NK cells enhance CAR-T cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cell fitness

1 BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access NK cells enhance CART cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CART cell fitness Mireia Bachiller,1 Lorena PerezAmill,1 Anthony Matthew Battram ,1 Sebastian Ciro Carné,1 Amer Najjar,2 Els Verhoeyen,3,4 Manel Juan ,5,6 Alvaro UrbanoIspizua,7,8 Beatriz MartinAntonio 9 To cite: BachillerM, PerezAmillL, BattramAM, etal. NK cells enhance CART cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CART cell fitness. Journal for ImmunoTherapy of Cancer 2021;9:e002866. doi:10.1136/ jitc-2021-002866 ►Additional supplemental material is published online only. To view, please visit the journal online (http:// dx. doi. org/ 10. 1136/ jitc2021002866). MB and LPA contributed equally. Accepted 28 July 2021 For numbered affiliations see end of article. Correspondence to Dr Beatriz MartinAntonio; beatriz. antonio@ quironsalud. es Original research © Author(s) (or their employer(s)) 2021. Reuse permitted under CC BYNC. No commercial reuse. See rights and permissions. Published by BMJ. ABSTRACT Background Chimeric 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 nonHodgkin’s lymphoma (NHL) and multiple myeloma (MM), present lower persistence of CART cells that might be due to decreased fitness of T cells acquired with aging. Moreover, cord blood derivedNK cells (CBNKs) and CARNK cells derived from CBNK can be used ‘offtheshelf’ 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 CART cell administration. In this setting, NK cells demonstrate an important immunoregulation of T cells that could be exploited to enhance the efficacy of CART cells. Methods A combinatorial treatment based on either CART and CARNK cells or CBNK and CART 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. Results We show that CARNK cells derived from CBNK are only effective at high doses (high E:T ratio) and that their activity rapidly decreases over time in comparison with CART cells. In comparison and to exploit the potential of ‘offtheshelf’ CBNK, we demonstrate that a low number of CBNK in the CART cell product promotes an early activation of CART cells and their migration to MM cells leading to enhanced antiMM efficacy. Moreover, cytokines related to CRS development were not increased, and importantly, CBNK enhanced the fitness of both CARpos and CARneg T cells, promoting lower levels of exhaustion and senescence. Conclusion This study demonstrates a relevant immunoregulatory role of CBNK collaborating with CART cells to enhance their antitumor activity. A novel and different approach to consider in CART cell immunotherapy studies is presented here with the goal to enhance the efficacy of the treatment. INTRODUCTION In recent years, adoptive cell immunotherapy approaches, particularly administering chimeric antigen receptor (CAR)- modified T cells, have been established as new methods of treatment for patients with hematological malignancies.1 To date, most clinical studies in patients with B cell malignancies administer CART cells in an autologous setting.2 3 However, there is a proportion of patients where the production of CART cells results in an insufficient amount required for the treatment, and unfortunately, patients do not receive the therapy. For this type of patient, a universal source of immune cells, such as CARmodified natural killer (NK) cells, are becoming an attractive option. However, whereas CARNK cells have demonstrated superiority over unmodified NK cells in preclinical studies4 5 and safety in clinical studies,6 their efficacy is still lower than that of CART cells.1 7 Specifically, CART cells directed to CD19 (CART19) have achieved outstanding responses in acute lymphoblastic leukemia (ALL) and nonHodgkin’s lymphoma (NHL) patients,3 8–10 and Bcell maturation antigen (BCMA) has emerged as the most promising target for CART cells used to treat multiple myeloma (MM) patients.11–16 However, whereas pediatric ALL patients treated with CART19 cells maintain durable responses that correlate with CART cell persistence,3 NHL patients present poorer responses.10 Moreover, in MM patients, CARTBCMA cells do not persist long, and patients end up relapsing despite achieving initial complete responses.15 16 These findings indicate the need to improve CART cell persistence and efficacy in NHL and MM patients to avoid relapses. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 2BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access Cord bloodderived NK cells (CBNKs) have been established as a source to obtain ‘offtheshelf’ NK cells for the treatment of cancer patients.17 18 CARNK cells obtained from CBNK have demonstrated improved antitumor activity compared with unmodified CBNK5 and have also demonstrated safety in patients.6 19 We previously observed that when CBNK come into contact with MM tumor cells, they secrete a variety of proinflammatory molecules that enhance the formation of tumor/immune cell clusters, bringing T lymphocytes and other immune cells into close contact and enhancing the antitumor activity of T lymphocytes.20 Therefore, we hypothesized that NK cells, either CBNK or CARNK cells, would enhance the efficacy of CART cells leading to more durable responses. Here, we first confirmed that at low immune cell numbers, CART cells are superior to CARNK cells. Then, to enhance the efficacy of CART cells, we designed a combinatorial treatment based on CART cells with either CARNK cells or CBNK. Two CARs directed against CD19 (ARI0001)2 and BCMA (ARI2h),14 which were previously developed by us and which have been administered to patients, were employed. We demonstrate that a combinatorial treatment for NHL with CART19 and CARNK19 cells does not improve the efficacy compared with the same total number of CART19 cells alone, suggesting that there is a minimum number of CART cells that is critical for the antitumor efficacy. However, a combinatorial treatment based on CART cells with the addition of half the dose of CBNK enhances the antitumor efficacy in a model of MM and ARI2h cells. This beneficial impact of CBNK over ARI2h cells is initiated at incredibly early time points of tumor/immune cell contact where CBNK facilitate earlier activation and enhanced migration of ARI2h cells to tumor cells. These events impact on the fitness of ARI2h cells in the long term by ameliorating exhaustion levels and the emergence of markers related to immunosenescence in ARI2h cells. METHODS Donors of immune cells CBNK and peripheral blood T cells were obtained from the Banc de Sang i Teixits de Barcelona (BST) from either cord blood (CB) or peripheral blood from healthy donors after obtaining informed consent and being approved by the BST. Tumor cell lines Ramos, RPMI8226, U266 and K562 cell lines were purchased from American Tissue Culture Collection (Manassas, Virginia, USA). ARP1 cell line was kindly provided by Multiple Myeloma Research Center (Little Rock, Arkansas, USA). RPMI8226, K562 and ARP1 were cultured in RPMI with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Pen/Strep) and U266 with 15% FBS. K562based artificial antigenpresenting cells expressing membranebound Interleukin (IL)-21 (K562mb2141BBL feeder cells) were cultured in RPMI with 10% FBS and 1% Pen/Strep. Tumor MM cell lines were modified to express GFPFireFlyLuciferase (GFPFFLuc) using the plasmids pLVMSCV_LucT2AGFP (kindly provided by Amer Najjar) coding for GFPFFLuc, and pMD2.G and psPAX2 coding for VSVG and gag/ pol, respectively. Mycoplasma testing in cell lines was performed every 2 months. Lentivirus production for CAR-T and CAR-NK cells For CART cells, either pCCLEF1aCAR19 or pCCLEF1aCARBCMA with packaging plasmids pMDLgpRRE, pRSVRev and envelope plasmid VSV were used. For CARNK cells, pCCLMSCVCAR19 with pMDLgpRRE, pRSVRev and envelope plasmid BaEVTR (provided by Dr Els Verhoeyen) were used for production of virus particles. Production of virus particles to transduce T cells was done in HEK293T cells using the same protocol previously described by us.14 Production of virus for CARNK cells was done in HEK293T cells that were transfected with transfer vector pCCLMCSVCAR19 with pMDLgpRRE, pRSVRev and BaEVTR plasmid. JetPEI (PolyPlus Transfection) was used as a transfection reagent. Transfection was performed using NaCl and JetPei following the manufacturer’s protocol. Viral supernatants were collected at 48 hours and concentrated with LentiX Concentrator (ClonTech) following the supplier’s protocol and stored at −80°C until use. CB-NK expansion K562mb2141BBL feeder cells were used to expand untransduced NK cells. They were irradiated at 100 Gy and added at a feeder cell:CBNK ratio of 2:1. CBNK were left to expand for 14 days, with new feeder cells added on day 7. T cells and CB-NK transduction T cells were obtained from buffy coats by Ficoll and magnetic T cell depletion (Miltenyi Biotec). T cells were expanded in Click’s media (50% RPMI, 50% Click’s (Irvine Scientific), 5% human serum, 1% Pen/Strep), activated with Dynabeads Human TActivator CD3/CD28 (Thermo Fisher Scientific) and IL2 (100 IU/mL) every other day. Experiments were performed after 8–10 days of T cell expansion. Viral transduction was performed after 48 hours of the expansion at a MOI of 10. NK cells were selected