Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor–T cell manufacture

Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)–T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.     

Remote controlling of CAR-T cells and toxicity management: Molecular switches and next generation CARs

Cell-based immunotherapies have been selected for the front-line cancer treatment approaches. Among them, CAR-T cells have shown extraordinary effects in hematologic diseases including chemotherapy-resistant acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). In this approach, autologous T cells isolated from the patient''s body genetically engineered to express a tumor specific synthetic receptor against a tumor antigen, then these cells expanded ex vivo and re-infusion back to the patient body. Recently, significant clinical response and high rates of complete remission of CAR T cell therapy in B-cell malignancies led to the approval of Kymriah and Yescarta (CD19-directed CAR-T cells) were by FDA for treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Despite promising therapeutic outcomes, CAR T cells also can elicit the immune-pathologic effects, such as Cytokine Release Syndrome (CRS), Tumor Lysis Syndrome (TLS), and on-target off-tumor toxicity, that hampered its application. Ineffective control of these highly potent synthetic cells causes discussed potentially life-threatening toxicities, so researchers have developed several mechanisms to remote control CAR T cells. In this paper, we briefly review the introduced toxicities of CAR-T cells, then describe currently existing control approaches and review their procedure, pros, and cons.     

Evaluation of piggyBac-mediated anti-CD19 CAR-T cells after ex vivo expansion with aAPCs or magnetic beads

Adoptive immunotherapy is a new potential method of tumour therapy, among which anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T cell), is a typical treatment agent for haematological malignancies. Previous clinical trials showed that the quality and phenotype of CAR-T cells expanded ex vivo would seriously affect the tumour treatment efficacy. Although magnetic beads are currently widely used to expand CAR-T cells, the optimal expansion steps and methods have not been completely established. In this study, the differences between CAR-T cells expanded with anti-CD3/CD28 mAb-coated beads and those expanded with cell-based aAPCs expressing CD19/CD64/CD86/CD137L/mIL-15 counter-receptors were compared. The results showed that the number of CD19-specific CAR-T cells with a 4-1BB and CD28 co-stimulatory domain was much greater with stimulation by aAPCs than that with beads. In addition, the expression of memory marker CD45RO was higher, whereas expression of exhausted molecules was lower in CAR-T cells expanded with aAPCs comparing with the beads. Both CAR-T cells showed significant targeted tumoricidal effects. The CAR-T cells stimulated with aAPCs secreted apoptosis-related cytokines. Moreover, they also possessed marked anti-tumour effect on NAMALWA xenograft mouse model. The present findings provided evidence on the safety and advantage of two expansion methods for CAR-T cells genetically modified by piggyBac transposon system.     

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells

BackgroundChimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study.MethodsA third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests.ResultsUp to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process.DiscussionCryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.     

Targeting CD276 by CAR-T cells induces regression of esophagus squamous cell carcinoma in xenograft mouse models

Esophageal cancer, including esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), has a poor prognosis and limited therapeutic options. Chimeric antigen receptor (CAR)-T cells represent a potential ESCC treatment. In this study, we examined CD276 expression in healthy and esophageal tumor tissues and explored the tumoricidal potential of CD276-targeting CAR-T cells in ESCC. CD276 was strongly and homogenously expressed in ESCC and EAC tumor lesions but mildly in healthy tissues, representing a good target for CAR-T cell therapy. We generated CD276-directed CAR-T cells with a humanized antigen-recognizing domain and CD28 or 4–1BB co-stimulation. CD276-specific CAR-T cells efficiently killed ESCC tumor cells in an antigen-dependent manner both in vitro and in vivo. In patient-derived xenograft models, CAR-T cells induced tumor regression and extended mouse survival. In addition, CAR-T cells generated from patient T cells demonstrated potent cytotoxicity against autologous tumor cells. Our study indicates that CD276 is an attractive target for ESCC therapy, and CD276-targeting CAR-T cells are worth testing in ESCC clinical trials.     

Co-infusion of haplo-identical CD19-chimeric antigen receptor T cells and stem cells achieved full donor engraftment in refractory acute lymphoblastic leukemia

Background

Elderly patients with relapsed and refractory acute lymphoblastic leukemia (ALL) have poor prognosis. Autologous CD19 chimeric antigen receptor-modified T (CAR-T) cells have potentials to cure patients with B cell ALL; however, safety and efficacy of allogeneic CD19 CAR-T cells are still undetermined.

Case presentation

We treated a 71-year-old female with relapsed and refractory ALL who received co-infusion of haplo-identical donor-derived CD19-directed CAR-T cells and mobilized peripheral blood stem cells (PBSC) following induction chemotherapy. Undetectable minimal residual disease by flow cytometry was achieved, and full donor cell engraftment was established. The transient release of cytokines and mild fever were detected. Significantly elevated serum lactate dehydrogenase, alanine transaminase, bilirubin and glutamic-oxalacetic transaminase were observed from days 14 to 18, all of which were reversible after immunosuppressive therapy.

