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BackgroundChimeric antigen receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In comparison with pediatric patients, where responses are maintained over many years, older patients, such as those with non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM), present lower persistence of CAR-T cells that might be due to decreased fitness of T cells acquired with aging. Moreover, cord blood derived-NK cells (CB-NKs) and CAR-NK cells derived from CB-NK can be used ‘off-the-shelf’ as immune cells with antitumor properties for the treatment of cancer patients. However, to date, clinical studies have only demonstrated the safety of these therapies but not optimal efficacy. To confront the shortcomings of each therapy, we devised a novel approach consisting of simultaneous (CAR-)NK cell and CAR-T cell administration. In this setting, NK cells demonstrate an important immunoregulation of T cells that could be exploited to enhance the efficacy of CAR-T cells.MethodsA combinatorial treatment based on either CAR-T and CAR-NK cells or CB-NK and CAR-T cells in two models of NHL and MM was performed. Antitumor efficacy was analyzed in vitro and in vivo, and parameters related to early activation, exhaustion and senescence of T cells were analyzed.ResultsWe show that CAR-NK cells derived from CB-NK are only effective at high doses (high E:T ratio) and that their activity rapidly decreases over time in comparison with CAR-T cells. In comparison and to exploit the potential of ‘off-the-shelf’ CB-NK, we demonstrate that a low number of CB-NK in the CAR-T cell product promotes an early activation of CAR-T cells and their migration to MM cells leading to enhanced anti-MM efficacy. Moreover, cytokines related to CRS development were not increased, and importantly, CB-NK enhanced the fitness of both CARpos and CARneg T cells, promoting lower levels of exhaustion and senescence.ConclusionThis study demonstrates a relevant immunoregulatory role of CB-NK collaborating with CAR-T cells to enhance their antitumor activity. A novel and different approach to consider in CAR-T cell immunotherapy studies is presented here with the goal to enhance the efficacy of the treatment.

Background Multiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells. Methods T cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice. Results The addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment. Conclusions Our results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

Simple Summary Here, we review the last pre-clinical and clinical studies published in the last five years where natural killer (NK) cells have been administered as an immunotherapy option for the treatment of cancer patients. We describe studies administering NK cells alone and in combination with monoclonal antibodies that either promote antibody-dependent cell cytotoxicity or block immune checkpoint receptors. We review the use of genetically modified NK cells including chimeric antigen receptor (CAR)-modified NK cells and other modifications that can enhance the anti-tumor activity of NK cells. Moreover, we describe studies related to the antimicrobial activity of NK cells as we believe they demonstrate important lessons that we can learn and apply to improve the anti-tumor activity of NK cells. All these studies are described with the aim to find tips to improve the success of NK cells as an immunotherapy option in cancer patients. Abstract Natural killer (NK) cells are potent anti-tumor and anti-microbial cells of our innate immune system. They are equipped with a vast array of receptors that recognize tumor cells and other pathogens. The innate immune activity of NK cells develops faster than the adaptive one performed by T cells, and studies suggest an important immunoregulatory role for each population against the other. The association, observed in acute myeloid leukemia patients receiving haploidentical killer-immunoglobulin-like-receptor-mismatched NK cells, with induction of complete remission was the determinant to begin an increasing number of clinical studies administering NK cells for the treatment of cancer patients. Unfortunately, even though transfused NK cells demonstrated safety, their observed efficacy was poor. In recent years, novel studies have emerged, combining NK cells with other immunotherapeutic agents, such as monoclonal antibodies, which might improve clinical efficacy. Moreover, genetically-modified NK cells aimed at arming NK cells with better efficacy and persistence have appeared as another option. Here, we review novel pre-clinical and clinical studies published in the last five years administering NK cells as a monotherapy and combined with other agents, and we also review chimeric antigen receptor-modified NK cells for the treatment of cancer patients. We then describe studies regarding the role of NK cells as anti-microbial effectors, as lessons that we could learn and apply in immunotherapy applications of NK cells; these studies highlight an important immunoregulatory role performed between T cells and NK cells that should be considered when designing immunotherapeutic strategies. Lastly, we highlight novel strategies that could be combined with NK cell immunotherapy to improve their targeting, activity, and persistence.

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.

