Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo

IntroductionNatural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary.MethodsIn this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence.ResultsWe demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model.ConclusionTogether with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins

Image and motor behavior for monitoring tumor growth in C6 glioma model.

The primary objective of this study is to monitor tumor growth by using image techniques and behavioral testing through general and specific motor activities (spontaneous movements and gait). Our sample includes male Wistar rats, 2 months old and weighing 250-300 g, that is categorized into three groups: control, sham, and experimental. The experimental group was anesthetized; the C6 cells with luciferase expression that were suspended in a culture medium were implanted into the right frontoparietal cortex of the rats. The sham group received implant only with culture medium without cells. Images and behavioral tests were evaluated at base time and at 7, 14, 21, and 28 days after induced tumor growth analysis. The tumor volume measured by magnetic resonance imaging (MRI) and quantitative bioluminescence imaging (BLI) signal showed a correlation coefficient of r = 0.96. The MRI showed that the mean tumor volume increased by approximately 10, 26, and 49 times according to a comparison of tumor volume on the seventh day with 14, 21, and 28 days, respectively. The quantification of the BLI signal was (4.12 ± 2.01) x 10(8), (8.33 ± 3.12) x 10(8), (28.43 ± 6.32) x 10(8), and (63.02 ± 10.53) x 10(8) photons/s at the seventh, fourteenth, twenty-first, and twenty-eighth day, respectively. After 14 days of tumor induction, both behavioral tests showed significant differences between tumor and sham or control groups. Our study showed a high correlation between MRI and BLI for tumor growth monitoring with complement aspects analysis in tumor volume. In addition, functional behavioral analysis displayed sensitivity to monitor tumor growth, as well as to detect early significant changes between groups, primarily in the tumor group. The results of gait analysis were more sensitive than general motor analysis

Spatial parameters of gait analysis by CatWalk test.

<p>(A–C) SL, stride length (cm); (D–F) mAC, maximum contact area (cm²); (G–I) PP, print positions (cm); (J–L) BOS, base of support (cm) for control, sham, and tumor groups (n = 4 per group) and each of the paws or side paws; (M–O) the basal and 28 days after induction spatial parameters are represented. <i>Abbreviations</i>: RF, right forepaw; RH, right hindpaw; LF, left forepaw; LH, left hindpaw; LS, left side; RS, right side; FP, forepaw; HP, hindpaw.</p

Estimated mean and 95% confidence interval of each group for spontaneous motor activity analysis.

<p>Estimated mean and 95% confidence interval of each group for spontaneous motor activity analysis.</p

Temporal parameters of gait analysis by CatWalk test.

<p>Stand (A–C), step cycle (D–F), cadence (steps/s) (G), duration (H), for control, sham, and tumor groups (n = 4 per group) and each of the paws or side paws. Abbreviations: s, seconds; RF, right forepaw; RH, right hindpaw; LF, left forepaw; LH, left hindpaw.</p

Dynamic parameters for gait analysis by CatWalk test.

<p>(A) Animal preview and automatic analysis of paws classification (RF, right forepaw in blue color; RH, right hindpaw in pink color; LF, left forepaw in yellow color; LH, left hindpaw in green color); (B) footprint spatial view (cm); (C) footprint timing view (s); (D) 2D footprint intensities measured in arbitrary units (range: 0–255 A.U.); (E) footfall patterns; (F) 3D footprint intensities (range: 0–255 A.U.); and (G) footprint dimensions of each paw (cm²). <i>Abbreviations</i>: PP, print positions; BOS, base of support; SL, stride length; SC, step cycle; mCA, maximum contact area; s, seconds; cm, centimeters.</p

The experiment-timeline design.

<p>The picture of the rat head represents the parameters used for tumor induction: 10µL of C6Luc solution were administered during 10 min, according to the following coordinates: AP 2 mm; LL 2 mm; D 2.5 mm from bregma. The experimental groups were divided as control, sham and tumor. <i>In vivo</i> experiments comprised behavioral tests, BLI, and MRI analysis, using the same animals of each group to perform all timepoints (X) (4 rats per group, totalizing 12 rats). <i>Ex vivo</i> experiments comprised BLI and histological analysis. A new group of 48 rats was used (4 rats per each group and each time point) until the 21st day after the induction (O). 1 rat per group/time was used in the BLI analysis, and the same group of rats was used in the BLI analysis to complete the number of 4 rats per group/ time used for histological analysis. The “X” into the red square represents that the same 12 animals of the in vivo experiments were used in the last time point of the ex vivo experiments (28th day).</p

Spontaneous locomotor analysis by Actimeter.

<p>The following parameters were analyzed in slow (S) and fast (F) thresholds: (A–B) Horizontal movement (MOV), (C–D) stereotypic movements (STE), and (E–F) rearing (REA) or vertical movement. Red line: tumor group (n = 4), blue line: sham group (n = 4), and black line: control group (n = 4).</p

Estimated mean and 95% confidence interval of each group in some temporal parameters of gait analysis.

<p>Estimated mean and 95% confidence interval of each group in some temporal parameters of gait analysis.</p