, and no patient died of liver injury. Conclusions: In BCMA-based CAR-T-cell therapy for RRMM, CRS is an important factor causing the evolution of liver indexes. The evolution of liver indexes after CAR-T-cell infusion is transient and reversible after treatment.

Clinical development of chimeric antigen receptor-T cell therapy for hematological malignancies / 中华医学杂志(英文版)

Cellular therapies have revolutionized the treatment of hematological malignancies since their conception and rapid development. Chimeric antigen receptor (CAR)-T cell therapy is the most widely applied cellular therapy. Since the Food and Drug Administration approved two CD19-CAR-T products for clinical treatment of relapsed/refractory acute lymphoblastic leukemia and diffuse large B cell lymphoma in 2017, five more CAR-T cell products were subsequently approved for treating multiple myeloma or B cell malignancies. Moreover, clinical trials of CAR-T cell therapy for treating other hematological malignancies are ongoing. Both China and the United States have contributed significantly to the development of clinical trials. However, CAR-T cell therapy has many limitations such as a high relapse rate, adverse side effects, and restricted availability. Various methods are being implemented in clinical trials to address these issues, some of which have demonstrated promising breakthroughs. This review summarizes developments in CAR-T cell trials and advances in CAR-T cell therapy.

Targeting the HIV reservoir: chimeric antigen receptor therapy for HIV cure / 中华医学杂志(英文版)

Although antiretroviral therapy (ART) can reduce the viral load in the plasma to undetectable levels in human immunodeficiency virus (HIV)-infected individuals, ART alone cannot completely eliminate HIV due to its integration into the host cell genome to form viral reservoirs. To achieve a functional cure for HIV infection, numerous preclinical and clinical studies are underway to develop innovative immunotherapies to eliminate HIV reservoirs in the absence of ART. Early studies have tested adoptive T-cell therapies in HIV-infected individuals, but their effectiveness was limited. In recent years, with the technological progress and great success of chimeric antigen receptor (CAR) therapy in the treatment of hematological malignancies, CAR therapy has gradually shown its advantages in the field of HIV infection. Many studies have identified a variety of HIV-specific CAR structures and types of cytolytic effector cells. Therefore, CAR therapy may be beneficial for enhancing HIV immunity, achieving HIV control, and eliminating HIV reservoirs, gradually becoming a promising strategy for achieving a functional HIV cure. In this review, we provide an overview of the design of anti-HIV CAR proteins, the cell types of anti-HIV CAR (including CAR T cells, CAR natural killer cells, and CAR-encoding hematopoietic stem/progenitor cells), the clinical application of CAR therapy in HIV infection, and the prospects and challenges in anti-HIV CAR therapy for maintaining viral suppression and eliminating HIV reservoirs.

Advances in Allogeneic Chimeric Antigen Receptor T Cells / 中国医学科学院学报

Autologous chimeric antigen receptor(CAR)T-cell therapy has improved the prognosis of hematological malignancies.Nevertheless,allogeneic CAR-T cells have potential advantages over the autologous approach available on the market.However,allogeneic CAR-T cells may cause life-threatening graft-versus-host disease(GVHD)or be rapidly eliminated by the host immune system.In this review,we analyze the different sources of T cells for optimal allogeneic CAR-T cell therapy,describe the different approaches,and introduce the gene editing measures to produce allogeneic CAR-T cells with limited potential for GVHD and improved anti-tumor effect.

Advances of Targeted Therapy for Acute Myeloid Leukemia--Review / 中国实验血液学杂志

Acute myeloid leukemia (AML) has highly heterogeneous clinical manifestations and poor prognosis, and traditional chemotherapy is the main treatment. In recent years, with the in-depth development of next-generation sequencing technology, the treatment of AML is gradually exploring the precise targeted therapy in the direction of molecular biology and immunophenotype. The advent of various small-molecule inhibitors and immune-targeted drugs has brought hope to patients who cannot tolerate intensive chemotherapy or with relapsed/refractory AML. Compared with traditional chemotherapy, targeted therapy has the advantages of significant curative effect and fewer adverse effects. This article reviews the latest research progress of targeted drug therapy for AML.

