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.

Biomarkers for chimeric antigen receptor T cell therapy in B cell malignancies can assess therapeutic efficacy and toxicity / 中华检验医学杂志

Chimeric antigen receptor (CAR)-T cell immunotherapy have developed for nearly two decades and achieved great clinical success in the treatment of hematological malignancies. Efficacy monitoring and toxicity management of CAR-T cell immunotherapy are essential steps to ensure safety and improve overall survival in multicenter clinical trials and commercialized treatments. CAR-T cell immunotherapy related biomarkers can be used as an indicator of patient baseline characteristics, tumor biology, and CAR-T cell function. Besides, side effects during treatment can also be assessed by the biomarkers.

Programming CAR T cells to enhance anti-tumor efficacy through remodeling of the immune system / 医学前沿

Chimeric antigen receptor (CAR) T cells have been indicated effective in treating B cell acute lymphoblastic leukemia and non-Hodgkin lymphoma and have shown encouraging results in preclinical and clinical studies. However, CAR T cells have achieved minimal success against solid malignancies because of the additional obstacles of their insufficient migration into tumors and poor amplification and persistence, in addition to antigen-negative relapse and an immunosuppressive microenvironment. Various preclinical studies are exploring strategies to overcome the above challenges. Mobilization of endogenous immune cells is also necessary for CAR T cells to obtain their optimal therapeutic effect given the importance of the innate immune responses in the elimination of malignant tumors. In this review, we focus on the recent advances in the engineering of CAR T cell therapies to restore the immune response in solid malignancies, especially with CAR T cells acting as cellular carriers to deliver immunomodulators to tumors to mobilize the endogenous immune response. We also explored the sensitizing effects of conventional treatment approaches, such as chemotherapy and radiotherapy, on CAR T cell therapy. Finally, we discuss the combination of CAR T cells with biomaterials or oncolytic viruses to enhance the anti-tumor outcomes of CAR T cell therapies in solid tumors.

A novel staining protocol based on protein L for detection of expression of chimeric antigen receptor by flow cytometry / 临床检验杂志

Objective To evaluate the feasibility of the novel staining protocol based on protein L in flow cytometry to detect the expression of chimeric antigen receptor (CAR) on the surface of T cells from patients.Methods The peripheral blood mononuclear cells(PBMCs) were collected from 2 patients with CD19 + lymphoma by hemapheresis and T cells were purified by magnetic bead selection.CAR was transfected with retroviruses targeting CD19 and CD22 into T cells to induce chimeric antigen receptor-modified T cells (CAR-T cells).The single-chain fragments of antibody molecules on the surface of CAR-T cells were labeled by biotinylated protein L-streptavidin-PE system.The proportions of activated CD19-CAR and CD22-CAR positive T cells in the culture were detected by flow cytometry.The results were compared with those detected by conventional flow cytometry method based on Anti-IgG staining.Results The expression of CAR on the surface of CAR-T cells was successfully detected by flow cytometry protocol based on the staining of single-chain variable fragment (scFv)-biotinylated protein L-streptavidin-PE.The percentages of CD19-CAR-T cells from the 2 patients were 71.6％ vs 64.2％ and 49.3％ vs 43.8％ in the protein L group and the Anti-IgG control group respectively,and the percentages of CD22-CAR-T cells were 53.1％ vs 46.3％ and 56.5％ vs 64.0％.The results of the both groups were similar.Conclusion The staining protocol based on protein L could be used as a routine staining method in flow cytometry for the detection of CAR-T cells.

Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

Targeting cancer stem cells by using chimeric antigen receptor-modified T cells: a potential and curable approach for cancer treatment

Cancer stem cells (CSCs), a subpopulation of tumor cells, have self-renewal and multi-lineage differentiation abilities that play an important role in cancer initiation, maintenance, and metastasis. An accumulation of evidence indicates that CSCs can cause conventional therapy failure and cancer recurrence because of their treatment resistance and self-regeneration characteristics. Therefore, approaches that specifically and efficiently eliminate CSCs to achieve a durable clinical response are urgently needed. Currently, treatments with chimeric antigen receptor-modified T (CART) cells have shown successful clinical outcomes in patients with hematologic malignancies, and their safety and feasibility in solid tumors was confirmed. In this review, we will discuss in detail the possibility that CART cells inhibit CSCs by specifically targeting their cell surface markers, which will ultimately improve the clinical response for patients with various types of cancer. A number of viewpoints were summarized to promote the application of CSC-targeted CART cells in clinical cancer treatment. This review covers the key aspects of CSC-targeted CART cells against cancers in accordance with the premise of the model, from bench to bedside and back to bench.

