Effects of polybrene and retronectin as transduction enhancers on the development and phenotypic characteristics of VHH-based CD19-redirected CAR T cells: a comparative investigation.

Chimeric antigen receptor T cells (CAR T cells) have improved the prognosis of patients with certain hematologic malignancies. However, broader clinical application of this type of therapy is dependent on production protocols. We characterized VHH-based CD19-redirected CAR T cells generated using the transduction enhancers (TEs) polybrene or retronectin. The proliferation rate of activated T cells transduced using polybrene concentrations > 6 mg/mL decreased compared with untreated group. There was a direct relationship between polybrene concentration and transduction efficacy. Moreover, we demonstrated the proliferation of retronectin-transduced T cells increased in a dose-dependent manner (4-20 µg/mL). Whereas, different retronectin concentrations did not mediate a significant increase in T cell transduction rate. Moreover, lentiviral transduction rate was also dependent on the concentration of lentiviruses. At optimized TE concentrations, multiplicity of infection (MOI) of > 10 decreased living T cell transduction rate. Additionally, we demonstrated that CAR T cell phenotype is highly affected by TE type. Naïve T cell differentiation to central memory T cell was observed in the beginning of the expansion process and effector memory T cells became the predominant subset in the second week of expansion. Importantly, retronectin increased the proliferation of CAR T cells alongside medicating higher transduction rates, resulting in more naïve and central memory T cells. We demonstrated that a higher percentage of CAR T cells were generated using retronectin (with a less differentiated phenotype) making retronectin a more effective TE than polybrene for long-term CAR T cell processing in preclinical or clinical studies.

Spinoculation and retronectin highly enhance the gene transduction efficiency of Mucin-1-specific chimeric antigen receptor (CAR) in human primary T cells.

BACKGROUND: Producing an appropriate number of engineered cells is considered as one of the influential factors in the successful treatments with chimeric antigen receptor (CAR) T cells. To this aim, the transduction rate of the viral vectors can play a significant role. In addition, improving transduction rates can affect the success rate of this treatment due to hard-transduced T lymphocytes. RESULTS: In this study, activated T cells were transduced using different transduction methods such as spinoculation, retronectin, polybrene, spinoculation + retronectin, and spinoculation + polybrene after selecting the most efficient transfection method to produce recombinant viral particles containing MUC1 CAR. PEI and lipofectamine with the amount of 73.72 and 72.53%, respectively, showed the highest transfection rates with respect to calcium phosphate (14.13%) for producing lentiviral particles. However, the cytotoxicity of transfection methods was not significantly different. Based on the results, spinoculation + retronectin leads to the highest transduction rates of T cells (63.19 ± 4.45%) relative to spinoculation + polybrene (34.6 ± 4.44%), polybrene (10.23 ± 0.79%), retronectin (10.37 ± 1.85%), and spinoculation (21.11 ± 1.55%). Further, the polybrene (40.02%) and spinoculation + polybrene (48.83% ± 4.83) increased cytotoxicity significantly compared to other groups. CONCLUSION: Improving transduction conditions such as using spinoculation with retronectin can ameliorate the production of CAR-T cells by increasing the rate of transduction, as well as the success rate of treatment.

Generation of CAR-T Cells by Lentiviral Transduction.

CAR-T cell therapy is one of the most successful cell-based therapies. T cells are the most common cells to be genetically modified for cancer therapy, not only because T cells have cytotoxicity but also because they are easily cultured ex vivo and genetically modified with viral vectors. Hence, for nonexperts, T cell engineering is an ideal starting point for mammalian cell engineering or for development of therapeutics. Here, we have described a basic procedure for lentiviral transduction of human primary T cells to generate a CAR-T cell and assays to confirm CAR expression and function.

Clinical value of neutrophil-to-lymphocyte ratio as a predictor of prognosis of RetroNectin®-activated cytokine-induced killer cell therapy in advanced non-small-cell lung cancer.

AIM: To assess the impact of neutrophil-to-lymphocyte ratio (NLR) on time to progression (TTP) and overall survival (OS) and explore the value of NLR as an indicator in patients with non-small-cell lung cancer (NSCLC) treated with RetroNectin®-activated cytokine-induced killer (R-CIK) cells. PATIENTS & METHODS: Using data gathered from a single center between January 2010 and June 2015, 201 patients with stage IIIB or IV NSCLC receiving at least four cycles of R-CIK cell therapy were included. Univariate and multivariate Cox regression analyses were performed to evaluate the associations of NLR with TTP and OS. RESULTS: The pretreatment NLR was correlated with TTP and OS. Multivariate analysis showed that NLR was an independent factor for survival. CONCLUSION: NLR was an independent indicator to predict benefit from R-CIK-based combination therapy.

Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells.

Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells, and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction, while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, this study investigated the effects of retronectin-mediated T-cell activation for CD19-specific CART cell production. Peripheral blood mononuclear cells of healthy donors and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Activation of peripheral blood mononuclear cells was performed by CD3/CD28, CD3/CD28/retronectin, or CD3/retronectin. Interleukin-7 and -15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype, and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naïve-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency, and cytokine production, particularly of CLL patient-derived CART cells. Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of healthy donor-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

Retroviral Transduction of Quiescent Murine Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation, cell and gene therapy applications, and the development of leukemia models for research. Because the hematopoietic progeny carries the genetic information of HSCs and replenishes the blood and immune system, corrective gene transfer into HSCs provides an ideal therapeutic approach for many monogenic hematological diseases and a useful tool for studies of HSC function and blood formation in normal and malignant hematopoiesis. However, the efficiency of gene transfer into HSCs has been limited by several features of viral vectors, viral titer, methods of viral transduction, and the property of stem cell quiescence. In this chapter, we describe the production of retrovirus using murine stem cell virus (MSCV)-based retroviral vectors and purification and transduction of murine HSCs.

The use of RetroNectin in studies requiring in vitro HIV-1 infection of human hematopoietic stem/progenitor cells.

Human immunodeficiency virus (HIV) causes damage, directly or indirectly, to the whole hematopoietic system, including CD34+ hematopoietic stem/progenitor cells (HSPCs). CXCR4-tropic strains of HIV-1 may affect the function of CD34+CXCR4+ progenitor cells either by infecting the cells or modifying the dynamics of more differentiated hematopoietic cells. However, CD34+ cells are known for their resistance to HIV-1 infection in vitro, which restricts any detailed analysis of the impact of HIV on HSPCs. We report the use of RetroNectin, a recombinant fibronectin fragment used for gene transfer with lentiviral vectors, to overcome the limitation associated with CD34+ cell resistance to HIV-1 infection. RetroNectin coating of plates improved in vitro HIV-1 infectivity on human CD34+ cells by 10 fold. This resulted in stable HIV-1 infection for 5 weeks in an OP9-DL1 coculture. These results suggest that RetroNectin may be a useful tool for long-term monitoring of in vitro HIV-infected CD34+ cells.

Frontline Science: Functionally impaired geriatric CAR-T cells rescued by increased α5ß1 integrin expression.

Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5ß1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-ß1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5ß1 integrin expression. Neutralization of α5ß1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5ß1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T.

Tumor Suppressor Analysis in CML.

Retroviral models have tremendously contributed to our understanding of CML development and have been indispensable for preclinical drug testing which facilitated the implementation of a targeted therapy. The retroviral insertion of Bcr-Abl into mice that are genetically depleted for a potential tumor suppressor is a tool to test for a specific gene function in Bcr-Abl disease. Here we describe how to generate a Bcr-Abl retrovirus that is subsequently used for infection of primary murine BM cells, which are genetically depleted for a potential tumor suppressor gene. We will suggest control experiments and outline further methods that are required to allow for assessment of disease development upon tumor suppressor knockout in CML.

Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells are a novel immunotherapy for cancer. To achieve anti-tumor efficacy, these cells must survive, expand, and persist after infusion into patients, functions that are reportedly best achieved by cells with a stem or central-memory rather than effector-memory phenotype. We have developed third-generation CAR T cells specific for the tumor-associated antigen GD2 for use in a phase I clinical trial. We investigated the optimal cell culture conditions for CAR T-cell production, and here we describe the relative effects of 3 activation and cytokine conditions on CAR T-cell expansion, effector function and phenotype. METHODS: Peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 or anti-CD3 and Retronectin. Activated cells were transduced with the CAR-encoding retroviral vector and expanded in either interleukin (IL)-2 or IL-7 and IL-15. Immune phenotype and expansion were tracked throughout the culture, and transduction efficiency, and subsequent GD2-specific effector functions were evaluated by flow cytometry and cytotoxic T lymphocytes assay. RESULTS: CD3/Retronectin stimulation with IL-2 resulted in poorer activation, expansion and Th1 cytokine secretion of CAR T cells than CD3/CD28 stimulation with either IL-2 or IL-7 and IL-15. However, CAR T cells cultured in CD3/CD28/IL7/IL-15 and CD3/Retronectin/IL-2 had superior cytotoxic T lymphocyte activity and a more stem-like phenotype. DISCUSSION: The combination of CD3 and CD28 with IL-7 and IL-15 gave the best balance of CAR T-cell expansion and potent GD2-specific effector functions while retaining a stem/memory phenotype, and these growth conditions will therefore be used to manufacture CAR T cells for our phase I clinical trial.