Conclusions

Our preliminary results suggest that co-infusion of haplo-identical donor-derived CAR-T cells and mobilized PBSCs may induce full donor engraftment in relapsed and refractory ALL including elderly patients, but complications related to donor cell infusions should still be cautioned.

Trial registration

Allogeneic CART-19 for Elderly Relapsed/Refractory CD19+ ALL. NCT02799550

     

Efficacy and toxicity for CD22/CD19 chimeric antigen receptor T-cell therapy in patients with relapsed/refractory aggressive B-cell lymphoma involving the gastrointestinal tract

Gastrointestinal (GI) tract is the most common site of extranodal involvement in non-Hodgkin lymphoma. Life-threatening complications of GI may occur because of tumor or chemotherapy. Chimeric antigen receptor (CAR) T-cell therapy has been successfully used to treat refractory/relapse B-cell lymphoma, however, little is known about the efficacy and safety of CAR-T cell therapy for GI lymphoma. Here, we reported the efficacy and safety of CAR-T cell therapy in 14 patients with relapsed/refractory aggressive B-cell lymphoma involving the GI tract. After a sequential anti-CD22/anti-CD19 CAR-T therapy, 10 patients achieved an objective response, and seven patients achieved a complete response. CAR transgene and B-cell aplasia persisted in the majority of patients irrespective of response status. Six patients with partial response or stable disease developed progressive disease; two patients lost target antigens. Cytokine release syndrome (CRS) and GI adverse events were generally mild and manageable. The most common GI adverse events were diarrhea (4/14), vomiting (3/14) and hemorrhage (2/14). No perforation occurred during follow-up. Infection is a severe complication in GI lymphoma. Two patients were infected with bacteria that are able to colonize at GI; one died of sepsis early after CAR-T cells infusion. In conclusion, our study showed promising efficacy and safety of CAR-T cell therapy in refractory/relapsed B-cell lymphoma involving the GI tract. However, the characteristics of CAR-T–related infection in GI lymphoma should be further clarified to prevent and control infection.     

CAR19/22 T cell cocktail therapy for B-ALL relapsed after allogeneic hematopoietic stem cell transplantation

B cell acute lymphocytic leukemia (B-ALL) patients who have relapsed after hematopoietic stem cell transplantation (HSCT) have a poor prognosis, and there is currently no standard approach available. Chimeric antigen receptor (CAR)-T cells induce high rates of initial response and long-term remission among patients with B-cell malignancies, especially B-ALL. Meanwhile, sequential infusion of CAR19/22 T cells has been proven to be effective at preventing tumor immune escape. In the present study, we retrospectively analyzed 23 B-ALL patients who relapsed after allogeneic (allo)-HSCT and underwent sequential infusion of CAR19/22 T cells, including nine donor-derived and 14 recipient-derived, in our center from July 2016 to July 2020, to evaluate the safety and efficacy of the cocktail of two single-specific CAR-T cells in B-ALL patients relapsed after transplantation. Except for one patient refusing evaluation, the remaining 22 patients achieved minimal residual disease (MRD)-negative complete remission within 30 days after CAR-T infusion. Most toxicities were slight and reversible. The estimated 12-month progression-free survival (PFS) rate was 59.2% (95% confidence interval [CI], 35.9% to 76.5%), and the estimated 12-month overall survival (OS) rate was 67.4% (95% CI, 43.2% to 83.1%). Only two patients had CD19-negative recurrence. In addition, early recurrence after transplantation, graft-versus-host disease (GVHD) and severe infection after CAR-T infusion were poor prognostic factors. Our results indicate that sequential infusion of CAR19/22 T cells is safe and effective for relapsed ALL patients after HSCT. This trial was registered at www.chictr.org.cn as #ChiCTR-OPN-16008526.     

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

Efficacy of B7-H3-Redirected BiTE and CAR-T Immunotherapies Against Extranodal Nasal Natural Killer/T Cell Lymphoma

Extranodal nasal natural killer (NK)/T cell lymphoma (ENKTCL) is a rare but highly aggressive subtype of non-Hodgkin lymphoma (NHL). Nevertheless, despite extensive research, the estimated 5-year overall survival of affected patients remains low. Therefore, new treatment strategies are needed urgently. Recent advances in immunotherapy have the potential to broaden the applications of chimeric antigen receptor-modified T (CAR-T) cells and the bispecific T-cell engaging (BiTE) antibody. Here, we screened a panel of biomarkers including the B7-H3, CD70, TIM-3, VISTA, ICAM-1, and PD-1 in NKTCL cell lines. As a result, we found for the first time that B7-H3 was highly and homogeneously expressed in these cells. Consequently, we constructed a novel anti-B7-H3/CD3 BiTE antibody and B7-H3-redirected CAR-T cells, and evaluated their efficacy against NKTCL cel lines both in vitro and in vivo. Notably, we found that both anti-B7-H3/CD3 BiTE and B7-H3-redirected CAR-T cells effectively targeted and killed NKTCL cells in vitro, and suppressed the growth of NKTCL tumors in NSG mouse models. Thus, B7-H3 might be a promising therapeutic target for treating patients with NKTCL tumors.     

