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We evaluated the administration of ARI-0001 cells (chimeric antigen receptor T cells targeting CD19) in adult and pediatric patients with relapsed/refractory CD19+ malignancies. Patients received cyclophosphamide and fludarabine followed by ARI-0001 cells at a dose of 0.4–5 × 106 ARI-0001 cells/kg, initially as a single dose and later split into 3 fractions (10%, 30%, and 60%) with full administration depending on the absence of cytokine release syndrome (CRS). 58 patients were included, of which 47 received therapy: 38 with acute lymphoblastic leukemia (ALL), 8 with non-Hodgkin’s lymphoma, and 1 with chronic lymphocytic leukemia. In patients with ALL, grade ≥3 CRS was observed in 13.2% (26.7% before versus 4.3% after the amendment), grade ≥3 neurotoxicity was observed in 2.6%, and the procedure-related mortality was 7.9% at day +100, with no procedure-related deaths after the amendment. The measurable residual disease-negative complete response rate was 71.1% at day +100. Progression-free survival was 47% (95% IC 27%–67%) at 1 year: 51.3% before versus 39.5% after the amendment. Overall survival was 68.6% (95% IC 49.2%–88%) at 1 year. In conclusion, the administration of ARI-0001 cells provided safety and efficacy results that are comparable with other academic or commercially available products. This trial was registered as ClinicalTrials.gov: NCT03144583 .

Introduciton Despite impressive preliminary efficacy of CAR-T cells in multiple myeloma (MM), NK cell engineering has emerged as a competitive and safer approach, as they entail an ‘off-the-shelf’ strategy with no graft-versus-host disease. NK-92 is a universal, cheap and fast obtainable cellular therapy previously used in clinical trials. Although modest responses with these cells have been reported in MM, their oncolytic potential can be enhanced by genetic modification. However, there are still reasonable doubts about the efficacy of irradiated NK-92 cells used in clinic. Thus, the aim of our study is to generate a safe and effective CAR NK-92 immunotherapy for MM treatment. Methods NK-92MI cells were transduced with a lentiviral vector expressing NKG2D or BCMA CAR. Single and dual CARs were generated, with same or different combinations of costimulator domains. CAR NK-92MI cells expressing a safety switch were generated by retroviral transduction with an SFGiCasp9.2A.ACD19 construct. All populations were purified by FACS sorting to obtain stable modified effectors. In vitro antitumor activity was analyzed against low and high target ligands expressing MM cell lines: U266, similar expression of both BCMA and NKG2DL; XG-1, BCMAhigh and NKG2DLlow; as well as a BCMA knock-out cell line generated by CRISPR-Cas9 system. For in vivo experiments, NSG mice were intravenously injected with 1×106 U266 ffLuc-GFP cells. 5×106 CAR NK-92MI cells were i.v. administered 48h later, once a week for three weeks. Results CAR NK-92MI cells showed specificity and higher in vitro antitumor activity compared to parental NK-92 cells, as well as lack of hematotoxicity. The combined expression of both NKG2D and BCMA CAR has demonstrated cytotoxic coverage against MM cell lines. In vivo, clinical 10 Gy irradiation dose completely abrogate the efficacy of CAR NK-92 cells in our treatment schedule. Besides, lower irradiation doses are not enough to eliminate NK cells. In order to obtain a safe allogeneic immunotherapy, CAR NK-92MI cells expressing a suicide gene therapy have been generated (99.9% purity) being susceptible to death (near 99% death) upon induction with Rimiducid (AP1903). Currently, iCasp9-CD19 CAR NK-92MI cells are being tested in vivo. Conclusions CAR NK-92MI effectors expressing single and dual CARs have been generated and they all show higher in vitro antitumor efficacy against different MM targets compared to parental NK-92MI cells. In vivo experiments show the inefficacy of irradiated CAR NK-92MI cells as therapeutic strategy in our MM model, leading to the necessity of a combination with a safety switch to ensure an effective and safe off-the-shelf NK immunotherapy for MM treatment.

