Document Type : Original Article


1 Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.

2 Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

3 Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

4 School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.

5 Department of Biological Science and Technology, Faculty of Nano and Bio Science and Technology, Persian Gulf University, Bushehr, Iran.


Background: CD38 is highly expressed on multiple myeloma (MM) cells and has been successfully targeted by different target therapy methods. This molecule is a critical prognostic marker in both diffuse large B-cell lymphoma and chronic lymphocytic leukemia.
Objective: We have designed and generated an anti-CD38 CAR-NK cell applying NK 92 cell line. The approach has potential application as an off-the-shelf strategy for treatment of CD38 positive malignancies.
Methods: A second generation of anti-CD38 CAR-NK cell was designed and generated, and their efficacy against CD38-positive cell lines was assessed in vitro. The PE-Annexin V and 7-AAD methods were used to determine the percentage of apoptotic target cells. Flow cytometry was used to measure IFN-γ, Perforin, and Granzyme-B production following intracellular staining. Using in silico analyses, the binding capacity and interaction interface were evaluated.
Results: Using Lentivirus, cells were transduced with anti-CD38 construct and were expanded. The expression of anti-CD38 CAR on the surface of NK 92 cells was approximately 25%. As we expected from in silico analysis, our designed CD38-chimeric antigen receptor was bound appropriately to the CD38 protein. NK 92 cells that transduced with the CD38 chimeric antigen receptor, generated significantly more IFN-γ, perforin, and granzyme than Mock cells, and successfully lysed Daudi and Jurkat malignant cells in a CD38-dependent manner.
Conclusion: The in vitro findings indicated that the anti-CD38 CAR-NK cells have the potential to be used as an off-the-shelf therapeutic strategy against CD38-positive malignancies. It is recommended that the present engineered NK cells undergo additional preclinical investigations before they can be considered for subsequent clinical trial studies.


  1. García-Guerrero E, Götz R, Doose S, Sauer M, Rodríguez-Gil A, Nerreter T, et al. Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab. Leukemia. 2021;35(1):201-14.
  2. Morandi F, Horenstein AL, Costa F, Giuliani N, Pistoia V, Malavasi F. CD38: A Target for Immunotherapeutic Approaches in Multiple Myeloma. Frontiers in Immunology. 2018;9.
  3. Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. The Lancet. 2015;385(9967):517-28.
  4. Markovic U, Romano A, Del Fabro V, Bellofiore C, Bulla A, Parisi MS, et al. Daratumumab as Single Agent in Relapsed/Refractory Myeloma Patients: A Retrospective Real-Life Survey. Frontiers in Oncology. 2021;11.
  5. June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359(6382):1361-5.
  6. Rezvani K, Rouce RH. The application of natural killer cell immunotherapy for the treatment of cancer. Frontiers in immunology. 2015;6:578.
  7. Ebrahimiyan H, Tamimi A, Shokoohian B, Minaei N, Memarnejadian A, Hossein-Khannazer N, et al. Novel insights in CAR-NK cells beyond CAR-T cell technology; promising advantages. International Immunopharmacology. 2022;106:108587.
  8. Wagner DL, Fritsche E, Pulsipher MA, Ahmed N, Hamieh M, Hegde M, et al. Immunogenicity of CAR T cells in cancer therapy. Nature reviews Clinical oncology. 2021;18(6):379-93.
  9. Carlsten M, Childs RW. Genetic manipulation of NK cells for cancer immunotherapy: techniques and clinical implications. Frontiers in immunology. 2015;6:266.
  10. Shin MH, Kim J, Lim SA, Kim J, Kim S-J, Lee K-M. NK cell-based immunotherapies in cancer. Immune network. 2020;20(2).
  11. Romee R, Leong JW, Fehniger TA. Utilizing cytokines to function-enable human NK cells for the immunotherapy of cancer. Scientifica. 2014;2014.
  12. Mullard A. FDA approves second BCMA-targeted CAR-T cell therapy. Nat Rev Drug Discov. 2022;21(4):249.
  13. Ali SA, Shi V, Maric I, Wang M, Stroncek DF, Rose JJ, et al. T cells expressing an anti–B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood, The Journal of the American Society of Hematology. 2016;128(13):1688-700.
  14. Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. New England Journal of Medicine. 2020;382(6):545-53.
  15. Wada F, Shimomura Y, Yabushita T, Yamashita D, Ohno A, Imoto H, et al. CD38 expression is an important prognostic marker in diffuse large B‐cell lymphoma. Hematological Oncology. 2021;39(4):483-9.
  16. Oppezzo P, Navarrete M, Chiorazzi N. AID in chronic lymphocytic leukemia: Induction and action during disease progression. Frontiers in Oncology. 2021;11:753.
  17. Konen JM, Fradette JJ, Gibbons DL. The good, the bad and the unknown of CD38 in the metabolic microenvironment and immune cell functionality of solid tumors. Cells. 2020;9(1):52.
  18. Gao L, Liu Y, Du X, Ma S, Ge M, Tang H, et al. The intrinsic role and mechanism of tumor expressed-CD38 on lung adenocarcinoma progression. Cell death & disease. 2021;12(7):1-13.
  19. Hogan KA, Chini C, Chini EN. The multi-faceted ecto-enzyme CD38: roles in immunomodulation, cancer, aging, and metabolic diseases. Frontiers in immunology. 2019;10:1187.
  20. Deaglio S, Aydin S, Grand MM, Vaisitti T, Bergui L, D'Arena G, et al. CD38/CD31 interactions activate genetic pathways leading to proliferation and migration in chronic lymphocytic leukemia cells. Mol Med. 2010;16(3-4):87-91.
  21. Wo YJ, Gan ASP, Lim X, Tay ISY, Lim S, Lim JCT, et al. The Roles of CD38 and CD157 in the Solid Tumor Microenvironment and Cancer Immunotherapy. Cells. 2019;9(1):26.
  22. Stikvoort A, van der Schans J, Sarkar S, Poels R, Ruiter R, Naik J, et al. CD38-specific chimeric antigen receptor expressing natural killer KHYG-1 cells: A proof of concept for an “Off the Shelf” therapy for multiple myeloma. HemaSphere. 2021;5(7).
  23. Gurney M, Stikvoort A, Nolan E, Kirkham-McCarthy L, Khoruzhenko S, Shivakumar R, et al. CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide. Haematologica. 2022;107(2):437.
  24. Gurney M, Stikvoort A, Nolan E, Kirkham-McCarthy L, Khoruzhenko S, Shivakumar R, et al. CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide. Haematologica. 2022;107(2):437-45.
  25. Sanchez-Martinez D, Allende-Vega N, Orecchioni S, Talarico G, Cornillon A, Vo D-N, et al. Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity against multiple tumors. Theranostics. 2018;8(14):3856.
  26. Andrea AE, Chiron A, Bessoles S, Hacein-Bey-Abina S. Engineering Next-Generation CAR-T Cells for Better Toxicity Management. International Journal of Molecular Sciences. 2020;21(22):8620.
  27. Tomasik J, Jasiński M, Basak GW. Next generations of CAR-T cells-new therapeutic opportunities in hematology? Frontiers in Immunology. 2022;13:1034707.
  28. Zhao X, Yang J, Zhang X, Lu X-A, Xiong M, Zhang J, et al. Efficacy and safety of CD28-or 4-1BB-based CD19 CAR-T cells in B cell acute lymphoblastic leukemia. Molecular Therapy-Oncolytics. 2020;18:272-81.
  29. Fujiwara K, Tsunei A, Kusabuka H, Ogaki E, Tachibana M, Okada N. Hinge and transmembrane domains of chimeric antigen receptor regulate receptor expression and signaling threshold. Cells. 2020;9(5):1182.
  30. Mirazee J, Jia D, Chen X, Achar S, Chien C, Pouzolles M, et al. 401 Hinge length: A novel method of predicting cytotoxicity of CAR constructs against antigen-low leukemia. BMJ Specialist Journals; 2022.
  31. Majzner R, Rietberg S, Sotillo E, Dong R, Vachharajani V, Labanieh L. Tuning the antigen density requirement for CAR T-cell activity. Cancer Discov. 2020; 10 (5): 702–723. doi: 10.1158/2159-8290. CD-19-0945.[Europe PMC free article][Abstract][CrossRef][Google Scholar].
  32. Xin L, Yu H, Hong Q, Bi X, Zhang X, Zhang Z, et al. Identification of strategic residues at the interface of antigen–antibody interactions by in silico mutagenesis. Interdisciplinary Sciences: Computational Life Sciences. 2018;10:438-48.
  33. Myung Y, Pires DE, Ascher DB. Understanding the complementarity and plasticity of antibody–antigen interfaces. Bioinformatics. 2023;39(7):btad392.
  34. Sela-Culang I, Kunik V, Ofran Y. The structural basis of antibody-antigen recognition. Front Immunol 4: 302. 2013.
  35. Faraji SN, Nejatollahi F, Tamaddon A-M, Mohammadi M, Aminsharifi AR. Generation and characterization of a specific single-chain antibody against DSPP as a prostate cancer biomarker: Involvement of bioinformatics-based design of novel epitopes. International Immunopharmacology. 2019;69:217-24.
  36. An N, Hou YN, Zhang QX, Li T, Zhang QL, Fang C, et al. Anti-multiple myeloma activity of nanobody-based anti-CD38 chimeric antigen receptor T cells. Molecular pharmaceutics. 2018;15(10):4577-88.
  37. Ramaraj T, Angel T, Dratz EA, Jesaitis AJ, Mumey B. Antigen–antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2012;1824(3):520-32.
  38. Liu Q, Graeff R, Kriksunov IA, Lam CM, Lee HC, Hao Q. Conformational closure of the catalytic site of human CD38 induced by calcium. Biochemistry. 2008;47(52):13966-73.
  39. Lee HT, Kim Y, Park UB, Jeong TJ, Lee SH, Heo YS. Crystal structure of CD38 in complex with daratumumab, a first-in-class anti-CD38 antibody drug for treating multiple myeloma. Biochem Biophys Res Commun. 2021;536:26-31.
  40. van de Donk NW, Janmaat ML, Mutis T, Lammerts van Bueren JJ, Ahmadi T, Sasser AK, et al. Monoclonal antibodies targeting CD 38 in hematological malignancies and beyond. Immunological reviews. 2016;270(1):95-112.
  41. Kinder M, Bahlis NJ, Malavasi F, De Goeij B, Babich A, Sendecki J, et al. Comparison of CD38 antibodies in vitro and ex vivo mechanisms of action in multiple myeloma. Haematologica. 2021;106(7):2004.
  42. Drent E, Groen RW, Noort WA, Themeli M, van Bueren JJL, Parren PW, et al. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma. haematologica. 2016;101(5):616.
  43. Müllbacher A, King N. Target Cell Lysis by Natural Killer Cells is Influenced by β2‐Microglobulin Expression. Scandinavian journal of immunology. 1989;30(1):21-9.
  44. Bald T, Krummel MF, Smyth MJ, Barry KC. The NK cell–cancer cycle: advances and new challenges in NK cell–based immunotherapies. Nature immunology. 2020;21(8):835-47.
  45. Ambrose AR, Hazime KS, Worboys JD, Niembro-Vivanco O, Davis DM. Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles. Proceedings of the National Academy of Sciences. 2020;117(38):23717-20.
  46. Althaus J, Nilius-Eliliwi V, Maghnouj A, Döring S, Schroers R, Hudecek M, et al. Cytotoxicity of CD19-CAR-NK92 cells is primarily mediated via perforin/granzyme pathway. Cancer Immunology, Immunotherapy. 2023:1-11.
  47. Drent E, Poels R, Mulders MJ, van de Donk NW, Themeli M, Lokhorst HM, et al. Feasibility of controlling CD38-CAR T cell activity with a Tet-on inducible CAR design. PloS one. 2018;13(5):e0197349.
  48. Dimopoulos MA, Oriol A, Nahi H, San-Miguel J, Bahlis NJ, Usmani SZ, et al. Daratumumab, lenalidomide, and dexamethasone for multiple myeloma. New England Journal of Medicine. 2016;375(14):1319-31.
  49. Usmani SZ, Nahi H, Plesner T, Weiss BM, Bahlis NJ, Belch A, et al. Daratumumab monotherapy in patients with heavily pretreated relapsed or refractory multiple myeloma: final results from the phase 2 GEN501 and SIRIUS trials. The Lancet Haematology. 2020;7(6):e447-e55.
  50. Attal M, Richardson PG, Rajkumar SV, San-Miguel J, Beksac M, Spicka I, et al. Isatuximab plus pomalidomide and low-dose dexamethasone versus pomalidomide and low-dose dexamethasone in patients with relapsed and refractory multiple myeloma (ICARIA-MM): a randomised, multicentre, open-label, phase 3 study. The Lancet. 2019;394(10214):2096-107.
  51. Martin T, Strickland S, Glenn M, Charpentier E, Guillemin H, Hsu K, et al. Phase I trial of isatuximab monotherapy in the treatment of refractory multiple myeloma. Blood cancer journal. 2019;9(4):41.
  52. Krejcik J, Casneuf T, Nijhof IS, Verbist B, Bald J, Plesner T, et al. Daratumumab depletes CD38+ immune regulatory cells, promotes T-cell expansion, and skews T-cell repertoire in multiple myeloma. Blood, The Journal of the American Society of Hematology. 2016;128(3):384-94.
  53. Mei H, Li C, Jiang H, Zhao X, Huang Z, Jin D, et al. A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma. Journal of Hematology & Oncology. 2021;14(1):1-17.
  54. Cui Q, Qian C, Xu N, Kang L, Dai H, Cui W, et al. CD38-directed CAR-T cell therapy: a novel immunotherapy strategy for relapsed acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Journal of Hematology & Oncology. 2021;14(1):1-5.
  55. Tang Y, Yin H, Zhao X, Jin D, Liang Y, Xiong T, et al. High efficacy and safety of CD38 and BCMA bispecific CAR-T in relapsed or refractory multiple myeloma. Journal of Experimental & Clinical Cancer Research. 2022;41(1):1-15.
  56. Guo Y, Feng K, Tong C, Jia H, Liu Y, Wang Y, et al. Efficiency and side effects of anti-CD38 CAR T cells in an adult patient with relapsed B-ALL after failure of bi-specific CD19/CD22 CAR T cell treatment. Cellular & Molecular Immunology. 2020;17(4):430-2.
  57. Hambach J, Riecken K, Cichutek S, Schütze K, Albrecht B, Petry K, et al. Targeting CD38-expressing multiple myeloma and burkitt lymphoma cells in vitro with nanobody-based chimeric antigen receptors (Nb-CARs). Cells. 2020;9(2):321.
  58. Gong J-H, 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(4):652-8.

59. Maki G, Klingemann H-G, Martinson JA, Tam YK. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. Journal of hematotherapy & stem cell research. 2001;10(3):369-83.