We investigated the cytolytic and mechanistic activity of anti-CD19 chimeric antigen receptor natural killer (CD19.CAR.NK92) therapy in lymphoma cell lines (diffuse large B-cell, follicular, and Burkitt lymphoma), including rituximab- and obinutuzumab-resistant cells, patient-derived cells, and a human xenograft model. CD19.CAR.NK92 therapy significantly increased cytolytic activity at E:T ratios (1:1–10:1) via LDH release and prominent induction of apoptosis in all cell lines, including in anti-CD20 resistant lymphoma cells. The kinetics of CD19.CAR.NK92 cell death measured via droplet-based single cell microfluidics analysis showed that most lymphoma cells were killed by single contact, with anti-CD20 resistant cell lines requiring significantly longer contact duration with NK cells. In addition, systems biology transcriptomic analyses of flow-sorted lymphoma cells co-cultured with CD19.CAR.NK92 revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, and immunoregulatory and chemokine signaling pathways. Furthermore, a 92-plex cytokine panel analysis showed increased secretion of granzymes, increased secretion of FASL, CCL3, and IL10 in anti-CD20 resistant SUDHL4 cells with induction of genes relevant to mTOR and G2/M checkpoint activation, which were noted in all anti-CD20 resistant cells co-cultured with CD19.CAR.NK92 cells. Collectively, CD19.CAR.NK92 was associated with potent anti-lymphoma activity across a host of sensitive and resistant lymphoma cells that involved distinct immuno-biologic mechanisms of cell death.
Subscribe to Journal
Get full journal access for 1 year
only $33.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Boissel L, Betancur M, Lu W, Wels WS, Marino T, Van Etten RA, et al. Comparison of mRNA and lentiviral based transfection of natural killer cells with chimeric antigen receptors recognizing lymphoid antigens. Leuk Lymphoma. 2012;53:958–65.
Boissel L, Betancur M, Wels WS, Tuncer H, Klingemann H. Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells. Leuk Res. 2009;33:1255–9.
Gong JH, Maki G, Klingemann HG. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia. 1994;8:652–8.
Klingemann H. Challenges of cancer therapy with natural killer cells. Cytotherapy. 2015;17:245–9.
Klingemann HG. Natural killer cell-based immunotherapeutic strategies. Cytotherapy. 2005;7:16–22.
Maki G, Klingemann HG, Martinson JA, Tam YK. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematother Stem Cell Res. 2001;10:369–83.
Suck G, Odendahl M, Nowakowska P, Seidl C, Wels WS, Klingemann HG, et al. NK-92: an ‘off-the-shelf therapeutic’ for adoptive natural killer cell-based cancer immunotherapy. Leukemia. 1994;8:652–8.
Tam YK, Martinson JA, Doligosa K, Klingemann HG. Ex vivo expansion of the highly cytotoxic human natural killer-92 cell-line under current good manufacturing practice conditions for clinical adoptive cellular immunotherapy. Cytotherapy. 2003;5:259–72.
Yan Y, Steinherz P, Klingemann HG, Dennig D, Childs BH, McGuirk J, et al. Antileukemia activity of a natural killer cell line against human leukemias. Clin Cancer Res. 1998;4:2859–68.
Swift BE, Williams BA, Kosaka Y, Wang XH, Medin JA, Viswanathan S, et al. Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model. Haematologica. 2012;97:1020–8.
Weitzman J, Betancur M, Boissel L, Rabinowitz AP, Klein A, Klingemann H. Variable contribution of monoclonal antibodies to ADCC in patients with chronic lymphocytic leukemia. Leuk Lymphoma. 2009;50:1361–8.
Boissel L, Betancur-Boissel M, Lu W, Krause DS, Van Etten RA, Wels WS, et al. Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. Oncoimmunology. 2013;2:e26527.
Zhang C, Oberoi P, Oelsner S, Waldmann A, Lindner A, Tonn T, et al. Chimeric antigen receptor-engineered NK-92 Cells: an off-the-shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Front Immunol. 2017;8:533.
Chavez JC, Bachmeier C, Kharfan-Dabaja MA. CAR T-cell therapy for B-cell lymphomas: clinical trial results of available products. Ther Adv Hematol. 2019;10:2040620719841581.
Maude SL, Teachey DT, Porter DL, Grupp SA. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood. 2015;125:4017–23.
Salles G, Barrett M, Foa R, Maurer J, O’Brien S, Valente N, et al. Rituximab in B-Cell hematologic malignancies: a review of 20 years of clinical experience. Adv Ther. 2017;34:2232–73.
Sarkar S, Sabhachandani P, Ravi D, Potdar S, Purvey S, Beheshti A, et al. Dynamic analysis of human natural killer cell response at single-cell resolution in B-cell non-Hodgkin lymphoma. Front Immunol. 2017;8:1736.
Jochems C, Hodge JW, Fantini M, Fujii R, Morillon YM 2nd, Greiner JW, et al. An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele. Oncotarget. 2016;7:86359–73.
Beheshti A, Benzekry S, McDonald JT, Ma L, Peluso M, Hahnfeldt P, et al. Host age is a systemic regulator of gene expression impacting cancer progression. Cancer Res. 2015;75:1134–43.
Ravi D, Beheshti A, Abermil N, Passero F, Sharma J, Coyle M, et al. Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC-dependent cell death in T-cell and Hodgkin lymphoma. Cancer Res. 2016;76:3319–31.
Beheshti A, Neuberg D, McDonald JT, Vanderburg CR, Evens AM. The impact of age and sex in DLBCL: systems biology analyses identify distinct molecular changes and signaling networks. Cancer Inform. 2015;14:141–8.
Vanherberghen B, Olofsson PE, Forslund E, Sternberg-Simon M, Khorshidi MA, Pacouret S, et al. Classification of human natural killer cells based on migration behavior and cytotoxic response. Blood. 2013;121:1326–34.
Hart A. Mann-Whitney test is not just a test of medians: differences in spread can be important. Br Med J 2001;323:391–3.
Assarsson E, Lundberg M, Holmquist G, Bjorkesten J, Thorsen SB, Ekman D, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS ONE. 2014;9:e95192.
Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015;1:417–25.
Ziegler-Heitbrock L, Lotzerich M, Schaefer A, Werner T, Frankenberger M, Benkhart E. IFN-alpha induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3. J Immunol. 2003;171:285–90.
Son YI, Dallal RM, Mailliard RB, Egawa S, Jonak ZL, Lotze MT. Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells. Cancer Res. 2001;61:884–8.
Carnevale G, Carpino G, Cardinale V, Pisciotta A, Riccio M, Bertoni L, et al. Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells. Sci Rep. 2017;7:14419.
Weichhart T, Hengstschlager M, Linke M. Regulation of innate immune cell function by mTOR. Nat Rev Immunol. 2015;15:599–614.
Klingemann HG, Wong E, Maki G. A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood. Biol Blood Marrow Transpl. 1996;2:68–75.
Tonn T, Schwabe D, Klingemann HG, Becker S, Esser R, Koehl U, et al. Treatment of patients with advanced cancer with the natural killer cell line NK-92. Cytotherapy. 2013;15:1563–70.
Tonn T, Becker S, Esser R, Schwabe D, Seifried E. Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92. J Hematother Stem Cell Res. 2001;10:535–44.
Arai S, Meagher R, Swearingen M, Myint H, Rich E, Martinson J, et al. Infusion of the allogeneic cell line NK-92 in patients with advanced renal cell cancer or melanoma: a phase I trial. Cytotherapy. 2008;10:625–32.
Boyiadzis M, Agha M, Redner RL, Sehgal A, Im A, Hou JZ, et al. Phase 1 clinical trial of adoptive immunotherapy using “off-the-shelf” activated natural killer cells in patients with refractory and relapsed acute myeloid leukemia. Cytotherapy. 2017;19:1225–32.
Williams BA, Law AD, Routy B, denHollander N, Gupta V, Wang XH, et al. A phase I trial of NK-92 cells for refractory hematological malignancies relapsing after autologous hematopoietic cell transplantation shows safety and evidence of efficacy. Oncotarget. 2017;8:89256–68.
Oelsner S, Friede ME, Zhang C, Wagner J, Badura S, Bader P, et al. Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma. Cytotherapy. 2017;19:235–49.
Liu E, Tong Y, Dotti G, Shaim H, Savoldo B, Mukherjee M, et al. Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia. 2018;32:520–31.
Omidvar N, Wang EC, Brennan P, Longhi MP, Smith RA, Morgan BP. Expression of glycosylphosphatidylinositol-anchored CD59 on target cells enhances human NK cell-mediated cytotoxicity. J Immunol. 2006;176:2915–23.
Wu N, Zhong MC, Roncagalli R, Perez-Quintero LA, Guo H, Zhang Z, et al. A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education. Nat Immunol. 2016;17:387–96.
Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J Immunol. 1998;161:571–7.
Song G, Cho WC, Gu L, He B, Pan Y, Wang S. Increased CD59 protein expression is associated with the outcome of patients with diffuse large B-cell lymphoma treated with R-CHOP. Med Oncol. 2014;31:56.
Treon SP, Mitsiades C, Mitsiades N, Young G, Doss D, Schlossman R, et al. Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies. J Immunother. 2001;24:263–71.
Woo J, Iyer S, Cornejo MC, Mori N, Gao L, Sipos I, et al. Stress protein-induced immunosuppression: inhibition of cellular immune effector functions following overexpression of haem oxygenase (HSP 32). Transpl Immunol. 1998;6:84–93.
Allen F, Bobanga ID, Rauhe P, Barkauskas D, Teich N, Tong C, et al. CCL3 augments tumor rejection and enhances CD8(+) T cell infiltration through NK and CD103(+) dendritic cell recruitment via IFNgamma. Oncoimmunology. 2018;7:e1393598.
AME, TK, DR supported by TUFTS NIH CTSI pilot funding UL1TR002544, NIH grants 1R33CA223908-01 and R01 GM127714-01A1.
Conflict of interest
Funding in part of this study for provided by Nantkwest to AME, other roles for AME advisory board (with honorarium): Bayer, Seattle Genetics, Affimed, Verastem, Pharmacyclics, Research to Practice, and Physician Education Resource; Research support: Takeda, Seattle Genetics, Merck, NIH/NCI, Leukemia and Lymphoma Society, and ORIEN. YC founder and consultant with financial interests in Oncomics LLC.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Ravi, D., Sarkar, S., Purvey, S. et al. Interaction kinetics with transcriptomic and secretory responses of CD19-CAR natural killer-cell therapy in CD20 resistant non-hodgkin lymphoma. Leukemia 34, 1291–1304 (2020). https://doi.org/10.1038/s41375-019-0663-x
Renaissance of armored immune effector cells, CAR-NK cells, brings the higher hope for successful cancer therapy
Stem Cell Research & Therapy (2021)
Advanced Drug Delivery Reviews (2021)
Trends in Pharmacological Sciences (2021)
Recent advances in chimeric antigen receptor natural killer cell therapy for overcoming intractable hematological malignancies
Hematological Oncology (2021)
Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
Journal for ImmunoTherapy of Cancer (2021)