from CB units by Ficoll and magnetic NK cell depletion (Miltenyi Biotec). To produce CARNK (ARI3 cells), NK cells from a CB unit were expanded with NK MACS Medium (Miltenyi Biotec), 1% NK MACS Supplement, 5% human serum, IL2 (500 IU/mL) and IL15 (140 IU/mL). On day 5, CBNK were transduced at a multiplicity of infection (MOI) of 5 with Vectofusin1 (10 mg/mL) following Vectofusin1 protocol (Miltenyi Biotec). Cells were washed 6–24 hours after transduction. On day 7, ARI3 cells were coexpanded with feeder cells adding IL2 (400 IU/mL) every other day for the next 7 days. Of note, throughout the manuscript, ‘ARI3 cells’ Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 3 BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access refers to CARNK cells and CBNK refers to untransduced NK cells. In vivo models Immunodeficient NSG mice were purchased from The Jackson Laboratory. Mice used for experiments were of the same sex and 8–12 weeks old. Mice were irradiated on day 1 at a dose of 2 Gy in a biological radiator with two sources of 137 Cs (J.L. Shepherd, model MARK 1, 30 serial number 1199) and received itravenous tumor cells modified to express GFPFFLuc on day 0. Different models of tumor burden were created for NHL depending on the dose of NHL cells administered. Thus, mice received 1×105, 3.5×105, or 5×105 NHL cells to create the low, medium, or high tumor burden models. Then, they received immune cells on day 7 (for NHL) or day 14 (for MM). The disease was followed weekly by bioluminescence as previously described.20 Bone marrow (BM) and spleen were harvested when mice were euthanized to analyze the presence of CART cells and MM cells by flow cytometry. Cytotoxicity assays Luciferase killing assays were performed by coculturing target tumor cells expressing GFPFFLuc with effector cells tested in each case in a whitewalled 96well plate. Dluciferin (20 mg/mL) was added 15 min prior to the bioluminescence reading in a Synergy HT Plate Reader (BioTek). The percentage of remaining tumor cells was calculated as (luminescence of sample/luminescence of tumor cells alone) ×100. Cytokine production Interferon Gamma (IFNγ), tumor necrosis factor alpha (TNFα) and IL2 were quantified by ELISA (ELISA MAX Deluxe Set, Biolegend) following the manufacturer’s protocol. Luminex assays were conducted with ProcartaPlex multiplex immunoassay kit (Thermo Fisher Scientific). Confocal microscopy RPMI8226 cell line modified to express GFP was cocultured with CART cells stained with Cell Tracker Blue CMAC Dye (Thermo Fisher Scientific). Either CBNK or nontransduced T cells (UT) were stained with CellTracker Deep Red Dye (Thermo Fisher Scientific) and added to the previously mentioned coculture. Images were acquired using a Leica SP5 microscope. 405, 488 and 633 lasers were used for excitation. For time lapse experiments, in vivo image acquisitions were performed every 30 s for 14 hours. Challenges ARP1GFPFFLuc (MM) cells were cocultured with CART cells at an effector:target ratio 0.5:1. In the condition with CBNK, CBNK were added at half the amount of CART cells, leaving a final ratio of CART:MM(:NK) 0.5:1(:0.25). The remaining tumor cells were assessed by fluorescent microscopy until no GFP was observed, and thus the challenge was over. T lymphocytes were then stained, and the phenotype was compared with that seen in unstimulated T cells (day 0) and CART cells before exposure to tumor cells. Flow cytometry CAR expression was detected with either a recombinant BCMAFc protein (Enzo Life Sciences) or a recombinant CD19Fc protein (Thermo Fisher Scientific) and an antihuman IgG FcBV421 (Biolegend, clone: M1310G05). For T cell phenotyping, PD1APC (clone: J105), TIM3FITC (clone: F382E2), TIGITPerCPCy5.5 (clone: A15153G), LAG3PE (clone: 11C3C65), CXCR3AlexaFluor 488 (Clone 1C6/CXCR3), CCR7PerCPCy5.5 (clone: 150503), CD45RAAPC (clone: HI100), CD27PE (clone: MT271), CD28FITC (Clone CD28.2), CD3PE (clone SK7), CD4APCH7 (clone L200) and CD8PECy7 (clone: RPAT8) antibodies were used. For NK cell phenotyping, CD16Alexa488 (Clone 3G8), NKP30PeCy7 (Clone P3015), NKG2DAPCCy7 (Clone 1D11), NKG2APacificBlue (Clone S19004C), NKG2CPe (Clone S19005E), KIR2DL1PE (Clone HPDM1), KIR2DL2/ L3FITC (Clone DX27), KIR3DL1BV421 (Clone DX9) and KIR2DL1/S1/S3/S5PerCp Cy5.5 (Clone HPMA4) were all from Biolegend. CD56APC (Clone REA196, Miltenyi) was used for NK cells. Staining was performed in FACS buffer. For T cell determination in BM or spleen at in vivo endpoint, mouse FcR blocking reagent (Miltenyi) was used. All experiments were read on a FACS Canto II (BD Biosciences) and analyzed with FlowJo software (Tree Star, Eugene, Oregon, USA). Graphs and statistical