Effects of RetroNectin on CIK cell proliferation and cytotoxicity / 实用肿瘤学杂志

Objective To investigate the recombinant human fibronectin (RetroNectin,RN)on prolifera-tion,immunological characteristics and cytotoxicity of cytokine -induced killer cells ( CIK) .Methods Peripheral blood mononuclear cells(PBMCs)were cultured in vitro by precoating with RetroNectin (R-CIK group),CD3Ab (C-CIK group),RetroNectin combined with CD3Ab(R+C-CIK group)and traditional method(CIK group) were to generate CIK.The changes of growth rate,characterization,cytotoxicity and apoptosis of CIK were deter-mined by cell counting ,LDH assay and flow cytometry respectively .Results R+C-CIK cell showed a higher proliferation rate than other three groups .The difference was statistically significant (P<0.05);The percentage of CD3/CD56 double positive cells in R +C-CIK and R-CIK group had a higher proportion than those in C -CIK and CIK group at day12 and day15(P<0.05);Cytotoxicity of CIK towards K562 cells in R+C-CIK group and R-CIK group were significantly higher than that in C -CIK and CIK group ,when effector-to-target ratio was 20∶1 and 40∶1(P<0.05).We also found that the cytotoxicity of CIK towards LOVO cells in R +C-CIK group and R-CIK group were higher than that in C -CIK group and CIK group(P<0.05).Conclusion RetroNectin and Anti-CD3-MAb synergistically promote antitumor efficiency of CIKs by increasing proliferation and cyto-toxicity.

Effect of RetroNectin on the Proliferation of CIK Cells and the Possible Mechanisms / 中国肿瘤临床

Objective: To investigate the effect of RetroNectin on CIKs cells and the related mechanisms. Methods: Peripheral blood mononuclear cells (PBMC) were collected from patients and divided into two groups: group Ⅰ and group Ⅱ. Samples in group Ⅰ were seeded into culture flask precoated with RetreNec-tin and CD3mAb to induce CIKs. While samples in group Ⅱ were seeded into common culture flask. The pro-liferation of CIKs was detected by cytometric analysis. The cytotoxic activity of CIKs was determined by LDH assays. The phenotype changes and cell cycle of CIKs were identified by flow cytometry. The apoptosis of cells was detected by Annexin V/PI. Western blot was employed to detect the level of protein Vav1. The CD49d and CD49e were blocked by anti-CD49d and anti-CD49e and the proliferation of cells was tested by cytometric analysis after the blockage. The phenotype changes of cells were identified by flow cytometry after the blockage. Results: RetroNectin enhanced the proliferation of CIKs (P<0.05). Flow cytometric analysis showed that RetroNectin significantly increased the number of CD25+ T cells (P<0.05). RN-CIK was more ac-tive than CIK in killing HCT-8 cell lines in vitro (P<0.05). RetroNectin could block the CIKs at G_1 phase (P<0.05) and resist apoptosis. There was no significant difference in the proliferation between the two groups af-ter the blockage with CD49d and CD49e (P>0.05). The expression of protein Vavl was associated with CD25+T cells. Conclusion: RetroNectin enhances the proliferation of CIKs by influencing the cell cycle, resist-ing apoptosis possibly through the site of CD49d and CD49e, and inducing T cell activation as the second sig-naling through Vav1.

The Effect of Recombinant Fibronectin Fragment (Retronectin) and Cytokines during deltaLNGFR Retroviral Transduction of CD34+ Cells / 대한혈액학회지

BACKGROUND: Hematopoietic stem cells (HSC) remain one of the most promising target cells for gene therapeutic approaches, but is currently limited by a number of issues, including the low gene transfer efficiency. METHODS: We evaluated the effect of recombinant fibronectin fragment (retronectin) and cytokines during retroviral transduction of CD34+ cells and analyzed the characteristics of transduced cells. To rapidly characterize and isolate transduced cells, we used a retroviral vector coding for the truncated form of the low-affinity nerve growth factor receptor (delta LNGFR). RESULTS: The total number of CD34+ cells (1.5~6.3-fold) and the amount of cycling (S+G2/M) cell fractions (2~6-fold) were increased after cytokine prestimulation, especially using thrombopoietin (TPO)-based cytokines. The immunophenotype of CD34+ cells showed no significant differences according to the cytokines. However, CD34+AC133+ cells were more effectively maintained in the presence of TPO. The transduction efficiency of CD34+ cells was significantly increased in the presence of retronectin (6.7+/-2.3% vs 23.1+/-4.4%). Immunomagnetic selection of the transduced cells allowed us to enrich these effectively (6.7+/-2.3% --> 86.3+/-6.5%). Compared with control, delta LNGFR transduction did not influence on the immunophenotype and cloning efficiency of CD34+ cells. Among the delta LNGFR+/-derived colonies, 85.0% were shown to contain an integrated delta LNGFR gene. CONCLUSION: These data show that retronectin and TPO-based cytokines can be used to facilitate retroviral transduction of CD34+ cells. Also, delta LNGFR transduced cells could be rapidly characterized by FACS analysis and effectively enriched by immunomagnetic selection method. Further improvement of transduction conditions for stimulation of HSC proliferation without differentiation and development of new vectors that obviate the requirement for cell division are likely to enhance transduction of primitive HSC.