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High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

ABSTRACT: Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.

SUBMITTER: Muller S

PROVIDER: S-EPMC7025537 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

Müller Stephan S Bexte Tobias T Gebel Veronika V Kalensee Franziska F Stolzenberg Eva E Hartmann Jessica J Koehl Ulrike U Schambach Axel A Wels Winfried S WS Modlich Ute U Ullrich Evelyn E

Frontiers in immunology 20200124

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeuti ...[more]

PMID: 32117200

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Project description:BackgroundRecent evidence suggests that resistance to CD19 chimeric antigen receptor (CAR)-modified T cell therapy may be due to the presence of CD19 isoforms that lose binding to the single-chain variable fragment (scFv) in current use. As such, further investigation of CARs recognize different epitopes of CD19 antigen may be necessary.MethodsWe generated a new CD19 CAR T (HI19α-4-1BB-ζ CAR T, or CNCT19) that includes an scFv that interacts with an epitope of the human CD19 antigen that can be distinguished from that recognized by the current FMC63 clone. A pilot study was undertaken to assess the safety and feasibility of CNCT19-based therapy in both pediatric and adult patients with relapsed/refractory acute lymphoblastic leukemia (R/R B-ALL).ResultsData from our study suggested that 90% of the 20 patients treated with infusions of CNCT19 cells reached complete remission or complete remission with incomplete count recovery (CR/CRi) within 28 days. The CR/CRi rate was 82% when we took into account the fully enrolled 22 patients in an intention-to-treat analysis. Of note, extramedullary leukemia disease of two relapsed patients disappeared completely after CNCT19 cell infusion. After a median follow-up of 10.09 months (range, 0.49-24.02 months), the median overall survival and relapse-free survival for the 20 patients treated with CNCT19 cells was 12.91 months (95% confidence interval [CI], 7.74-18.08 months) and 6.93 months (95% CI, 3.13-10.73 months), respectively. Differences with respect to immune profiles associated with a long-term response following CAR T cell therapy were also addressed. Our results revealed that a relatively low percentage of CD8+ naïve T cells was an independent factor associated with a shorter period of relapse-free survival (p = 0.012, 95% CI, 0.017-0.601).ConclusionsThe results presented in this study indicate that CNCT19 cells have potent anti-leukemic activities in patients with R/R B-ALL. Furthermore, our findings suggest that the percentage of CD8+ naïve T cells may be a useful biomarker to predict the long-term prognosis for patients undergoing CAR T cell therapy.Trial registrationClinicalTrials.gov : NCT02975687; registered 29 November, 2016. https://clinicaltrials.gov/ct2/keydates/NCT02975687.

Project description:BackgroundChimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering.MethodsTwo second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5+ malignant cell lines, primary CD5+ malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5+ T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo.ResultsBoth BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5+ malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK.ConclusionsAnti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

Project description:Relapsed and chemotherapy-refractory B-cell acute lymphoblastic leukemia (B-ALL) remain significant causes of cancer-associated morbidity and mortality for children and adults. Development of new molecularly targeted treatment strategies for patients with high-risk B-ALL is thus a major preclinical and clinical priority. Adoptive cellular therapy with patient-derived human T cells genetically engineered to express CD19 redirected chimeric antigen receptors (CD19 CAR T cells) is one immunotherapeutic modality that has recently demonstrated remarkable efficacy in re-inducing remission in patients with multiply relapsed B-ALL. Investigative teams at several major cancer centers are currently conducting phase I clinical trials in children and/or adults with relapsed/refractory B-ALL to assess the safety and to identify the maximally tolerated dose of each group's CD19 CAR T-cell product. All groups have reported major clinical toxicities associated with CD19 CAR T-cell treatment, including cytokine release syndrome (CRS) and macrophage activation syndrome, neurologic dysfunction and aplasia of normal B lymphocytes, while CD19 CAR T cells persist in vivo. Toxicities have generally been transient or manageable with supportive care measures. Some patients with life-threatening CD19 CAR T-cell induced sequelae have received anti-cytokine receptor antibody treatment to diminish CRS symptoms and/or corticosteroids to terminate CAR T-cell proliferation. Remarkably, 67-90% of children and adults with B-ALL treated with CD19 CAR T cells in these trials have achieved morphologic leukemia remission with many patients also in molecular remission. The duration of CD19 CAR T cell persistence in vivo has varied appreciably among treated patients and likely reflects differences in the CD19 CAR constructs utilized at each institution. CD19-positive and CD19-negative B-ALL relapses after CD19 CAR T-cell treatment have occurred in some patients. Phase II trials to assess the efficacy of CD19 CAR T-cell immunotherapy in larger cohorts of patients with relapsed/refractory B-ALL are ongoing or planned.

Dataset Information

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

Publications

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

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