Phase I study of chimeric antigen receptor modified T cells in treating HER2-positive advanced biliary tract cancers and pancreatic cancers

This phase I clinical trial (NCT01935843) is to evaluate the safety, feasibility, and activity of chimeric antigen receptor-engineered T cell (CART) immunotherapy targeting human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancers (BTCs) and pancreatic cancers (PCs). Eligible patients with HER2-positive (>50%) BTCs and PCs were enrolled in the trial. Well cultured CART-HER2 cells were infused following the conditioning treatment composed of nab-paclitaxel (100-200 mg/m) and cyclophosphamide (15-35 mg/kg). CAR transgene copy number in the peripheral blood was serially measured to monitor the expansion and persistence of CART-HER2 cells in vivo. Eleven enrolled patients received 1 to 2-cycle CART-HER2 cell infusion (median CAR T cell 2.1 × 10/kg). The conditioning treatment resulted in mild-to-moderate fatigue, nausea/vomiting, myalgia/arthralgia, and lymphopenia. Except one grade-3 acute febrile syndrome and one abnormal elevation of transaminase (>9 ULN), adverse events related to the infusion of CART-HER2 cells were mild-to-moderate. Post-infusion toxicities included one case of reversible severe upper gastrointestinal hemorrhage which occurred in a patient with gastric antrum invaded by metastasis 11 days after the CART-HER2 cell infusion, and 2 cases of grade 1-2 delayed fever, accompanied by the release of C-reactive protein and interleukin-6. All patients were evaluable for assessment of clinical response, among which 1 obtained a 4.5-months partial response and 5 achieved stable disease. The median progression free survival was 4.8 months (range, 1.5-8.3 months). Finally, data from this study demonstrated the safety and feasibility of CART-HER2 immunotherapy, and showed encouraging signals of clinical activity.

Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment

Chimeric antigen receptor (CAR) is a recombinant immunoreceptor combining an antibody-derived targeting fragment with signaling domains capable of activating cells, which endows T cells with the ability to recognize tumor-associated surface antigens independent of the expression of major histocompatibility complex (MHC) molecules. Recent early-phase clinical trials of CAR-modified T (CAR-T) cells for relapsed or refractory B cell malignancies have demonstrated promising results (that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia (B-ALL)). Given this success, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.

Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body's immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS.

Chimeric antigen receptor T cell based therapeutic modality in solid tumors / 中国肿瘤临床

The CD19-targeted chimeric antigen receptor (CAR) T-cell treatment has achieved clinical success in treating B-cell lineage hematopoietic malignancies. This accomplishment involved the precise and efficient elimination of tumor cells by tumor-associated an-tigen-redirected immune cells. As a result, the reinitiation of several CAR T-cell (CART) based clinical trials in solid tumors was promot-ed. However, developing a feasible and efficient CAR T based therapeutic modality for solid tumors is urgently needed given the differ-ential properties between liquid and solid tumors. In this review, we discuss the advances in the management of cytotherapeutic mo-dality for solid tumors. The aspects considered include toxicity management, target selection, and primer or conditioning treatment.

Effects of conditioned media for rat bone marrow-derived mesenchymal stem cells on palmitic acid-induced insulin resistance in HepG2 cells / 中华内科杂志

Objective To study the effect of conditioned media for rat bone marrow mesenchymal stem cells (BMSCs-CM) on palmitic acid (PA)-induced insulin resistance (IR) in HepG2 cells and its underlying molecular mechanisms.Methods HepG2 cells were treated with or without BMSCs-CM and L-DMEM in the presence or absence of PA.Glucose utilization in HepG2 cells were detected with PAS,glucose and glycogen measurements.Western blotting was used to assess the expression of phospho-insulin receptor substrate (p-IRS),phosphatidylinositol 3-kinase (PI3 K) and p-AKT.Results (1) Incubation of HepG2 cells with 0.25 mmol/L PA for 24 hours significantly increased the glucose concentration and decreased the glycogen content (P ＜ 0.05) in the media.(2) Treatment with BMSCs-CM significantly ameliorated the glucose and glycogen alteration in cells pretreated with PA (P ＜ 0.05),however,no obvious effect of BMSCs-CM on the cell glucose and glycogen production.(3) BMSCs-CM treatment also increased protein expression of p-IRS,PI3K and p-AKT in PA incubated HapG2 cells (P＜ 0.05).The effect of BMSCs-CM on PI3K and p-AKT expression could be mimicked upon addition of 740Y-P,a PI3K agonist,but abolished by LY294002,a PI3K specific inhibitor.Conclusions BMSCs-CM could improve the insulin sensitivity in HepG2 cells pretreated with PA through upregulation of insulin signaling component expression.

