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.

Progress of gene-mediated precision immunotherapy in the treatment of acute myeloid leukemia / 中国药科大学学报

@#Acute myeloid leukemia (AML) is a disease caused by abnormal cloning of hematopoietic stem cells in the bone marrow, which leads to accumulation of a large number of abnormally differentiated myeloid cells. It is difficult to cure by traditional treatment. The successful application of chimeric antigen receptor T cell (CAR-T) immunotherapy indicates that the treatment of hematological tumors has entered a new stage of precision immunotherapy. However, CAR-T immunotherapy has been found to have many problems in clinical applications, including long treatment cycle, expensive prices, off-target effects, cytokine release syndrome, etc. Therefore, it is necessary to expand the application of CAR or adopt improved measures to enhance the therapeutic effect. This article reviews the new strategies for genetic engineering modification of CAR immune cells and the research progress and application of in situ programming to generate CAR-T, and besides, briefly introduces the new methods about the delivery of gene drugs in vivo, aiming to provide new ideas and theoretical basis for expanding and improving the application of precision immunotherapy in AML.

Control effect of Dasatinib on the treatment of acute myeloid leukemia and adverse events of CD123 targeting CAR-T:a case report and literature review / 陆军军医大学学报

Objective To preliminarily explore the efficacy of chimeric antigen receptor T cells(CAR-T)targeting CD 123 in the treatment of acute myeloid leukemia(AML)and the role of dasatinib in the treatment of CD123 targeting CAR-T induced side effects.Methods Clinical data of 1 patient with relapsed AML admitted to No.920 Hospital of PLA Joint Logistic Support Force in September,2019 were collected.The patient relapsed after previous multi-line chemotherapy and was treated with CD123 targeting CAR-T therapy.The routine blood changes of the patient after treatment were observed.Dasatinib was used when agranulocytosis occurred,40 mg orally 3 times per day,and was stopped when agranulocytosis was relieved.Changes in blood cells,CAR-T amplification,and disease control were observed.The patient was followed up for over 1 year.Results Flow cytometry for bone marrow showed that minimal residual disease negative result was observed in 30 d after infusion.The patient remained disease-free for over 1 year.After CD 123 CAR-T cells infusion,significant expansion of CAR-T cells was observed,accompanied by granulocyte deficiency and cytokine release syndrome(CRS).After using dasatinib,inhibition of CAR-T cell expansion was observed,accompanied by blood cell recovery,and CRS symptoms were alleviated.After stop of dasatinib,CAR-T cells expanded again and blood cells decreased again.Conclusion CAR-T cells targeting CD 123 have certain efficacy in the treatment for relapsed AML.Dashatinib has a blocking effect on the amplification and function of CAR-T,which can alleviate bone marrow suppression caused by CD 123 targeting CAR-T and avoid severe CRS.

Study on the anti-tumor effect of CD30 CAR-T cells based on multi-chain structure / 安徽医科大学学报

Objective @#To develop a CD30-targeted CAR-T cell drug based on the multi-chain chimeric antigen re- ceptor T cells (CAR-T) of the bridging protein DAP12 , and to study the in vitro and in vivo preclinical efficacy of CD30 CAR-T on Hodgkin lymphoma tumor cells .@*Methods @#Through gene synthesis and molecular cloning tech- niques , a CAR plasmid targeting CD30 was designed and constructed , and the obtained lentivirus was packaged . The T cells were transfected with the lentivirus , where the multi-chain CAR-T targeting CD30 was the CD30 - KIRS2/Dap12-BB group , the single-chain second-generation CAR-T was the CD30-41BBζgroup , and the T cells without virus infection were the NTD group . The positive rate of CAR was detected by flow cytometry , the cytotoxic- ity of the cells was detected by lactate dehydrogenase (LDH) release assay , the secretion level of the cytokine in- terferon γ(IFN-γ) was detected by enzyme-linked immunosorbent assay ( ELISA) , and the antitumor activity of CD30 CAR-T in mice was further detected by a mouse xenograft tumor model . @*Results @#A comparison was made between the multi-chain CAR-T targeting CD30 and the single-chain second-generation CAR-T. It was found that the antitumor effect of the multi-chain CAR-T was similar to that of the single-chain CAR-T. However , it was worth noting that the IFN-γsecretion level of the multi-chain CAR-T was higher (P < 0 . 001) . More importantly , in the mouse tumor model experiment , the multi-chain CAR-T achieved complete tumor regression . @*Conclusion @#The multi-chain CAR-T targeting CD30 is superior to the traditional single-chain CAR-T in terms of antitumor activity .

