The cytotoxic activity of CIK + Blina, combined with the two different CARCIK-CD19 products, were significantly higher against both targets (up to 58% and 70%) in comparison to CIKs alone (18% and 35, respectively), with higher efficacy observed at higher E:T ratios, needlessly to say (Figure 3A andSupplementary Figure S1A)

The cytotoxic activity of CIK + Blina, combined with the two different CARCIK-CD19 products, were significantly higher against both targets (up to 58% and 70%) in comparison to CIKs alone (18% and 35, respectively), with higher efficacy observed at higher E:T ratios, needlessly to say (Figure 3A andSupplementary Figure S1A). CARCIK-MNZ, CARCIK-BG2, and CIK + Blina against Compact disc19+focus on cells, suggesting equivalent efficiency. All effectors shaped an increased number of synapses, activated NFAT and NFkB, and secreted IL-2 and IFN- upon Diprotin A TFA encountering targets. CIK + Blina displayed strongest NFAT and IFN- induction, whereas CARCIK-BG2 demonstrated superior synapse formation. All the effectors have shown therapeutic activity in vivo against the CD19+Daudi tumor model, with CARCIK cells showing a more durable response compared to CIK + Blina, likely due to the short half-life of Blina in this model. Keywords:chimeric antigen receptor, bispecific antibody, cytokine-induced killer, B-cell neoplasia, CAR signaling == 1. Introduction == Cytokine-induced killer cells (CIKs) are CD3+CD56+T lymphocytes expanded in vitro that have cytotoxicity and tumor-homing capacity [1,2,3,4,5], without significant graft-versus-host disease (GvHD) [6,7,8,9]. Allogenic CIKs have been used in several clinical trials to treat hematological and solid cancers [10]. However, CIKs alone have shown therapeutic activity mostly in a low-tumor burden context, underscoring the need for strategies aimed at increasing their cytotoxic activity and tumor specificity. The activity of CIKs can be redirected towards tumor targets by the combination with bispecific antibodies (BsAbs) simultaneously targeting a CIK surface antigen (e.g., CD5 or CD3) along with a tumor antigen, such as the T-cell engager CD3xCD19 blinatumomab [11,12,13,14,15,16,17]. Another approach involves the genetic modification of CIKs to express chimeric antigen receptors (CARs). Several T-cell-based products modified by viral infection with anti-CD19 CARs have been approved for B-cell acute lymphoblastic leukemia (B-ALL) and B-cell non-Hodgkin lymphoma (B-NHL) treatment [18,19,20,21,22,23]. However, manipulation of viral vectors requires high containment levels and extensive quality controls for safety. Moreover, patient-derived T-cells can fail to expand in vitro when derived from heavily pre-treated patients. To overcome these hurdles, another approach, proposed by our collaborators in Monza [24,25], employs allogenic CIKs as effectors and the Sleeping Beauty (SB) transposon system for stable non-viral cell modification. The phase I/IIa clinical trials are demonstrating that CARCIK-CD19 cells (utilizing a third generation anti-CD19 CAR equipped with CD28 and OX40 domains) can expand and persist in vivo in B-ALL patients and achieve anti-leukemic activity without severe toxicities (FT01CARCIK and FT03CARCIK; Eudract n. 2017-000900-38 and 2020-005025-85) [26]. To better define the mechanism of action and relative efficacies of the different approaches available to enhance CIK cell activity, our study aimed at evaluating and comparing the in vitro and in vivo functional activities of CIKs, either combined with soluble CD3xCD19 bispecific antibody blinatumomab (CIK + Blina) or modified with two different anti-CD19 CAR molecules carrying different signaling modules but sharing the same anti-CD19 moiety. Specifically, CAR-MNZ is the same anti-CD19 CAR used by our groups in clinical trials [24], whereas CAR-BG2 recapitulates the Tisagenlecleucel product (Novartis) [27], Diprotin A TFA albeit cloned in a transposon vector. == 2. Materials and Methods == == 2.1. Cell Lines and Primary Cells == The B-cell SAPK lines REH and Daudi, and T-cell lines Jurkat and HuT 78 were maintained in culture in RPMI1640 medium (Euroclone, Wetherby, West Yorkshire, UK) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Euroclone), 2 mM L-Glutamine (Euroclone), and 100 M gentamycin (PHT Pharma, Milano, Italy). Peripheral blood mononuclear cells (PBMCs) from normal donors buffy coats were obtained by Ficoll Hypaque (Cedarlane, Burlington, Canada) gradient centrifugation. Written informed consent was obtained Diprotin A TFA from all participants prior to sample collection and the study protocol was approved from the ethical committee of Bergamo, Hospital ASST Papa Giovanni XXIII (Project: Development of novel strategies to redirect immune cells towards tumors, using bispecific antibodies or CAR, approved on 13 November 2018). == 2.2. Transposon Plasmids == The anti-CD19 CAR-MNZ transposon plasmid expresses the human third generation anti-CD19-CD28-OX40-CD3 CAR under the pTMNDU3 promoter. The CAR coding sequence is flanked by the recognition.