Background Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed. Methods Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products. Results All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (LILRB1), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell– and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdcscid Il2rdtm1Wjl/SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients. Keywords: chimeric antigen receptor, CD19, leukemia, lymphoma, immunotherapy, 4-1BB, T cell, preclinical studies

In recent years, the relevance of the immune system to fight cancer has led to the development of immunotherapy, including the adoptive cell transfer of immune cells, such as natural killer (NK) cells and chimeric antigen receptors (CAR)-modified T cells. The discovery of donor NK cells' anti-tumor activity in acute myeloid leukemia patients receiving allogeneic stem cell transplantation (allo-SCT) was the trigger to conduct many clinical trials infusing NK cells. Surprisingly, many of these studies did not obtain optimal results, suggesting that many different NK cell parameters combined with the best clinical protocol need to be optimized. Various parameters including the high array of activating receptors that NK cells have, the source of NK cells selected to treat patients, different cytotoxic mechanisms that NK cells activate depending on the target cell and tumor cell survival mechanisms need to be considered before choosing the best immunotherapeutic strategy using NK cells. In this review, we will discuss these parameters to help improve current strategies using NK cells in cancer therapy. Moreover, the chimeric antigen receptor (CAR) modification, which has revolutionized the concept of immunotherapy, will be discussed in the context of NK cells. Lastly, the dark side of NK cells and their involvement in inflammation will also be discussed.

Natural Killer (NK) cells are antitumoral, antiviral and antimicrobial cells. The first antitumor mechanism described for NK cells was the "missing self" recognition which happens between the inhibitory "Killer Immunoglobulin-like Receptors" (KIRs) on NK cells and the HLA-I present in all nucleated cells. Thus, down-regulation or absence of HLA-I in tumor cells leads to "missing self" recognition activating NK cells. As a consequence, NK cells deliver Granzyme-B activating apoptotic cell death. However, NK cells can mediate Granzyme-B and Caspase-independent cell death against tumor cells expressing HLA-I, such as multiple myeloma (MM) cells. The cytotoxic mediators mediating non-apoptotic cell death remain unknown. Mechanisms mediating NK cell antimicrobial activity have been less studied, being recognized the role of Granulysin mediating Caspase independent cell death. Cord blood (CB) derived NK cells (CB-NK) is a clinically applicable strategy for the generation of highly functional NK cells which can be used to treat MM and potentially other haematological malignancies. We have demonstrated that CB-NK cytotoxicity against MM is Granzyme B and Caspase-independent. This CB-NK cytotoxicity is transmissible among MM cells, meaning that the initial MM recipient cells (termed primary MM cells or 1ºMM cells)-which formed direct contact with CB-NK are able to transfer lipid-protein vesicles to neighbouring unexposed MM cells (termed secondary MM cells or 2ºMM cells) causing a transmissible cytotoxicity to 2ºMM cells, which have not been in direct contact with CB-NK. We aimed to determine the key cytotoxic mediators transferred from CB-NK to 1ºMM and afterwards to 2ºMM cells that mediate secondary cytotoxicity. To do that, we performed TRANS-SILAC proteomics to determine proteome trafficking between CB-NK and MM cells. TRANS-SILAC proteomics demonstrated increased cell-cell communication between MM cells after CB-NK exposure, with secondary communication between MM cells to transfer the previously received cytotoxic CB-NK material. This secondary transfer represents a dilution of the CB-NK cytotoxic material. At the same time, MM cells transferred their own proteome to neighboring MM cells. Histones were among the CB-NK transferred proteins to 1ºMM, and 2ºMM cells. We selected the Histone H2AZ variant 1 (H2AZ) for validation. Time lapse in vitro confocal microscopy with CB-NK over-expressing H2AZ confirmed dynamic transfer of H2AZ from CB-NK to MM cells. Transfer occurred through vesicles and large intercellular structures. Extracellular DNA staining confirmed that these structures with H2AZ were accompanied by DNA. At the extracellular level, Histones exhibit significant antimicrobial activity, being the main antimicrobial effectors of neutrophils. The multiple positive charges and hydrophobic residues present in Histones allow them to bind to and invade negatively-charged cell membranes which are present in both microbial pathogens and tumor cells and interact with the DNA. We determined the cytotoxic role of H2AZ. H2AZ over-expression caused Caspase independent cell death in four different MM cell lines (p Disclosures No relevant conflicts of interest to declare.