Research progress in targeted therapies of chronic lymphocytic leukemia / 上海交通大学学报（医学版）

Chronic lymphocytic leukemia(CLL)is one of small B-cell lymphomas and leukemias,characterized as a clonal disease of mature B cells.The disease is remarkably heterogeneous,with the majority of patients having an indolent course,yet they are currently incurable.Abnormal signaling pathways are indispensable in the pathogenesis of CLL.In CLL,the common abnormalities of signaling pathways include B-cell receptor(BCR)signaling,apoptosis,nuclear factor kappa B(NF-κB)signaling and Notch signaling.According to the target in signaling pathways,a series of targeted drugs,such as Bruton's tyrosine kinase(BTK)inhibitors(ibrutinib,zanubrutinib),phosphorylate phosphoinositide 3-kinase(PI3K)inhibitor(duvelisib)and B-cell leukemia/lymphoma 2(BCL2)inhibitor(venetoclax),which have significantly changed the prognosis of patients in clinic.Other targeted drugs,such as fenebrutinib,nemtabrutinib and umbralisib,as well as chimeric antigen receptor T-cell(CAR-T)therapy developed in the field of immuno-oncology and T cell engineering,are currently under trial,with more personalized treatment modalities being explored,which may become potential drug targets in the future.In this paper,relevant literature of CLL was reviewed,and recent research progress in molecular pathogenesis and targeted therapies of chronic lymphocytic leukemia was reviewed.

Care of a child with human herpesvirus-6B encephalitis after chimeric antigen receptor T-cell therapy / 中国实用护理杂志

Summarizing the nursing experience of a child with HHV-6B encephalitis after umbilical cord blood transplantation and CAR-T therapy. The child was 4 years old and was diagnosed with acute T lymphocytic leukemia on May 28, 2021. Nursing points: meticulously observe symptoms for early diagnosis and treatment; develop a specialized management plan, implement individualized care; enhance medication management to improve the quality of care; establish a shared decision-making communication model to prevent hospital-acquired infections; provide patient-centered care for lumbar puncture; assess the needs of the child and family, alleviate negative emotions; improve pre-discharge preparation, emphasize continuity of care. With proactive treatment and careful nursing, the child′s condition improved, and they were discharged. Follow-up for six months showed the child in a sustained remission state with no adverse sequelae, and normal life resumed.

Study on the relationship between serum cytokines levels after chimeric antigen receptor (CAR)-T cell immunotherapy and the prognosis of bridged allogeneic hematopoietic stem cell transplantation in acute B lymphoblastic leukemia patients / 中华检验医学杂志

