CD19-positive Acute Lymphoblastic Leukemia Research Articles

Non-Viral Sleeping Beauty Transposon Engineered CD19-CAR-NK Cells Show a Safe Genomic Integration Profile and High Antileukemic Efficiency

Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity. Recently, we and others showed that virally transduced NK cells equipped with a synthetic chimeric antigen receptor (CAR) targeting CD19 induced enhanced killing of acute lymphoblastic leukemia (ALL) cells. Here, we demonstrate for the first time that primary NK cells can be engineered using the non-viral Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-CAR with a safe genomic integration profile and high anti-leukemic efficiency in vitro and in vivo.Methods: Primary NK cells were isolated from PBMCs from healthy donors. SB transposons vectorized as minicircles (MC), which encode either a Venus fluorescent protein or a CD19-CAR together with truncated EGFR (tEGFR) as a marker, were introduced in combination with the hyperactive SB100X transposase into primary NK cells via nucleofection. The genetically engineered NK cells were expanded using IL-15 cytokine stimulation under feeder-cell free conditions. Vector integration sites were mapped by analyzing the genomic region around each insertion site in genomic DNA from long-term cultivated gene-modified NK cells, engineered ether by lentiviral (LV) or SB-based technology. Stable gene delivery and biological activity were monitored by flow cytometry and cytotoxicity of CD19-CAR NK cells against CD19-positive ALL and CD19-negative cell lines.Results:Applying a protocol optimized with respect to nucleofection pulses, time points and plasmid ratios, primary NK cells showed long-lasting Venus expression (up to 50%) upon SB-mediated gene delivery with similar viability as non-treated (NT) NK cells during feeder-cell free ex-vivo expansion using IL-15. Likewise, SB transposon-engineered CD19-CAR NK cells displayed high viability, durable transgene expression (Fig 1 A), and no significant change in the NK cell phenotype profile.Next, we assessed vector integration into genomic safe harbors (GSH). GSH are defined as regions of human chromosomes that fulfill the following five criteria: not ultraconserved, >300 kb away from miRNA genes, >50 kb away from transcriptional start sites (TSS), >300 kb away from genes involved in cancer and outside transcription units. CD19-CAR NK cells generated using SB100X showed a significantly higher frequency of vector integration into GSH compared to LV-transduced CAR-NK cells and a significantly more-close to random nucleotide frequency (computer-generated random positions in the genome map to GSHs; random 43.68%; LV 14.78%, SB100X 23.99%; p<0.05) (Fig 1 B).MC.CD19-CAR NK cells generated with the SB platform demonstrated significantly higher cytotoxicity compared to NT NK cells against CD19-positive Sup-B15 ALL cells after long-term cultivation for two to three weeks and no loss of natural intrinsic cytotoxicity. After 4-hour co-culture, significantly enhanced specific tumor cell lysis was found for MC.CD19-CAR NK cells vs NT NK cells at all effector to target cell ratios (E:T) tested (E:T 20:1 83.88% vs 43.13%; E:T 10:1 75.18% vs 31.32%; E:T 5:1 67.38 vs 32.22%; E:T 1:1 42.54 vs 10.19%; p<0.05) (Fig 1 C). With regard to intrinsic natural cytotoxicity of NK cells, no significant decrease in cell killing was overserved for SB-gene-modified CD19-CAR NK cells compared to NT NK cells against CD19-negative K562 cells (E:T 5:1 83%; p<0.05) (Fig 1 D).Significantly enhanced antitumor potential of SB-generated CD19-CAR NK cells was confirmed in a systemic CD19-positive lymphoma xenograft model (NSG-Nalm-6/Luc) in vivo. After injection of 0.5x10 6 tumor cells per mouse and lymphoma engraftment, animals were treated with a single dose of 10x10 6 SB-modified CD19-CAR NK cells pooled from three different donors with a mean tEGFR/CAR expression of 34%. MC.CD19-CAR NK cell therapy resulted in rapid lymphoma eradication in all treated mice (n=4; p<0.05), whereas mice receiving similar amounts of NT NK cells showed progressive lymphoma growth comparable to untreated control mice (Fig 1 E-F).Conclusion: Taken together, the Sleeping Beauty transposon system represents an innovative gene therapy approach for non-viral engineering of safe, highly functional and relatively cost-efficient CAR-NK cells that may not only be suitable for ALL therapy but also for a broad range of other applications in cancer therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of Donor-Derived CD19.CAR-T Cells in Treating Patients That Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation

Background The Chimeric Antigen Receptor T (CAR-T) cells with strong anti-leukemia role can treat relapsed/refractory CD19-positive acute lymphoblastic leukemia (CD19+-ALL) with good outcome. The allogeneic CAR-T cells receives activation signals from both T cell receptor (TCR) and CAR, which may possess stronger activity in anti-leukemia cells. However, the infusion of allogeneic CAR-T cells may cause graft-versus-host disease, which could limit its application after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It is still unclear that the role of the donor-derived CAR-T cells in treating relapsed patients after allo-HSCT. In this study, the prospective study was performed to investigate the role of donor-derived CAR-T cells on relapsed patients after allo-HSCT. Methods From April 2016 to March 2019, relapsed patients after allo-HSCT with CD19+-ALL and a Karnofsky score greater than or equal to 60 were enrolled in this study. The donor underwent apheresis for mononuclear cells to construct the CAR-T cells. The bone marrow aspiration every month after CART- cells infusion was carried out for the assessment of disease status by follow cytometry. The chimerism was detected every month after CAR-T cells treatment. Results Eighteen patients enrolled in this study. The median number of infused CAR-T cells was 1.825Í106/Kg. Thirteen patients (13/18=72.22%) reached complete remission (CR) after CAR-T cells treatment. Four patients (4/18=22.22%) had ineffectiveness. One patient died from b uncontrolled bleeding because of low platelet. The patients with blast cells &lt;5% had higher CR. The full chimerism achieved after CART- cells treatment for all patients with the decrease of chimerism at the time of relapse. The median time of follow-up was seven months (ranged from three months to twenty-five months). Three patients with decreased CAR-T cells or chimerism was underwent allogeneic hematopoietic stem cell transplantation or relapsed within six months. The other eleven patients were complete remission with full chimerism or the continual proliferation of CAR-T cells without the second allo-HSCT during our follow-up period. Seventeen patients observed cytokine release syndrome in which six patients with degree III-IV. Two patients developed GVHD in skin and intestinal tract. All patients recovered after management. No other severe complications and death were observed. Conclusion Our results showed that the treatment by donor-derived CAR-T cells for relapsed patients after allo-HSCT is safe and effective. No second transplantation was needed for relapsed patients after allo-HSCT with the treatment of donor-derived CAR-T cells that with good chimerism and continual proliferation of CAR-T cells. However, further clinical trials should be performed to investigate this protocol with larger cases. Disclosures No relevant conflicts of interest to declare.

Abstract IA07: Engineering T cells for the immunotherapy for pediatric solid tumors

Abstract Engineering T cells for the immunotherapy for pediatric solid tumorsImmunotherapy with genetically modified T cells holds the promise to improve outcome for pediatric cancer patients who currently cannot be cured. In addition, T-cell therapy has the potential to reduce treatment-related complication for all patients. Cell therapy with T cells, genetically modified to express chimeric antigen receptors (CARs), had dramatic success for pediatric patients with recurrent/refractory CD19-positive acute lymphoblastic leukemia (ALL), resulting in the recent FDA approval of CD19-CAR T cells for recurrent/refractory ALL. In contrast, CAR T-cell therapies thus far have been less successful for pediatric solid tumors. In my talk, I will review current challenges of CAR T-cell therapies for solid tumors, highlighting our clinical studies targeting HER2 (Ahmed et al., JCO 2015 and JAMA Oncol 2017). In addition, I will review genetic engineering approaches that are being developed to improve the efficacy of CAR T cells for pediatric solid tumors. Citation Format: Stephen Gottschalk, Chris DeRenzo, Meena Hegde, Melinda Mata, Phoung Nguyen, Giedre Krenciute, Nabil Ahmed. Engineering T cells for the immunotherapy for pediatric solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA07.

CD19-Targeted Nanodelivery of Doxorubicin Enhances Therapeutic Efficacy in B-Cell Acute Lymphoblastic Leukemia.

Nanomedicine has advanced to clinical trials for adult cancer therapy. However, the field is still in its infancy for treatment of childhood malignancies such as acute lymphoblastic leukemia (ALL). Nanotherapy offers multiple advantages over conventional therapy. It facilitates targeted delivery and enables controlled release of drugs to reduce treatment-related side effects. Here, we demonstrate that doxorubicin (DOX) encapsulated in polymeric nanoparticles (NPs) modified with targeting ligands against CD19 (CD19-DOX-NPs) can be delivered in a CD19-specific manner to leukemic cells. The CD19-DOX-NPs were internalized via receptor-mediated endocytosis and imparted cytotoxicity in a CD19-dependent manner in CD19-positive ALL cells. Leukemic mice treated with CD19-DOX-NPs survived significantly longer and manifested a higher degree of agility, indicating reduced apparent systemic toxicity during treatment compared to mice treated with free DOX. We suggest that targeted delivery of drugs used in childhood cancer treatment should improve therapeutic efficacy and reduce treatment-related side effects in children.