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.
Highlights
Treatment of refractory and relapsed B-cell acute lymphoblastic leukemia (B-ALL) is challenging and relies on therapies such as chemotherapy, monoclonal antibodies, and hematopoietic stem cell transplantation [1]
Natural killer (NK) cells isolated from peripheral blood mononuclear cell (PBMC) by NK cell enrichment or CD3/CD19 depletion were pre-activated for 4 days with low dose IL15 (10 ng/ml) prior to lentiviral transduction comparing two transduction enhancers, afterwards the gene modified NK cells were continuously cultured in interleukin 15 (IL-15) for at least 3 days when phenotyping was performed (Figure 1A)
NK cells modified to express a CD19-specific chimeric antigen receptor-modified (CAR) represent an allogenic alternative to CD19-CAR-T cells for the therapy of relapsed and refractory B-ALL
Summary
Treatment of refractory and relapsed B-cell acute lymphoblastic leukemia (B-ALL) is challenging and relies on therapies such as chemotherapy, monoclonal antibodies, and hematopoietic stem cell transplantation [1]. Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy against relapsed and refractory B-ALL [4,5,6,7,8]. The usage of CAR-T cells is restricted to an autologous setting as allogenic T cells, even if HLA-matched, carry the risk of inducing graft vs host disease (GvHD) [9, 10]. Generation of relevant doses of CAR-T cells for heavily pretreated patients suffering from lymphopenia may be impracticable. An allogenic product for those patients might offer a suitable alternative
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