Objective:To investigate the relationship between the levels of serum cytokines and chemokines and the prognosis of patients with acute B-ALL after receiving chimeric antigen receptor (CAR)-T cell immunotherapy and acute graft-versus-host disease (aGVHD) in patients after bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:According to the case-control principle, Forty-two patients with B-ALL who received CD19-CAR-T cell immunotherapy bridged to allo-HSCT at Heibei Yanda Ludaopei Hospital from September 18, 2019 to May 9, 2022 were enrolled. Mann-Whitney U test was used to compare the changes of aGVHD-related cytokines and chemokine levels between CAR-T cell immunotherapy and bridging transplantation in different patients at the same time. Their plasma levels of cytokines and chemokines related to aGVHD were monitored at the day before CAR-T therapy and after CAR-T treatment at day 4, 7,14,21,28. The receiver operating characteristic curve was drawn to evaluate the predictive value of cytokines and chemokines in predicting the occurrence and the death of aGVHD patients. Kaplan-Meier method and Log-rank tests were used for Overall survival (OS) analysis. Results:Twenty-four of total 42 patients had aGVHD, of which 11 patients died and 31 patients survived. There was no significant difference in cytokines and chemokines between the aGVHD group and the non-aGVHD group on the day before CAR-T cell treatment. According to statistical analysis, the serum Elafin levels of aGVHD group was higher than that of non-aGVHD group at the 21st day [4 482 (2 811, 6 061) ng/L vs 2 466 (1 948, 3 375) ng/L, Z=3.145, P=0.001] and the 28st day [4 391 (2 808, 5594) ng/L vs 2 463 (1 658, 2 830) ng/L, Z=2.038, P=0.048] separately. At the 14th day, serum cytokines and chemokines levels between the two group were as follows,MIP-1 α [21.02 (12.36, 30.35) ng/L vs 5.56 (3.64, 10.79) ng/L], sCD25 [422.47 (257.99, 1 233.78) IU/ml vs 216.11 (133.75,457.39) IU/ml], Elafin [4 101 (2 393, 5 006) ng/L vs 2 155 (1 781, 3 033) ng/L], IL-6 [119.08 (23.97, 183.43) ng/L vs 8.39 (2.91, 17.42) ng/L] and IL-8 [13.56 (12.50, 24.52) ng/L vs 2.83 (1.73,6.87) ng/L] were at higher levels ( Z=2.653, P=0.007; Z=2.176, P=0. 030; Z=2.058, P=0.041; Z=3.329, P<0.001; Z=3.162, P=0.001). The KM survival curve showed that the cumulative survival rates of patients with higher serum levels of MIP-1α, sCD25, Elafin, IL-6 and IL-8 were lower than those with low levels at day 14, and the difference was statistically significant (χ 2=12.353, 4.890, 6.551, 10.563, 20.755, P<0.05). Conclusion:The outcomes of patients treated with CAR-T cell therapy bridged to allo-HSCT was correlated with serum MIP-1α, sCD25, Elafin, IL-6 and IL-8 levels after receiving CAR-T therapy. High concentrations of MIP-1α, sCD25, Elafin, IL-6 and IL-8 suggest poor prognosis and can be used as biomarkers to suggest appropriate clinical selection of therapy.

Targets of chimeric antigen receptor T-cell in breast cancer and the application progress / 肿瘤研究与临床

Breast cancer is the most common malignant tumor in women. It is particularly important to seek targeted therapy other than surgery, chemoradiotherapy, endocrine therapy. With the continuous exploration of tumor immunotherapy, more and more therapeutic targets have been discovered. This paper reviews therapeutic targets of chimeric antigen receptor T-cell (CAR-T) and the application in breast cancer.

Advance in chimeric antigen receptor-T cell immunotherapy for multiple myeloma / 中国生物制品学杂志

@#In recent years,considerable progress has been made in the treatment of multiple myeloma(MM).However,despite the current improved prognosis of this malignancy,it always ends in relapse and therefore new therapeutic approaches are urgently needed to overcome it.The chimeric antigen receptor(CAR)-T cells targeting B cell maturation antigen(BCMA),cluster of differentiation 19(CD19),cluster of differentiation 38(CD38) and kappa light chains have been evaluated,and have achieved remarkable results in clinical trials.However,even when MM is treated with CAR-T cell therapy,most patients eventually relapse,which is the greatest limitation of this therapy.This paperreviewedthe research progress,limitations and optimization of CAR-T cell immunotherapy in the treatment of MM.

Research and application progress in adoptive re-transfusion of regulatory cells in organ transplantation / 器官移植

Rejection and adverse reactions caused by long-term use of immunosuppressants severely affect the survival rate and quality of life of organ transplant recipients. Immune tolerance induction plays a key role in improving the survival rate and quality of life of organ transplant recipients. In recent years, tremendous progress has been achieved in adoptive re-transfusion of regulatory cells. In this article, research progress in regulatory T cell (Treg), myeloid-derived suppressor cell (MDSC) and regulatory B cell (Breg) in animal experiment and clinical application was reviewed, and the main clinical problems of adoptive re-transfusion of regulatory cells, the application of chimeric antigen receptor Treg and the concept of cell therapy in immune evaluation were summarized, aiming to deepen the understanding of regulatory cell therapy, promote the application of regulatory cells in immune tolerance of organ transplantation, and improve clinical efficacy of organ transplantation and the quality of life of recipients.