Evaluation of a clinical-grade,cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34⁺ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP).MethodscGMP VPA-mediated expansion was initiated with CD34⁺ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts.ResultsThe authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products.ConclusionsThe authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34⁺ cells for clinical utilization.     

Validation of a flow cytometry-based method to quantify viable lymphocyte subtypes in fresh and cryopreserved hematopoietic cellular products

Background aimsAdoptive cellular therapy with immune effector cells (IECs) has shown promising efficacy against some neoplastic diseases as well as potential in immune regulation. Both inherent variability in starting material and variations in cell composition produced by the manufacturing process must be thoroughly evaluated with a validated method established to quantify viable lymphocyte subtypes. Currently, commercialized immunophenotyping methods determine cell viability with significant errors in thawed products since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte immunophenotypes in fresh and cryopreserved hematopoietic cellular products.MethodsUsing fresh or frozen cellular products and stabilized blood, we report on the validation parameters accuracy, uncertainty, precision, sensitivity, robustness and contamination between samples for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3–CD56+CD16+/– NK cells, CD19+ B cells and CD14+ monocytes of relevance to fresh and cryopreserved hematopoietic cellular products using the Cytomics FC500 cytometer (Beckman Coulter).ResultsThe acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of ≤20% at three different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons) and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% coefficient of variation). Furthermore, the method can accurately report on the efficacy of cell depletion since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µL, respectively). The method complies with Foundation for the Accreditation of Cellular Therapy (FACT) standards for IEC, FACT-Joint Accreditation Committee of ISCT-EBMT (JACIE) hematopoietic cell therapy standards, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) and International Organization for Standardization 15189 standards. Furthermore, it complies with Ligand Binding Assay Bioanalytical Focus Group/American Association of Pharmaceutical Scientists, International Council for Standardization of Hematology/International Clinical Cytometry Society and European Bioanalysis Forum recommendations for validating such methods.ConclusionsThe implications of this effort include standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection and in-process quality controls or dosing of IECs. This method also complies with all relevant standards, particularly FACT-JACIE standards, in terms of enumerating and reporting on the viability of the “clinically relevant cell populations.”     

Characterization of cryopreserved human Langerhans cells

Epidermal Langerhans cells are potent antigen-presenting cells in the epidermis. The establishment of a cryopreservation method for human Langerhans cells would greatly contribute to our ability to successfully conduct various experiments dealing with Langerhans cells. Since Langerhans cells are known to be sensitive to cold injury, there have been no reports concerning the cryopreservation of Langerhans cells. We have investigated the effect of cryopreservation on the function and phenotype of human Langerhans cells. Langerhans cells from human foreskins were isolated with the immunomagnetic microbead method using monoclonal antibodies for CD1a. Langerhans cells were cryopreserved in the presence of dimethylsulfoxide (DMSO) 10% and fetal calf serum 90%. Cryopreserved Langerhans cells were phenotypically assessed by flowcytometry using monoclonal antibodies to HLA-DR and CD1a. The ultrastructures of the Langerhans cells were compared using electron microscopy. An autologous T cell stimulation test was performed to compare the functions of cryopreserved Langerhans cells and fresh Langerhans cells. The viability of the cryopreserved Langerhans cells was able to be maintained at more than 90%. Cryopreserved Langerhans cells expressed high levels of HLA-DR and CD1a antigens and stimulated autologous T cells to an extent almost identical to that obtained from fresh Langerhans cells. These findings indicate that the cryopreservation of human Langerhans cells could lead to a breakthrough in various experiments dealing with human Langerhans cells.     

Outcome of adrenal tissue fragments allotransplantation: The impact of cryopreservation

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4⁺ and CD8⁺ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.     

