Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma – assessing tumor and T cell interaction

A novel disialoganglioside 2 (GD2)-specific chimeric antigen receptor (CAR)-modified T cell therapy against retinoblastoma (RB) were generated. GD2-CAR consists of a single-chain variable fragment (scFv) derived from a monoclonal antibody, hu3F8, that is linked with the cytoplasmic signaling domains of CD28, 41BB, a CD3ζ, and an inducible caspase 9 death fusion partner. GD2 antigen is highly expressed in Y79RB cell line and in several surgical RB tumor specimens. In vitro co-culture experiments revealed the effective killing of Y79RB cells by GD2-CAR T cells, but not by control CD19-CAR T cells. The killing activities of GD2-CAR T cells were diminished when repeatedly exposed to the tumor, due to an attenuated expression of GD2 antigen on tumor cells and upregulation of inhibitory molecules of the PD1 and PD-L1 axis in the CAR T cells and RB tumor cells respectively. This is the first report to describe the potential of GD2-CAR T cells as a promising therapeutic strategy for RB with the indication of potential benefit of combination therapy with immune checkpoint inhibitors.     

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z⁺ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1⁺ or 19z1-CD80⁺ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z⁺ T cells had 10-fold reduced tumor progression compared to those treated with 19z1⁺ or 19z1-CD80⁺ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80⁺ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z⁺ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.     

Tandem CAR-T cells targeting FOLR1 and MSLN enhance the antitumor effects in ovarian cancer

Given the heterogeneity of solid tumors, single-target CAR-T cell therapy often leads to recurrence, especially in ovarian cancer (OV). Here, we constructed a Tandem-CAR targeting two antigens with secretory activity (IL-12) to improve the effects of CAR-T cell therapy. Twenty coexpressed upregulated genes were identified from the GEO database, and we found FOLR1 (folate receptor 1) and MSLN (mesothelin) were specifically and highly expressed in cancer tissues and only 11.25% of samples were negative for both antigens. We observed an increased proliferation rate for these three CAR-T cells, and Tandem CAR-T cells could efficiently lyse antigen-positive OV cells in vitro and secrete higher levels of cytokines than single-target CAR-T cells. More importantly, in vivo experiments indicated that Tandem CAR-T cells markedly decreased tumor volume, exhibited enhanced antitumor activity, and prolonged mouse survival. Furthermore, the infiltration and persistence of T cells in the Tandem-CAR group were higher than those in the MSLN-CAR and Control-T groups but comparable to those in the FOLR1-CAR group. Collectively, this study demonstrated that Tandem CAR-T cells secreting IL-12 could enhance immunotherapeutic effects by reducing tumor antigen escape and increasing T cell functionality, which could be a promising therapeutic strategy for OV and other solid tumors.     

Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide

Background aimsAdoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38^dim phenotype occurring during long-term cytokine stimulation of primary NK cells.MethodsPrimary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38^dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays.ResultsWe verified the functionality of the αCD38-CAR-NK cells against CD38⁺ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo.ConclusionsOverall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.     

P21-activated kinase 4 overexpression in metastatic gastric cancer patients

INTRODUCTION: P21-activated kinase 4 (PAK), a subfamily of serine/threonine kinases originally known as a regulator of cytoskeletal dynamics and cell motility, has recently been revealed to play a key role in oncogenic signaling pathways. We studied the frequency and clinical features of PAK4-overexpressed metastatic gastric cancer. PATIENTS AND METHODS: PAK4 overexpression was screened by Western blot in 18 human gastric cancer cell lines. Immunohistochemical staining of PAK4 protein was performed in tumor specimens of 49 metastatic gastric cancer patients who received palliative capecitabine/cisplatin as first-line treatment. RESULTS: PAK4 protein overexpression was detected strongly in five gastric cell lines (AGS, MGK-28, MKN-74, SNU-216, SNU-601) and weakly in four cell lines (KATOIII, MKN-1, SNU-620, and SNU-719). PAK4 knockdown by small interfering RNA induced apoptosis in PAK4-overexpressed AGS gastric cancer cells. Immunohistochemical staining revealed PAK4 overexpressions in 4 (8.1%) of 49 metastatic gastric cancer specimens. None of the four patients with PAK4(+) responded to capecitabine/cisplatin chemotherapy, and PAK4(+) gastric cancer patients had a trend of poorer survival compared with PAK(-)(P = .876). CONCLUSIONS: We demonstrated PAK4 overexpression in a subset of gastric cancer patients, implicating a role in gastric cancer tumorigenesis. Its prognostic significance and efficacy as a drug target should be further studied.     