In previous work we have demonstrated that CB-NK exert a cytotoxicity towards MM cells which involves lipid-protein vesicle transfer, which it is secondarily transferred from recipient MM cells to neighboring MM cells. This transmissible cytotoxicity causes a 5-7% of neighboring MM cell death (CDD 2015;22(1):96-107). However, this transmission between MM cells could have a diluent effect of the CB-NK cytotoxicity which could be also a tumor cell survival mechanism. To further analyze this mechanism, we determined the proteins transferred between cells and the role of lipids in this transfer. We performed TRANS-SILAC proteomics using two different approaches: 1) Labeling CB-NK cells with heavy amino-acids (hAA) to identify both CB-NK proteins transferred to MM (1ºMM) and secondary CB-NK proteins transferred from 1ºMM to neighboring MM cells (2ºMM). 2) Labeling MM cells, before CB-NK contact, with hAA to identify transferred proteins from 1ºMM to 2ºMM cells, and from 1ºMM to CB-NK. We found that 1ºMM cells acquired 9.5% of CB-NK proteins, and that these proteins were transferred to 2ºMM cells. As a consequence, 1ºMM cells diluted the CB-NK proteins from 9.5% to 3.8%; which represented 7.2% of proteins in 2ºMM cells. In the second approach, we observed that, MM cells transferred to neighboring MM cells only 1.9% of MM proteins in resting conditions. However, in the presence of CB-NK, this transfer was increased up to 7.7%. Furthermore, CB-NK cells acquired 7.3% of 1ºMM proteins. These findings demonstrate a secondary CB-NK protein transfer between MM cells, which represents a CB-NK protein dilution content from 1ºMM to 2ºMM, and an increased transfer of 1ºMM proteins to 2ºMM (Fig.1A). Proteomic analysis showed that transferred proteins were involved in FAS signaling, apoptosis, inflammation, chromatin organization, glycolysis, spliceosome, and rRNA metabolic process. Among these proteins we focused on histones and the 14-3-3 family which are associated to both cell death and cell survival. Confocal microscopy confirmed transfer of histones and 14-3-3 proteins. Protein transfer occurred within neutral lipid vesicles-structures. We also observed that MM cells were inter-connected with a much higher number of neutral lipid structures (nanotubes and bigger structures similar to bags) than in K562 cells (Fig. 1B). Thus, we next analyzed the role of these neutral lipids in MM cell-cell communication. The cholesterol synthesis and lipid transport inhibitor U18666A significantly decreased the number of these lipid connective structures between MM cells, and it was toxic for MM cells. Furthermore, breaking the lipid connection between MM cells with U18666A increased CB-NK cytotoxicity (p Last, we analyzed apoptotic levels of 1ºMM cells after co-culture with 2ºMM cells, and the apoptotic levels of the remaining 2ºMM cells. Both populations became early apoptotic, however, after 3-5 days they recovered from early apoptosis, decreasing from 36% of early apoptotic cells to 3%, which was their apoptotic basal levels, and most importantly, without causing cell death, as no difference in the cell number in comparison to control MM cells was observed. Therefore, our data demonstrate that this diluent transmission effect between MM cells might be a new cell mechanism that contributes to tumor cell survival. Importantly, because lipid structures mediate this diluent transmission effect, this tumor cell survival mechanism might be eliminated by using lipid metabolism inhibitors. In conclusion, cell-cell communication increases when MM cells are stressed because of CB-NK presence. Although this causes a transmissible cytotoxicity in a small % of MM cells, a diluent effect also occurs between cells helping MM cells to recover and promoting MM cell survival. Lipid structures play an important role in this cell-cell communication, which may represent a new therapeutic target for MM treatment. Disclosures No relevant conflicts of interest to declare.