Viability,proliferation and phenotype maintenance in cryopreserved human iliac apophyseal chondrocytes

Cryopreservation preserves cells at low temperature and creates a reserve for future use while executing the clinical translation. Unlike articular chondrocyte, cryopreservation protocol and its outcome are not described in iliac apophyseal chondrocytes, a potential source of chondrocytes in cartilage engineering. This study for the first time describes the cryopreservation of human iliac apophyseal chondrocytes. Four cartilage samples were procured from iliac crests of children undergoing hip surgery after consent. The total chondrocyte yield was divided into two groups. First group was grown as monolayer while second group was cryopreserved following the slow cooling method in the medium containing 10 % Dimethyl sulfoxide for 3 months. Group two cells were also grown as a monolayer following thawing. Viability, time to confluence, population doubling time and phenotype maintenance were compared for both the groups. Viability was 65.75 % after 3 months of cryopreservation at ?196 °C, as compared to 94.19 % for fresh chondrocytes (p = 0.001). Fresh and cryopreserved cells reached confluence on 10th and 15th day of culture respectively. Population doubling time was significantly more in fresh than cryopreserved chondrocytes on 10th (p = 0.0006) and 15th day (p = 0.0002) in culture. Both fresh and cryopreserved cells maintain their chondrocyte phenotype as assessed by immunocytochemistry. Relative gene expression by real time polymerase chain reaction showed similar upregulation of mRNA of Collagen 2, SOX 9, Aggrecan and Collagen 1 in cryopreserved chondrocyte as compared to fresh chondrocyte. Iliac apophyseal chondrocytes cryopreserved for 3 months maintained the phenotype successfully 2 weeks after thawing in culture. The viability and proliferation rates after thawing were adequate for a clinical translation of these cells.     

Outcome of adrenal tissue fragments allotransplantation: The impact of cryopreservation

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4⁺ and CD8⁺ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.     

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML.MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells.ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells.ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.     

稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定

实体瘤缺乏明确的嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)治疗靶点。因此,通过慢病毒将已经明确的靶点分子CD19带入实体瘤细胞系,研究CD19 CAR-T细胞对其的杀伤,能够为CAR-T细胞针对实体瘤的治疗提供潜在的支撑。本研究利用三质粒慢病毒系统构建了稳定表达CD19、萤火虫荧光素酶(firefly luciferase, FLUC)和绿色荧光蛋白(green fluorescent protein, GFP)的结肠癌CT26细胞系CT26-CD19-FLUC-GFP。该细胞系与CT26细胞系的生长活性一致。通过流式细胞术检测不同代次CT26-CD19-FLUC-GFP细胞,证实了CT26-CD19-FLUC-GFP细胞连续传代至第5、10、22代后CD19及GFP的稳定表达。进一步证实,连续传代至第22代的CT26-CD19-FLUC-GFP细胞中的CD19 mRNA及FLUC表达水平显著高于对照组CT26细胞。与T细胞相比,CD19 CAR-T细胞能够显著杀伤CT26-CD19-FLUC-GFP细胞及MC38-CD19细胞。CT26-CD19-FLUC-GFP细胞腹腔植入小鼠体内1周后,通过活体成像仪可以检测到腹腔区域的FLUC表达。上述结果表明,成功构建了稳定表达CD19-FLUC-GFP的CT26细胞系,且该细胞系能够被CD19 CAR-T细胞特异性杀伤。     

Cryopreservation of hematopoietic cells using a pre-constituted,protein-free cryopreservative solution with 5% dimethyl sulfoxide

Background aimsAdequate cryopreservation techniques are critical to ensure optimal recovery of functional progenitor cells in hematopoietic cell (HC) transplantation, minimize risk of contamination and prevent infusion-related adverse events (irAEs). In this article, we provide graft function and infusion safety results observed by decreasing the concentration of dimethyl sulfoxide (DMSO) in cryopreservative media and by minimizing processor-dependent formulation.MethodsTen HC products, collected after standard mobilization of multiple myeloma patients, were cryopreserved with PRIME-XV FreezIS (FreezIS) and compared with products previously cryopreserved with media formulated in-house to achieve a final DMSO concentration of 10% (Std10) and 5% (Std5). At infusion, HCs were analyzed for recovery of CD34+ cells and viability; irAEs and time to engraftment of neutrophils and platelets were also monitored.ResultsMedian CD34+ cell recovery for HC cryopreserved with Std10, Std5 and FreezIS was 38%, 78% and 68%, respectively (P = 0.0002). There were less frequent irAEs with Std5 and FreezIS (10%) compared with Std10 (80%) (P ≤ 0.0001). Median time to neutrophil engraftment was comparable (11 days) for all three groups, while platelet engraftment occurred at a median of 20, 19 and 17 days, respectively (p-values not significant).ConclusionsFreezIS, a Good Manufacturing Practice-grade, pre-constituted cryopreservative with low DMSO content, maintains functional viability of the HC product while reducing the incidence of irAEs compared with 10% DMSO solutions. The pre-constituted nature of this agent also decreases processor-dependent handling, hence decreasing the risk of variability and infection.