B ackground: ARI0002h is an academic autologous CAR T-cell product with a 4-1BB co-stimulatory domain and a humanized single chain variable fragment targeting BCMA, which is being tested in the CARTBCMA-HCB-01 clinical trial for patients (pts) with RRMM (NCT04309981). A fractionated infusion of 3x10 6 CAR+ cells (ARI0002h)/kg (10/30/60%) was administered after lymphodepletion regimen (LR). A second dose of 3x10 6 CAR+ cells/kg was planned starting at 4 months after the first dose, with or without LR according to persistence of CAR-T in peripheral blood (PB). Clinical results have been submitted in a separate abstract (#147188); here we present the correlative biological studies performed and related to disease response. Methods: ARI0002h production data were obtained and analyzed, including % CAR transduction in the final product (FP) by flow cytometry (FC). PB samples were obtained at inclusion and sequentially after infusion. Bone marrow (BM) samples were obtained at inclusion, at d28, d100 and at 6 months (m), to evaluate BCMA expression on plasma cells (PCs) and minimal residual disease (MRD) by next generation flow (NGF) with 10 -6 sensitivity. Apheresis, FP, and PB/BM samples on d28 were analyzed for T-cell subpopulations and surface markers. Persistence of ARI0002h was evaluated in PB after infusion by PCR. If a second infusion was administered, CART detection after 3 and 7d was also checked. Plasmatic soluble BCMA (sBCMA) was determined by ELISA prior to infusion, at d28 and if a relapse was observed. Human anti-human antibodies (HAHAs) were evaluated at d100 (using FC on transduced HEK cells). Clinical and response data were collected for analysis. Results: Mean BCMA molecules/cell on malignant BM PC at inclusion were 1306 (SD ±889) with non-significant (NS) correlation with response at d100. Thirty-three apheresis were obtained and transduced, with a median manufacturing time of 11d (range 9-14). All FP were successfully obtained at first attempt but one, which could be produced with a second apheresis. The mean % transduction of ARI0002h on autologous T cells was 56% (SD ±25). A higher CD4/CD8 ratio in the apheresis was correlated with achievement of stringent complete response (sCR) (p=0.003); this association was not observed in the FP. Moreover, T-cell subpopulations in the FP did not correlate with response (Figure 1A). A significant increase of PD-1 (p=0.0001), TIGIT (p=0.037) and LAG3 (p=0.003) expression was observed in CD4 CAR-T cells, as well as PD-1 in CD8 (p=0.0001), between the FP and d28 PB paired samples; similar results were observed in BM CAR-T cells at d28, except for LAG3 (Figure 1B). ARI0002h CAR-T cells showed maximum PB expansion at 14d (range 7d - 5m). Among pts with 3 and 6 m follow-up, 54% and 24% had evidence of CAR+ T cells in PB, with a median persistence of 4m (range 2-not reached). Expansion levels by PCR and PB persistence did not correlate with MM response. 22 out of 27 eligible pts (81%) received a 2 nd dose, with a subsequent CART expansion in 10 of 20 (50%) evaluable pts; LR was readministered in only 30% of these 10 pts. B-cell aplasia was observed in all pts concurring with CART expansion (Figure 1C). Also, a decline in B-cell counts was observed in some pts after reinfusion, with no association with response. Patients with detectable sBCMA at inclusion became sBCMA negative in all the subsequent tested samples at d28 and d100. None of these pts have relapsed. 27 pts had MRD assessed at day +100. In 3, the BM was severely hemodiluted and inadequate for MRD testing. Of 24 MRD-evaluable pts, 22 (92%) were MRD-negative (59% were negative at a sensitivity of 1x10 -6, 36% at 1x10 -5 and 5% at 1x10 -4), and 2 were MRD positive (one BCMA positive and another with BCMA undetermined result). Only 1 of 27 (4%) evaluable pts had positive HAHAs at day 100. CAR-T cells were undetectable at day 70 in this patient, but after receiving a 2 nd dose at m4 a short-lasted expansion was observed. Despite that, the patient deepened the response from partial response (PR) (d100) to very good PR (m6). Conclusion: ARI0002h production is fast and feasible. The peak of CART expansion occurs after 14d of infusion and also in some pts after reinfusion, regardless of LR, which suggests that LR may not be mandatory to allow further expansion after booster dose. Responses obtained are deep and construct immunogenicity seems to be low. Figure 1 Figure 1. Disclosures Gonzalez-Calle: BMS, Janssen, Amgen: Honoraria. Cabañas: Sanofi: Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Reguera: Janssen, Kite/Gilead, Novartis: Speakers Bureau; BMS-Celgene, Novartis: Membership on an entity's Board of Directors or advisory committees. Rodríguez-Otero: Regeneron: Honoraria; Celgene-BMS, Janssen, Amgen, Sanofi, GSK, Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosinol: Janssen, Celgene, Amgen and Takeda: Honoraria. Moraleda: Pfizer: Other: Educational Grants, Research Funding; Sanofi: Other: Educational Grants, Research Funding; MSD: Other: Educational Grants, Research Funding; ROCHE: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Takeda: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Sandoz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Educational Grants, Research Funding; NovoNordisk: Other: Educational Grants, Research Funding; Janssen: Other: Educational Grants, Research Funding; Celgene: Other: Educational Grants, Research Funding; Amgen: Other: Educational Grants, Research Funding. Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding. Mateos: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene - Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sea-Gen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria; GSK: Honoraria; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees. Paiva: Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. Fernandez de Larrea: GSK: Honoraria; Takeda: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Honoraria, Research Funding.

Background Multiple myeloma (MM) remains incurable despite the number of novel therapies that have become available in recent years. Occasionally, a patient with MM will develop a light-chain (AL) amyloidosis due to the deposition of amyloidogenic light chains causing organ dysfunction. Chimeric antigen receptor T-cell (CART) therapy has become the most promising approach in treating cancer patients, especially hematologic malignancies. Our institution has developed a second-generation B-cell maturation antigen (BCMA) CART which is currently being tested in a clinical trial for relapsed/refractory MM. Methods A 61-year-old woman diagnosed with an IgA-lambda symptomatic MM in 2014, with several prior lines of treatment presented with edema and significant non-selective albuminuria (24-hour proteinuria of 2626 mg with urinary M-protein of 307 mg, serum albumin 28 g/L) with preserved renal function (creatinine 0.6 mg/dL) and an increase in serum M protein, with a bone marrow (BM) infiltration by 23% plasma cells. There was no evidence of extramedullary disease by PET-CT and no CRAB signs were found. A subcutaneous fat aspiration and a renal biopsy established the diagnosis of systemic AL amyloidosis without cardiac involvement. At this point AL amyloidosis was the main reason to treat the patient, who received a fractioned dose of 3×106/kg BCMA-CAR T cells after lymphodepletion, developing a grade I cytokine release syndrome and treatment-related cytopenias (grade 4 neutropenia and grade 2 thrombocytopenia), with no neurotoxicity. Results On day +28, the patient had already obtained a deep hematologic response with negative measurable residual disease by flow cytometry in the BM. After 3 months, the patient maintained the hematologic complete response and achieved renal response. After 1 year follow-up, the patient remains in hematologic complete remission and renal response with a decrease in proteinuria of 70%. Conclusions Here, we present the first reported case, to our knowledge, of a patient with AL amyloidosis and renal involvement in the course of a MM, successfully treated with CART therapy targeting BCMA. This case suggests that concomitant AL amyloidosis in the setting of MM can benefit from CART therapy, even in patients in which predominant symptoms at the time of treating are caused by AL amyloidosis.

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.

Multiple myeloma (MM) remains incurable despite the number of novel therapies that have become available in recent years. Occasionally, a patient with MM will develop an amyloid light-chain (AL) amyloidosis with organ dysfunction. Chimeric antigen receptor T-cell (CART) therapy has become a promising approach in treating hematological malignancies. Our institution has developed a second-generation B-cell maturation antigen (BCMA)–CART which is currently being tested in a clinical trial for relapsed/refractory MM.We present the first reported case, to our knowledge, of a patient with AL amyloidosis and renal involvement in the course of an MM, successfully treated with CART therapy targeting BCMA. The patient received a fractioned dose of 3×106/kg BCMA–CARTs after lymphodepletion. At 3 months from infusion, the patient had already obtained a deep hematological response with negative measurable residual disease by flow cytometry in the bone marrow. After 12 months, the patient remains in hematological stringent complete remission and has achieved an organ renal response with a decrease of 70% of proteinuria.This case suggests that concomitant AL amyloidosis in the setting of MM can benefit from CART therapy, even in patients in which predominant symptoms at the time of treating are caused by AL amyloidosis.