Novel CD19-KIRS2/Dap12-BB CAR-T Treatment for 3 Patients with Relapsed and Refractory B-Cell Tumors / 中国实验血液学杂志

OBJECTIVE@#To investigate the safety and efficacy of novel CD19-KIRS2/Dap12-BB chimeric antigen receptor T cells (CAR-T cells) in the treatment of relapsed/refractory B-cell malignancy (R/R BCM).@*METHODS@#Three patients with R/R BCM treated with novel CD19-KIRS2/Dap12-BB CAR-T cells from June 2020 to November 2020 were enrolled, including 1 case of B-cell acute lymphoblastic leukaemia (B-ALL) and 2 cases of non-Hodgkin's lymphoma (NHL), and the efficacy and adverse reactions were observed.@*RESULTS@#After CAR-T cells infusion, patient with B-ALL achieved complete remission (CR) and minimal residual disease (MRD) turned negative, and 2 patients with NHL achieved partial remission (PR). Grade 2 cytokine release syndrome (CRS) occurred in B-ALL patient, grade 1 CRS occurred in 2 NHL patients, and grade II to IV hematologic adverse reactions occurred in 3 patients, all of which were controllable and reversible. The progression-free survival (PFS) of the 3 patients was 143, 199, and 91 days, and overall survival (OS) was 282, 430, and 338 days, respectively.@*CONCLUSION@#The novel CD19-KIRS2/Dap12-BB CAR-T cells in treatment of 3 patients with R/R BCM have significant short-term efficacy and controllable adverse reactions, but the long-term efficacy needs to be further improved.

Progress in Research and Application of CAR-T Cell Therapy in T-Lymphocyte Tumors --Review / 中国实验血液学杂志

T-lymphocyte tumors are a group of diseases containing various types of lymphatic system tumors, with strong heterogeneity and poor clinical outcomes. Chimeric antigen receptor T (CAR-T) cell therapy, as a new immune cell therapy, has made a breakthrough in the field of B-lymphocyte tumors. People are interested in the application prospect of this technique in the field of T-lymphocyte tumors. Some studies have shown that CAR-T cell therapy has made some progress in the treatment of T-lymphocyte tumors, and CAR-T for some targets has entered the stage of clinical trials. However, due to the characteristics of T cells, there are also many challenges. This article reviews the research and application of CAR-T cell therapy in T-lymphocyte tumors.

Research Progress of Chimeric Antigen Receptor Modified NK Cells in the Treatment of Acute Myeloid Leukemia --Review / 中国实验血液学杂志

Chimeric Antigen Receptor (CAR) is a research hotspot in the field of cellular immunotherapy in recent years, and CAR-T cells therapy are developing rapidly in hematological malignant tumors, but their clinical application is still limited by related risks. It is particularly important to find more optimized immunoreactive cells. Natural killer (NK) cells, as key effector cells of innate immunity, can kill tumor or infected cells quickly without prior sensitization. Autologous or allogeneic NK cell infusion has become an effective cell therapy for acute myeloid leukemia (AML). CAR-NK cells combine the advantages of CAR targeting tumor specific antigens and enhancing immune cells activity with the innate antitumor function of NK cells to enhance the targeting and lytic activity of NK cells to AML primordial cells. At present, most of the CAR-NK treatments for AML are still in the stage of specific target screening and verification. This article reviews the latest research progress of CAR-NK cell therapy in the field of AML therapy.

Application of TARP luciferase reporter system in function identification of CAR-T cells / 细胞与分子免疫学杂志

Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR-T cells by inserting T cell activity-responsive promoter (TARP) nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor (CAR). Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques. The lentivirus was packaged and was infected with human primary T lymphocytes. Flow cytometry was used to detected the positive rate of lentivirus-infected T cells. The functional characterization of CAR-T cells was identified by luciferase reporter gene system, Western blot, flow cytometry, and small animal live imaging techniques. Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed, and flow cytometry displayed the normal preparation of CAR-T cells. This system could dynamically respond to the activation of CAR-T cells by luciferase reporter gene system. The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR-T cells, and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR-T cells in mice. Conclusion TARP nanoluciferase reporter gene system provides a more convenient, sensitive and quantitative method for evaluating CAR-T cells activation level, exhaustion phenotype and tracing.

Research progress in leveraging biomaterials for enhancing NK cell immunotherapy / 浙江大学学报·医学版

NK cell immunotherapy is a promising antitumor therapeutic modality after the development of T cell immunotherapy. Structural modification of NK cells with biomaterials may provide a precise, efficient, and low-cost strategy to enhance NK cell immunotherapy. The biomaterial modification of NK cells can be divided into two strategies: surface engineering with biomaterials and intracellular modification. The surface engineering strategies include hydrophobic interaction of lipids, receptor-ligand interaction between membrane proteins, covalent binding to amino acid residues, click reaction and electrostatic interaction. The intracellular modification strategies are based on manipulation by nanotechnology using membranous materials from various sources of NK cells (such as exosome, vesicle and cytomembranes). Finally, the biomaterials-based strategies regulate the recruitment, recognition and cytotoxicity of NK cells in the solid tumor site in situ to boost the activity of NK cells in the tumor. This article reviews the recent research progress in enhancing NK cell therapy based on biomaterial modification, to provide a reference for further researches on engineering NK cell therapy with biomaterials.