Progress of immunotherapy for NK/T cell leukemia/lymphoma / 白血病·淋巴瘤

NK/T cell leukemia/lymphoma is a type of malignancy originating from T cells or natural killer cells with low incidence and poor clinical prognosis. There is still no effective treatment strategy. In recent years, targeted therapy has made great progress in the treatment of hematological malignancies, including monoclonal antibody and chimeric antigen receptor T cells (CAR-T), among which CD30, CD7, CD5, CD52, CCR4 and other target antigens are effective in the treatment of NK/T cell leukemia/lymphoma, but its widespread application still faces a great challenge. This article reviews the progress of immunotherapy for NK/T cell leukemia/lymphoma.

Prevention and treatment of virus infection in chimeric antigen receptor T cell therapy / 白血病·淋巴瘤

The widespread application of chimeric antigen receptor T (CAR-T) cell immunotherapy in clinical practice presents the challenges for management and prevention of virus infection during the therapy. The paper reviews the risk factors of virus infection for patients during CAR-T cell immunotherapy, summarizes virus infection after treatment and proposes the strategies for infection prevention and treatment.

Auto-HSCT bridging to CAR-T for follicular lymphoma transformed to B-lymphoblastic leukemia/lymphoma: report of 1 case and review of literature / 白血病·淋巴瘤

Objective:To investigate the effect of autologous hematopoietic stem cell transplantation (auto-HSCT) bridging to chimeric antigen receptor T cell (CAR-T) immunotherapy for follicular lymphoma (FL) transformed to B-lymphoblastic leukemia/lymphoma (B-LBL).Methods:The diagnosis and treatment of 1 patient with FL transformed to B-LBL admitted to the First Hospital of Soochow University in August 2020 were retrospectively analyzed and the literature was reviewed.Results:The male patient was 65 years old, and was diagnosed as FL (stage Ⅳ group A, FL international prognostic index -1 score 3 points, high-risk group) in August 2020. And then he was given 6 courses of RB (rituximab combined with bendamustine) regimen, with complete remission (CR) at mid-term and end-stage PET-CT, followed by regular maintenance therapy with rituximab every 2 months, and disease progressed after 4 courses of maintenance therapy. According to the results of histopathology and bone marrow aspiration in December 2021, he was diagnosed B-LBL involving the bone marrow. Partial remission was achieved after induction therapy with zanubnulindb combined with hyper CVAD (cyclophosphamide + doxorubicin + vindesine + dexamethasone) regimen, followed by auto-HSCT bridging to CAR-T treatment targeting CD19 and CD22, which proceeded smoothly with cytokine release syndrome grade 0, immune effector cell-associated neurotoxicity syndrome grade 0. The patient successfully underwent hematopoietic reconstruction and orally taken ibrutinib after discharge. PET-CT indicated CR 2 months after transplantation and he was still in disease-free survival state.Conclusions:The prognosis of FL transformed to B-LBL is poor, and auto-HSCT bridging to CAR-T can improve the prognosis and prolong the survival time of patients who cannot undergo allogeneic hematopoietic stem cell transplantation.

Progress of CD99 as a potential therapeutic target for leukemia / 白血病·淋巴瘤

CD99 gene encodes a transmembrane protein and participates in cell differentiation, adhesion, migration and protein transport. The expression of CD99 is generally low in most normal tissues and cells, and CD99 is differentially expressed in bone marrow and the surface of lymphatic hematopoietic cells. This article reviews the research progress of CD99 in hematological diseases, explores the role of CD99 in myeloid and lymphocytic leukemia, and the significance of CD99 as a therapeutic target for hematological malignancies.

Research progress on the role of neutrophils in progression and treatment of cancer / 国际生物医学工程杂志

Neutrophils are the most abundant population of white blood cells in the human circulation. They play a key role in the protection of the host against microbial infections as well as taking a critical part in inflammatory processes. Recently, several studies showed that neutrophils play an active role in the immune response during cancer development. Neutrophils and various immune cells constitute a significant part of the tumor immune microenvironment. Research has found that different subpopulations of neutrophils may promote or limit tumor growth, indicating that people should be more concerned about the potential of neutrophils. In this paper, the various functions of neutrophils in the progression and treatment of cancer and the possibility of using these functions as a new model of immunotherapy were reviewed.

Advances in application of regulatory T cells in transplant immune tolerance: from basic to clinical research / 器官移植

Regulatory T cells (Treg) are important inhibitory immune cells to establish immune tolerance, which play a pivotal role in regulating excessive immune response and autoimmune diseases of the host. Previous studies related to transplant immune tolerance have confirmed that increasing the number of Treg in vivo or enhancing the function of Treg serve as a therapeutic strategy to induce transplant immune tolerance. At present, Treg-based induction methods for transplant immune tolerance include adoptive infusion of Treg, in vivo amplification of Treg and utilization of antigen-specific Treg. In this article, the characteristics and mechanism of Treg, the latest research progress on basic experiments and clinical practice of Treg related to transplant immune tolerance at home and abroad were reviewed, and future challenges and development of Treg therapy were prospected, aiming to unravel the significance and application prospect of Treg in transplant immune tolerance, explore the advantages and limitations of Treg therapeutic strategies, and provide reference and evidence for subsequent research in this field.

Research progress of CAR-T immunotherapy in solid tumors combined with new strategies / 中国药科大学学报

@#In recent years, the chimeric antigen receptor T-cell (CAR-T) therapy has achieved breakthrough progress in the treatment of hematologic malignancies. However, when it comes to solid tumors, numerous challenges persist.These include limited CAR-T cell infiltration, susceptibility to T cell exhaustion, off-target effects, and more.Thus, novel therapeutic strategies are imperative to enhance the efficacy of CAR-T therapy for solid tumors. In comparison to standalone CAR-T approaches, the combination of CAR-T with other tumor treatment modalities has demonstrated remarkable effectiveness in both preclinical and clinical research.This review article summarizes the advancements in combining CAR-T with various solid tumor treatments: antibody drugs, oncolytic viruses, tumor vaccines, and nanomedicines.The objective is to furnish a theoretical foundation and novel perspectives for the development of innovative CAR-T combination strategies tailored for solid tumor therapy.

Treatment progress of relapsed/refractory follicular lymphoma / 白血病·淋巴瘤

Follicular lymphoma (FL) is the most common indolent B-cell lymphoma. The outcome of relapsed/refractory FL patients after multi-therapy is poor. The 64th American Society of Hematology annual meeting in 2022 announced the latest updates on relapsed/refractory FL, including targeted therapy, bio-specific antibodies and chimeric antigen receptor T-cell. This review provides an overview of these updates.

Diagnosis and treatment progress of primary central nervous system lymphoma / 白血病·淋巴瘤

Primary central nervous system lymphoma (PCNSL) is a rare lymphoma type. The prognosis of PCNSL patients after treated by traditional therapy regimen is very poor. The way to evaluate the prognosis of PCNSL and to increase therapeutic efficacy have become the clinical problem. The 64th American Society of Hematology (ASH) annual meeting reported the latest research progress of diagnosis and treatment of PCNSL, including image examination, genetic sequencing, targeted therapy, chimeric antigen receptor T-cell (CAR-T) therapy and autologous hematopoietic stem cell transplantation (ASCT). This paper reviews the latest progress of PCNSL in the 64th ASH annual meeting.

Progress of chimeric antigen receptor T-cell in treatment of multiple myeloma / 白血病·淋巴瘤

Chimeric antigen receptor T-cell (CAR-T) immunotherapy targeting B-cell maturation antigen (BCMA) has shown significant efficacy in relapsed/refractory multiple myeloma. With the rapid development of CAR-T therapies, CAR-T therapies targeting BCMA continue to be optimized, and new products such as new targets, dual-targets and universal CAR-T are arriving successively. Data from several relevant clinical studies were updated at the 64th American Society of Hematology Annual Meeting.