Bone cement injection for the treatment of thoracolumbar osteoporotic fractures：Common problems in the mature technology / 中国组织工程研究

10.3969/j.issn.2095-4344.2013.26.022

Construction of a LRP16 gene targeting vector and screening of homologously recombinant clone of embryonic stem cells / 中国组织工程研究

BACKGROUND: Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells.OBJECTIVE: To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP16 gene.DESIGN: Constructing an inserting inactivation target by inserting SA-RIES-β geo expression cassette.SETTING: Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA.MATERIALS: The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from lnvitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen Biotech Corp. pcDNA3.1(+) vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University.METHODS: The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction (PCR) screening. The SA-RIES-β geo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis.MAIN OUTCOME MEASURES: Clones with homologous recombination.RESULTS: The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES-β geo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted.CONCLUSION: A LRP16 gene targeting vector is constructed and a homologous recombinant is obtained.

Application of Proteomics in the Sperm Protein Research / 中国生物工程杂志

Proteomics was important for the coherent study of human reproduction and animal breeding including human infertility,sperm-egg binding and mutual recognition of the mechanism.It was well known that proteomics had become one of the main branches of life sciences in the future.This provides the technological means and theoretical foundation for the individual dynamic changes in the protein.At the same time,it plays an important role in the drug development,the mechanism of life activities and in the field of livestock breeding.

The influence of anti-sense hTERT on the expression of VEGF and Flt in SGC7901 cells / 解放军医学杂志

Objective To investigate the effects of anti-sense hTERT on the expression of VEGF and the receptor in SGC7901 cells, and study the influence in angiogenesis and progression in gastric carcinoma. Methods SGC7901 cell line was transfected by the recombinant virus containing sense and anti-sense hTERT cDNA. Then the expression of VEGF-C and Flt-4 was observed with RT-PCR, distribution of cell cycles was determined with flow cytometry. The expression of VEGF-C and Flt-4 protein was assessed with immunohistochemistry. Result The distribution of cell cycle of antisense hTERT transfected SGC7901 cells was changed, and the mRNA and protein expression of VEGF-C and Flt-4 was significantly down-regulated. Conclusion Anti-sense hTERT can act as an agent for inhibiting VEGF-C and Flt-4 mRNA and protein level, and it blocks tumor angiogenesis and lymphogenous metastasis.

RNA interference directed by small interference RNA of hTERT expressed in lung carcinoma cell A549 / 解放军医学杂志

Objective To investigate the inhibitory effect of siRNA human telomerase transcriptase (hTERT) on activity of telomerase and proliferation of lung carcinoma cell A549. Methods A plasmid including U6 promoter and siRNA of hTERT was designed and constructed. The plasmid was transfected into A549 cell line. The telomerase activity was tested by telomerase repeat amplification protocol ELISA (TRAP-ELISA). MTT assay was used to assay the cell proliferation activity,and hTERT expression was assessed by Western blot. Result The U6 expression plasmid that was constructed for hTERT gene 745 showed obvious interfering effect. hTERT-siRNA could down-regulate the expression of hTERT protein,inhibit telomerase activity and proliferation of A549 cells. Conclusion siRNA of hTERT can inhibit the expression of human telomerase and proliferation of A549 cells. It may open a new approach to the use of siRNA as a new tool to study gene function in cancer cell lines,and may be developed to be a new gene therapeutic agent for cancer.

Inhibitory effect of FHL2 on Id1-mediated suppression of proliferation and invasive capacity of breast cancer cells MCF-7 / 解放军医学杂志

Objective To investigate the inhibitory effect of four and a half LIM protein 2(FHL2) on inhibitor of differentiation 1(Id1)-mediated suppression of transcriptional regulation activity and Id1-promoted invasive growth of human breast cancer cells MCF-7.Methods The effect of FHL2 on Id1-mediated transcriptional repression in MCF-7 cells was determined by cotransfection and relative biluciferase assay.The cell proliferation was determined by MTT assay,and the invasive capacity of MCF-7 cells was determined by Transwell assay.Results The transcriptional repression effect of Id1 on basic helix-loop-helix(bHLH) factor E47-mediated transcription activity in MCF-7 cells,and Id1-promoted proliferation and invasive growth of MCF-7 cells were significantly suppressed by FHL2.Conclusion FHL2 can inhibit the proliferation and invasive growth of human breast cancer cells via repressing Id1-mediated functional activity.The results provide a basis for further investigating the functional roles of FHL2-Id1 signal pathway in the carcinogenesis and development of human breast cancer.