analysis Data were presented using GraphPad Prism software, and statistical analysis was performed using SPSS software V.20 using Student’s ttest to compare between two groups. RESULTS CAR-NK cells demonstrate higher in vitro activity than CAR-T cells only at high E:T ratios, requiring IL2 to maintain efficacy at low E:T ratios, which can be provided by CAR-T cells We first optimized a method to obtain a high number of CARNK cells directed against CD19 (ARI3 cells) starting from CBNK. We used a CAR directed against CD19 with 41BB as costimulatory domain previously produced by our institution termed ARI0001 (ARI1 from now on).21 Plasmids coding for ARI1 and ARI3 differed in the promoter (online supplemental figure 1A) as CBNK presented higher transduction efficiencies using the MCSV promoter instead of the EF1a promoter. Another difference between ARI1 and ARI3 was the packaging plasmid that was used in the transduction. The use of a Vesicular Stomatitis Virus (VSV) packaging plasmid for virus production led to extremely low transduction efficiencies of CBNK. Interestingly, Bari et al22 demonstrated that exchanging VSV for Baboon Envelope (BaEV) packaging plasmid Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 4BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access improved efficiencies of NK cell transduction. Therefore, VSV and BaEV were compared confirming improved efficiency of CAR transduction with BaEV (online supplemental figure 1online supplemental figure 1B). Then, two different expansion methods were compared with BaEV (figure 1A). In the first method (A.1), CBNK were expanded with IL2 and IL15 over 5 days. On day 5, CAR transduction was performed. On day 7, K562mb2141BBL feeder cells were added to CBNK, and they were left to expand until day 14 with the addition of IL2 every other day. In the second method (A.2), CBNK were expanded with IL2 and feeder cells for 5 days. On day 5, CAR transduction was performed. On day 7, CBNK were left to expand until day 14 with the addition of fresh feeder cells and IL2 every other day. Method A.1 achieved a higher percentage of ARI3 + cells than method A.2 (figure 1B), and therefore, method A.1 was selected for all ARI3 cell productions. The antiNHL activity of both CART cells and CARNK cells directed against CD19 (ARI1 for CART cells and ARI3 for CARNK cells: online supplemental figure 1A was compared in vitro. At higher effector:target (E:T) ratios, ARI3 cells presented higher antitumor activity than untransduced CBNK or ARI1 cells (figure 1C). However, at low E:T ratios, ARI3 efficacy was significantly lower to that of ARI1 cells (figure 1D), being this difference even more pronounced at longer times (24 hours vs 48 hours). Lack of efficacy of NK cells at longer times might reflect their requirement for IL2, which is not present in these assays. Therefore, we confirmed that addition of exogenous IL2 improved the in vitro efficacy of ARI3 cells, especially at later time points (figure 1D). In terms of cytokine production, at higher E:T ratios, ARI3 cells produced a higher amount of IFNγ than CBNK, with this production being increased further with the addition of IL2. However, production of IFNγ by ARI3 cells with and without IL2 was always lower than that of ARI1 cells (figure 1E). Regarding TNFα, this cytokine is rapidly produced at early time points and decays fast.14 While ARI3 cells produced higher TNFα levels than CBNK, the highest TNFα production was detected for ARI1 cells (figure 1F). ARI3 cells required IL2 at low E:T ratios to kill tumor cells, and interestingly, CART cells produce high amounts of IL2. Moreover, we previously demonstrated that CBNK can enhance the antitumor activity of untransduced (UT) T cells.20 Therefore, we hypothesized that ARI1 CART cells would boost the activity of ARI3 CARNK cells, and vice versa, which would lead to an overall enhanced antitumor efficacy. Thus, combination of ARI1 and ARI3 cells, where the total number of immune cells was the same as ARI1 cells alone (0.5+0.5 vs 1), demonstrated increased antiNHL activity (figure 1G) without impacting in higher IFNγ (figure 1H) and TNFα production (online supplemental figure 1C). Of note, when the total number of immune cells combining ARI1 and ARI3 was double that of ARI1 cells alone (1+1 vs 1), a higher antiNHL activity and IFNγ production (figure 1G and H) was observed with hardly any changes for TNFα (online supplemental figure 1C). Last, longterm in vitro cytotoxicity assays performed with a low E:T ratio demonstrated superiority of this combinatorial treatment (ARI1 +ARI3) versus ARI1 cells alone when the total number of immune cells was the same in both