Conditional control of chimeric antigen receptor T-cell activity through a destabilizing domain switch and its chemical ligand

Background aimsDespite the impressive efficacy of chimeric antigen receptor (CAR) T-cell therapy, adverse effects, including cytokine release syndrome and neurotoxicity, impede its therapeutic application, thus making the modulation of CAR T-cell activity a priority. The destabilizing domain mutated from Escherichia coli dihydrofolate reductase (DHFR) is inherently unstable and degraded by proteasomes unless it is stabilized by its chemical ligand trimethoprim (TMP), a Food and Drug Administration-approved drug. Here the authors reveal a strategy to modulate CAR T-cell activity at the protein level by employing DHFR and TMP as a chemical switch system.MethodsFirst, the system was demonstrated to work in human primary T cells. To introduce the system to CAR T cells, DHFR was genetically fused to the carboxyl terminal of a third-generation CAR molecule targeting CD19 (CD19-CAR), constructing the CD19-CAR-DHFR fusion.ResultsThe CD19-CAR-DHFR molecule level was shown to be modulated by TMP. Importantly, the incorporation of DHFR had no impact on the recognition specificity and normal function of the CAR molecule. Little adverse effect on cell proliferation and apoptosis was detected. It was proved that TMP could regulate cytokine secretion and the in vitro cytotoxicity of CD19-CAR-DHFR T cells. Furthermore, the in vivo anti-tumor efficacy was demonstrated to be controllable through the manipulation of TMP administration. The approach to control CD19-CAR also succeeded in 19-BBZ(71), another CD19-targeting CAR with a different structure.ConclusionsThe proposed approach based on DHFR and TMP provides a facile strategy to bring CAR T-cell therapy under conditional user control, and the strategy may have the potential to be transplantable.     

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

CCR7 expression in CD19 chimeric antigen receptor-engineered natural killer cells improves migration toward CCL19-expressing lymphoma cells and increases tumor control in mice with human lymphoma

Background aimsChimeric antigen receptor (CAR) T-cell therapy can be associated with significant toxicities. CAR-engineered natural killer (NK) cells provide a safer alternative while maintaining anti-tumor effects. Activated NK (aNK) cells are a clinical-grade cellular product obtained from the NK-92 cell line that have demonstrated both safety and potent cytotoxicity toward a wide range of cancers in phase 1 trials. Genetically engineered variants of aNK cells expressing a high-affinity Fc receptor (haNK) or co-expressing a CAR (t-haNK) are currently in phase 1/2 clinical trials. A key factor in the efficacy of cellular immunotherapies is biodistribution and tumor infiltration, which affect the local effector:target ratio. The chemokines CCL19 and CCL21 can drive recruitment of CCR7 receptor-expressing immune cells to secondary lymphoid organs.MethodsSince NK-92 cells do not spontaneously express CCR7, clinical-grade aNK cells were transfected with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR and a high-affinity CD16 Fc receptor.ResultsCCR7-engineered CD19 t-haNK showed significant migration in vitro toward K562 cells engineered to secrete CCL19. This observation was confirmed in a NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mouse model in which subcutaneous tumors of CCL19-expressing K562 cells displayed a higher number of infiltrating CCR7_CD19 t-haNK cells than CCR7-negative CD19 t-haNK cells. In NSG mice inoculated either intravenously or subcutaneously with CCL19-secreting Raji cells, treatment with CCR7_CD19 t-haNK improved survival and tumor control compared with CD19 t-haNK or vehicle.ConclusionsExpression of CCR7 receptor by off-the-shelf t-haNK cells improves their homing toward lymph node chemokines both in vitro and in vivo, resulting in superior tumor control.     

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC.

     

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19⁺ tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19⁺ human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2^−/−γc^−/− immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.     

RASAL1 influences the proliferation and invasion of gastric cancer cells by regulating the RAS/ERK signaling pathway

The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promoted apoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.     

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion.MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays.ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion.ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.     

Effect of epiberberine from Coptis chinensis Franch on inhibition of tumor growth in MKN-45 xenograft mice

Background and purposeGastric cancer is one of the major malignancies worldwide. Epiberberine (EPI) is a major alkaloid from Coptis chinensis Franch and the antitumor property of EPI remains poorly understood.MethodThe inhibition on gastric cancer cells was observed by MTT assays and colony formation experiments. The apoptosis, cell cycle, and reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in gastric cancer cells were analyzed by Flow cytometry. The anti-tumor effect of EPI was evaluated with the MKN-45-beraring nude mice, and the potential mechanisms were explored by RNA-seq, qPCR, siRNA silencing and western blotting.ResultsEPI inhibited the proliferation of human gastric cancer cell lines MKN-45 (harboring wild-type p53) and HGC-27 (harboring mutant p53) in a dose dependent manner. EPI induced the apoptosis and cell cycle arrest in these two cell lines, of which MKN-45 cells are more sensitive to EPI than HGC-27 cells. Further experiments indicated that EPI induced the accumulation of ROS and decreased of ΔΨm in MKN-45 cells. The significant differentially expressed genes obtained by RNA-seq were distinctly enriched in the p53 signaling pathway. The apoptosis induced by EPI in MKN-45 cells would be effectively inhibited with the treatment of p53 siRNA and p53 inhibitor PFT-α. Western blotting demonstrated that EPI diminished the expression of Bcl-2 and XIAP, and increased those of p53, Bax, p21, p27, Cytochrome C and Cleaved-caspase 3. Animal experiments confirmed that EPI significantly alleviated tumor growth in MKN-45 xenograft mice via p53/Bax pathway.ConclusionsThese data indicated that EPI could be a novel anti-tumor candidate against MKN-45-related gastric cancer via targeting p53-dependent mitochondria-associated pathway.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Simplified process for the production of anti–CD19-CAR–engineered T cells

Background aimsAdoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells.MethodsT cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days.ResultsThe method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution.ConclusionsWe developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR–transduced T cells to support an ongoing clinical trial.     

Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a “suicide gene” relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20⁺ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.     

Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice

Genetically modified CD8⁺ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer.