Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

Abstract

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

Keywords: adoptive cellular immunotherapy; chimeric antigen receptor; genetic engineering; human NK cells; lentiviral vectors (LVS); transduction.

Figure 1

Systematic improvement of primary human NK cell transduction. Primary human NK cells were isolated and cultured as described in the body of the manuscript. (A) After 7 to 10 days of expansion, NK cells were transduced with serial dilutions of EGFP-expressing lentiviral particles pseudotyped with BaEV-Rless, BaEV-TR, RD114-TR, VSV-G, or GALV-TM. Three to four days after transduction, EGFP expression was analyzed by flow cytometry. Data are represented as mean ± SEM of four biological replicates. (B) Primary human NK cells were transduced with serial dilutions of EGFP-expressing lentiviral particles pseudotyped with BaEV-Rless, BaEV-TR, or GALV-TM, using Retronectin, Vectofusin, or no transduction enhancer, respectively. Three to four days after transduction, EGFP expression was analyzed by flow cytometry. Data are represented as mean ± SEM of five biological replicates. (C) Primary human NK cells were transduced with serial dilutions of lentiviral particles pseudotyped with BaEV-Rless, expressing EGFP under the control of the wild-type or modified SFFV, the wild-type or optimized EF1α, the hPGK, or the MPSV promoter. Three to four days after transduction, the expression of EGFP was analyzed. From the serial dilutions, samples with gene transfer rates of between 5% and 10% were analyzed for their EGFP expression intensity [mean fluorescence intensities (MFIs)]. Data are represented as mean ± SEM of four biological replicates.

Figure 2

CD34-hinged CAR expression and enrichment in primary human NK cells. Primary human NK cells were transduced with the lentiviral vectors delineated in panels (A, B) encoding BFP in cis with CD34-hinged CD33, CD123, CD19, or Cetux CARs. (B) Schematic representation of CAR construct including CD34-derived C6 hinge for microbead interactions. (C) Three to four days after lentiviral CAR transduction, NK cells were analyzed for BFP and CAR (via CD34-PE staining) expression by flow cytometry. Representative dot plots are shown. (D, E) CD34-hinged CAR NK cells were enriched via magnetic cell sorting using CD34 microbeads and three fractions (before MACS, flowthrough, and after MACS) were analyzed for the content of NK cells expressing BFP by flow cytometry. The histogram blots show representative data. Data are represented as mean ± SEM of at least four biological replicates.

Figure 3

Cytotoxic potency of activated primary human NK cells against acute leukemia. Killing of various leukemic cell lines by activated primary human NK cells was analyzed by co-culture of NK cells and target cells at several ratios. Activated primary human NK cells were incubated with blocking antibodies against activation receptors (as stated) and co-cultured with NOMO-1, MOLM-14, REH, THP-1, CMK, and BV-173 cells at various ratios to determine cytotoxic activity of NK cells. After 16 h of co-incubation at 37°C, the cytotoxicity of the treated and untreated NK cells was analyzed via flow cytometry, and the degree of the cell lysis was analyzed as described in the Material and Methods. The graphs represent mean ± SEM of four biological replicates.

Figure 4

Cytotoxic activity of CAR NK cells against leukemic cells. (A) AML cells NOMO-1 and MOLM-14, and ALL cells REH were analyzed for their surface expression of CD19, CD33, and CD123. (C) Primary pediatric M4 and M5 AML blasts were analyzed for their surface expression of CD33, CD123, and EGFR using conjugated antibodies by flow cytometry. CAR-transduced and microbead-enriched primary human NK cells were blocked as described previously and co-cultured with NOMO-1, MOLM-14, and REH cells (B) or primary pediatric M4 and M5 AML blasts (D) at various effector-to-target cell ratios to determine the specific cytotoxic activity of CAR NK cells. The graphs represent mean ± SEM of at least five (cell lines) or three (AML blasts) biological replicates.

Figure 5

Visualization of the immunological synapses formed between CD19 CAR NK cells and CD19+ REH cells. (A) The immunological synapse between CD19 CAR NK cells and CD19+ REH cells was analyzed by fluorescence microscopy, visualizing the CAR expression (CD34-PE-conjugated antibody, red) on MACS-enriched CAR NK cells (BFP expression, blue) in relation to REH cells (EGFP expression, green). (B) MACS-enriched CD33 CAR NK cells served as negative controls. Contrast settings are uniform for both images for the channels: brightfield, 377/447 and 470/525 nm. The contrast in channel 545/620 nm was adjusted to maximize the visibility of the cell-to-cell contacts.

Figure 6

NK cells rely on endogenous IL15 production for in vivo persistence in NSG mice. (A) Schematic representation of the vector maps of the IL15 constructs used for NK cell transduction. (B) Primary human NK cell preparations consisting of 50% EGFP/IL15 or EGFP/IL15-IL15R and 50% untransduced NK cells (or 100% untransduced cells as control group) were injected into busulfan-preconditioned mice (3 animals per group). On days 5, 10, 20, and 27, NK cell persistence in the bloodstream was analyzed by mouse CD18, human CD45, and CD56 staining as well as EGFP expression. (C) Example of gating strategy showing representative data from day 10. Values indicate mean ± SEM of all three animals. (D) NK cell persistence in the blood including ratios of transduced to untransduced NK cells. Data are represented as mean ± SEM. Significances of p<0.05 are indicated by an asterisk (*).

Figure 7

CAR NK cells rely on endogenous IL15 production for leukemia control in an ALL xenograft model. (A) Schematic representation of vector maps of CAR constructs used for NK cell transduction. (B) Primary human NK cell preparations consisting of 50% BFP/CD19 CAR, IL15/CD19 CAR, or IL15-IL15R/CD19 CAR and 50% non-transduced NK cells were injected 2 days after xenotransplantation of REH^LucEG into busulfan-preconditioned NSG mice (6 animals per group, 7 animals as untreated control). (C) On days 7, 15, 22, and 27, leukemia progression was analyzed by luminescence imaging in a Caliper device. (D) Kaplan–Meier survival curves for the three treatment groups and the untreated control animals. (E) REH^LucEG and (F) NK cell persistence in the bloodstream was analyzed by CD19, CD34, CD45, and CD56 staining as well as EGFP expression by flow cytometry. Data are represented as mean ± SEM. Significances of p<0.05 are indicated by an asterisk (*).