treatments (figure 1I). Of note, IFNγ levels were not increased (figure 1J). CAR-NK cells do not maintain in vivo efficacy and enhance the migration of tumor cells to secondary lymphoid tissues in a model of NHL The impact of IL2 in the activity of ARI3 cells and the combinatorial treatment were further evaluated in vivo in different NHL tumor burden models. In a model of highly aggressive NHL, none of the immune cells administered (CBNK, ARI1 or ARI3) were able to prevent disease progression at all (online supplemental figure 2A and 2B). Of interest, analysis of mice tissues demonstrated that ARI3 cell treatment maintained the bone marrow (BM) with the lowest levels of disease in comparison with untreated mice (online supplemental figure 2C). However, mice treated with either CBNK or ARI3 cells presented a higher percentage of tumor cells in the spleen compared with both untreated and ARI1treated animals (online supplemental figure 2C). In a model of low tumor burden, treatment with either CBNK or ARI3 cells alone was not able to stop disease progression (figure 2A–2C). ARI3 cells maintained the BM free of disease (figure 2D), but as previously observed in the high tumor burden model (online supplemental figure 2C), a high number of NHL cells were detected in the spleen (figure 2D). The addition of exogenous IL2 to ARI3 cells was highly detrimental by causing NHL progression, as likely due to the fact that IL2 also induces B cell proliferation,23 with this group showing a dramatic increase in the number of NHL cells in the BM compared with ARI3 cells without exogenous IL2 and a high number of NHL cells in the spleen (figure 2D). These findings were in agreement with the role of IL2 promoting the presence of B cells in the BM and also in the spleen but to a lesser degree.24 In all cases, the high number of NHL cells in the spleen correlated with splenomegaly (figure 2E). Moreover, NK cells were detected in the spleen in all groups treated with NK cells, so the presence of tumor cells in the spleen was not due to lack of NK cell migration (figure 2F). Treatment with either ARI1 cells alone or combined with ARI3 cells did not demonstrate differences in this model of low tumor burden (figure 2A–2D) as both treatments completely prevented NHL progression. However, higher IFNγ production was observed in the combinatorial treatment at 31 days. No increase was seen at 24 days (figure 2G). Finally, even though, in these two groups, there were low numbers of humanderived T cells in the BM and the spleen (figure 2H), the presence of CAR + cells could be detected in both groups Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 5 BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access Figure 1 CARNK cells demonstrate higher in vitro activity than CART cells only at high E:T ratios, requiring IL2 to maintain efficacy at low E:T ratios, which can be provided by CART cells (see also online supplemental figure 1). (A) Diagram showing two different protocols (A.1 and A.2) used to obtain CARNK cells directed against CD19 (ARI3) from CBNK. (B) Efficiencies of CBNK transduction with both methods shown in figure part A. (C) Cytotoxicity assays of immune cells (untransduced T cells: UT, CART cells against CD19 (ARI1), CBNK and ARI3) against RamosNHL cell line performed at effector:target (E:T) ratio=10:1 at 24 hours and 48 hours. (D) Cytotoxicity assays of immune cells (UT, Ari1, CBNK, ARI3 and ARI3+ IL2) against Ramos performed at different E:T ratios at 24 hours and 48 hours. (E) IFNγ and (F) TNF-α production of the cytotoxicity assays in (D). (G) Cytotoxicity assays at 24 and 48 hours against Ramos at different E:T ratios where immune cells are added either alone UT (1), ARI1 (1), or in combination of ARI1 and ARI3 cells, where the combination could have in comparison with ARI alone, half the dose of ARI1 and ARI3 (0.5+0.5) or the same dose of Ari1 and ARI3 cells (1+1). (H) IFNγ production of the cytotoxicity assays in (G). (I) Cytotoxicity assay versus Ramos cells over 7 days performed at 0.25:1 E:T ratio adding immune cells alone or the combination of ARI1+ARI3 (0.5+0.5) where the total number of immune cells is the same as in the other conditions. (J) IFNγ production of the cytotoxicity assays in (I). *P<0.05. **P<0.0001. Statistic in figure parts D, E and F is performed comparing to ARI3. Statistic in figure parts G–J is performed comparing ARI3 (1) or ARI1+ARI3 (0.5+0.5) to ARI1 (1). CAR, chimeric antigen receptor; CBNK, cord bloodderived NK cells; NK, natural killer. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 6BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access with no significant difference observed between groups (figure 2I). The number of CAR-T cells, but not CAR-NK cells, is critical in the prevention of NHL progression A third in vivo model of NHL with medium levels of tumor burden was performed to find out whether the combinatorial treatment based on ARI1 +ARI3 was better than ARI1 cells alone. In this model of disease, the highest number of ARI1 cells was determinant to prevent disease progression, as groups treated with ARI1 cells alone retained the disease progression for longer time than the combinatorial treatment with half dose of ARI1 cells, leading to a higher survival of the mice (figure 3A–3C). In the peripheral blood (PB), 15 days after CAR treatment, a slight tendency for a higher production of IFNγ was detected for the combinatorial treatment, although it was not significant, and there were no differences in the number of T cells present in the PB, although it must be Figure 2 CARNK cells do not maintain in vivo efficacy and enhance the migration of tumor cells to secondary lymphoid tissues in a model of NHL. (A) In vivo efficacy in a model of low tumor burden of NHL of ARI (1) and ARI3 (1) cells alone or combined where the total number of immune cells is the same (ARI1+ARI3 (0.5+0.5)). The exogenous addition of IL2 was also included (ARI3 (1)+IL2) and treatment with CBNK as control. (B) Quantification of disease progression by weekly bioluminescence imaging and (C) overall survival of the different group of mice shown in figure part A. (D) Flow cytometry of bone marrow (BM) and spleen of mice at the end of the experiment showing the presence of NHL cells. (E) Spleen of mice treated with NK cells (CBNK, ARI3 and ARI3+IL2). (F) Presence of NK cells in BM and spleen of mice treated with NK cells. (G) IFNγ levels detected in mice serum at the time points indicated in the different groups of mice. (H) Percentage of total T cells and (I) CART cells in BM and spleen in mice treated with ARI1 or the combinatorial treatment based on ARI1+ARI3 (0.5+0.5) with the same total number of immune cells. (J) Ramos cell line was used as NHL cells. *P<0.05. Statistic shown in figure parts D and G is performed comparing to CBNK. CAR, chimeric antigen receptor; CBNK, cord bloodderived NK cells; NHL, nonHodgkin’s lymphoma; NK, natural killer. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 7 BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access Figure 3 The number of CART cells, but not CARNK cells, is critical in the prevention of NHL progression. (A) In vivo efficacy in a model of medium tumor burden of NHL of ARI (1) cells alone or combined with ARI3 cells where the total number of immune cells is the same (ARI1+ARI3 (0.5+0.5)). Treatment with untransduced T cells (UT) was added as control. (B) Quantification of disease progression by weekly bioluminescence imaging and (C) overall survival of the different group of mice shown in figure part A. (D) IFNγ levels detected in mice serum at the time points indicated in the different groups of mice. (E) Flow cytometry of bone marrow (BM) and spleen of mice at the end of the experiment showing the presence of NHL cells and (F) of T cells. (G) Flow plot showing the presence of CART cells in BM in mice treated with ARI1 cells. (H) Representative flow plot showing presence of NHL cells based on GFP and CD19 expression in BM in spleen in the different groups of mice. For the group of UT T cells, the mouse with the highest number of GFP+ cells is shown. Ramos cell line was used as NHL cells. *P<0.05. CAR, chimeric antigen receptor; NHL, nonHodgkin’s lymphoma; NK, natural killer; NS, not significant. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 8BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access noted that the combinatorial treatment had half the dose of T cells (figure 3D). Analysis of mice tissues demonstrated that both treatments maintained the BM free of disease. In addition, tumor cells were not detected in the spleens of the ARI1 alone group, but a very low number of NHL cells was detected in the spleen of mice receiving the combinatorial treatment (figure 3E). In both groups, NK cells could not be detected, and the presence of T cells in both the BM and the spleen was extremely low (figure 3F), although CART cells were still present (figure 3G). Of note, mice receiving the combinatorial treatment did not present a clear NHL population in the tissues. However, a population with low expression of GFP and CD19 (figure 3H), suggestive of a dying NHL cell population, was present, which might explain the higher bioluminescence signal detected in the combinatorial treatment (figure 3A and B). A small number of CB-NK improves the antitumor efficacy of CAR-T cells (ARI2h) in a model of MM Our results determined that ARI3 cells presented much lower efficacy than ARI1 cells at low E:T ratios, which translated into low in vivo efficacy (figures 1 and 2). Moreover, the combinatorial treatment based on ARI1 and ARI3 cells showed that the number of ARI1 cells and the starting tumor cell burden was determinant to prevent disease progression (figure 3). Nevertheless, we previously determined that CBNK are able to increase the antitumor activity of UT T cells by bringing UT T cells and MM cells into close proximity.20 Thus, we hypothesized that addition of CBNK to CART cells would increase the antitumor efficacy of CART cells. Of note, patients receiving CART cells receive a previous lymphodepleting treatment based on fludarabine and cyclophosphamide that removes NK cells. Moreover, NK cells represent around 10% of PB leukocytes, a much lower value than for T lymphocytes.25 Thus, we decided to design a treatment that includes NK cells, specifically with half the dose of CBNK with respect to that of CART cells. This hypothesis was tested in a model of MM and humanized CART cells directed against BCMA (ARI2h cells). Addition of CBNK to ARI2h cells increased the antiMM in vitro efficacy. Of interest, this beneficial impact was enhanced when the E:T (ARI2h:MM(:NK)) ratios were lower (1:1(:0.5)) vs 0.5:1(:0.25)) in figure 4A. Moreover, UT T cells and ARI2h cells did not augment tumor killing to the same extent as with CBNK when they were added at the same ratios, demonstrating that the highest beneficial effect occurred with the addition of CBNK (figure 4A), especially at earlier time points (figure 4B). Of interest, IFNγ, TNFα and IL2 levels in the supernatant were not increased with the addition of CBNK, being in some cases even slightly lower (figure 4C). Specifically, IFNγ production by either CD4 + or CD8+ T cells in these coculture assays did not increase when CBNK were present (figure 4D). Furthermore, longterm cytotoxicity assays performed at a very low E:T ratio (1:8), confirmed the beneficial impact of CBNK which even prevented the regrowth of MM cells (figure 4E). Of note, the pool of ARI2h cells that patients receive contains UT T cells. Therefore, we analyzed and confirmed that CBNK also enhanced the antiMM activity of UT T cells (figure 4F). Moreover, at low E:T ratios, the antiMM activity of either UT or CBNK alone was barely detectable and was much higher when both populations were combined (figure 4F). The beneficial impact of CBNK over ARI2h cells was confirmed in an in vivo model for MM in terms of lower disease progression and higher survival (figure 5A–5C). Despite the beneficial impact obtained with CBNK and ARI2h cells, mice treated only with CBNK showed a high proportion of MM cells in the BM and the spleen (figure 5D). Moreover, IFNγ levels and total T cells in the PB of mice 28 days after treatment (figure 5E), and the presence of T cells in the BM and the spleen (figure 5F), did not differ between the ARI2h and ARI2 +CBNK groups. CB-NK promotes the activation of ARI2h cells leading to enhanced migration of ARI2h cells to MM cells NK cells, as part of the innate immune system, perform their activity at earlier time points than T cells,1 which was concordant with the highest impact of CBNK over ARI2h cells observed at early time points (figure 4B). Therefore, we performed timelapse in vivo imaging to visualize differences in the activity of ARI2h cells in the presence or absence of CBNK over a period of 14 hours. As a control, UT T cells were added at the same number and in place of CBNK. In vivo imaging showed that CBNK accelerated the migration of ARI2h cells to MM cells, an effect that was not observed with UT T cells (online supplemental movies, figure 6A–6C). Specifically, CBNK migrate incredibly quickly towards tumor cells (within the first 22 min in figure 6B) leading to enhanced formation of immune/tumor cell clusters. In turn, this promoted the migration of ARI2h cells to MM cells (figure 6C). Moreover, whereas CBNK came into close proximity with tumor cells remarkably quickly, they started to distance from these clusters when tumor cells had already been eliminated (from 382 min onward in figure 6B). In parallel, movements of UT T cells added to ARI2h cells were hardly visible (online supplemental movies, figure 6B). The enhanced migration of CART cells to MM cells due to the presence of CBNK suggested a faster activation of CART cells at earlier time points. Therefore, CD69 expression, a marker of T cell activation, was measured demonstrating a higher activation of CD4 + T cells in the presence of CBNK (figure 6D and E). Furthermore, the secretion of cytokines in these cocultures was analyzed at 6 hours demonstrating a different secretome in the presence of CBNK (figure 6F) and that cytokines involved in T cell migration, such as CCL3 and CCL5, were increased in the presence of CBNK (figure 6G). Of interest, cytokines related to CRS development, such as TNFα, were not increased in the presence of CBNK (figure 6F). Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 9 BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access CB-NK improves the fitness of CARpos and CARneg T cells exposed to MM cells Last, we analyzed how the impact of CBNK on ARI2h cells observed at early time points (figure 6) would influence ARI2h cells exposed to MM cells in longterm challenge assays. These longterm assays were performed at low E:T ratios adding cells highly diluted so that the killing would take longer (figure 7A). These experiments were performed with six different donors for T cells and CBNK from three different CB units. Of interest, the impact of CBNK over the activity of T cells was not related to differences observed in the expression of some NK cell receptors (online supplemental figure 5). We observed that after encountering MM cells, the total number of CARpos and CARneg CD8 + cells was higher in the presence of CBNK, suggesting either a higher expansion or a lower cell death (figure 7B and C). The transition of memory stages of T cells was analyzed after production of ARI2h cells and after challenging to MM cells. In the naïve/stem cell memory (SCM) compartment, a decrease in the proportion of naïve CARneg CD4 and CD8 T cells was observed during the expansion, with no changes observed in the SCM compartment (online supplemental figure 4A). In the central memory (CM) and effector memory (EM) compartments, at day 0, before production of ARI2h cells, there was a lower proportion of CM in comparison with EM T cells in both CARpos and CARneg T cells (figure 7D Figure 4 A small number of CBNK improves the in vitro antitumor efficacy of CART cells (ARI2h) in a model of MM (see also online supplemental figure 3). (A) Cytotoxicity assays against ARP1 (MM cell line) adding as effectors immune cells alone (CBNK and ARI2h) or ARI2h cells combined with half the dose of: CBNK (ARI2h+CBNK), UT T cells (ARI2h+UT) or ARI2h cells (ARI2h+ARI2 hour). Assays were performed at different E:T ratios. (B) Fold change increased killing with the addition of half the dose of CBNK to ARI2h cells of cytotoxicity assay shown in figure part A at the E:T ratio of 0.5:1(:0.25) for CART:MM(:NK). (C) IFNγ, TNFα and IL2 production of the cytotoxicity assays in figure part A. (D) Flow cytometry analysis of IFNγ production by CD4+ and CD8+ T cells at the timepoints shown adding ARI2h:MM(:NK) at (0.25:1(:0.125)) ratio. (E) Cytotoxicity assay versus ARP1 and U266 MM cells over 7 days performed adding ARI2h:MM(:NK) at (1:8(:0.5)) ratio. In parallel, the same condition in the absence of CBNK was performed. (F) Cytotoxicity assays against ARP1 (MM cell line) adding as effectors immune cells untransduced T (UT T) cells combined with half the dose of CBNK (UT+CBNK). As controls, UT T cells, NK or ARI2h cells alone were compared. Assays were performed at different (E:T) ratios. *P<0.05 and **p<0.0001. Statistic in figure parts A and C is performed comparing to ARI2h. Statistic in figure part F is performed comparing UT T cells. CAR, chimeric antigen receptor; CBNK, cord bloodderived NK cell; MM, multiple myeloma; NK, natural killer. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 16 BachillerM, etal. J Immunother Cancer 2021;9:e002866. doi:10.1136/jitc-2021-002866 Open access 52 Lucas F, Pennell M, Huang Y, etal. T cell transcriptional profiling and immunophenotyping uncover LAG3 as a potential significant target of immune modulation in multiple myeloma. Biol Blood Marrow Transplant 2020;26:7–15. 53 Lozano E, Mena MP, Díaz T, etal. Nectin2 expression on malignant plasma cells is associated with better response to TIGIT blockade in multiple myeloma. Clin Cancer Res 2020;26:4688–98. 54 Castella M, Fernández de Larrea C, MartínAntonio B. Immunotherapy: a novel era of promising treatments for multiple myeloma. Int J Mol Sci 2018;19. doi:10.3390/ijms19113613. [Epub ahead of print: 15 Nov 2018]. 55 ZelleRieser C, Thangavadivel S, Biedermann R, etal. T cells in multiple myeloma display features of exhaustion and senescence at the tumor site. J Hematol Oncol 2016;9:116. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2021-002866 on 25 August 2021. Downloaded from https://jitc.bmj.com on 28 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies.