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329 Redirecting NK cell cytotoxicity by innate cell engagers: a differentiated and innovative approach compared to CAR-NK cells
  1. Sonya Ioana Ciulean1,
  2. Julia Uhlig1,
  3. Thea Eichenberg1,
  4. Joe Fischer1,
  5. Michael Albers2,
  6. Christian Breunig2,
  7. Jan Schmollinger2,
  8. Jens Pahl2,
  9. Ivica Fucek2,
  10. Christoph Bach1,
  11. Paul Franz1,
  12. Anna Dünkel1,
  13. Stephan Fricke1,
  14. Thomas Grunwald1,
  15. Ulrike Köhl1,3 and
  16. Joachim Koch2
  1. 1Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Sachsen, Germany
  2. 2Affimed GmbH, Heidelberg, Baden-Wurttemberg, Germany
  3. 3Institute of Clinical Immunology, Leipzig, Sachsen, Germany
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background The use of bispecific Innate Cell Engagers (ICE®) has become a successful strategy for immune cell activation and killing of tumor cells through antibody-dependent cellular cytotoxicity (ADCC).1 Combination of adoptive NK cell therapy with ICE® molecules significantly improved tumor-targeting and has shown unprecedented clinical response rates in heavily pretreated cancer patients.2 Alternatively, as NK cells alone show short time clinical activity only, genetic modification with chimeric antigen receptors (CAR) has demonstrated improved clinical success in patients with CD19+ haematological disorders.3 4 To compare both approaches, we have evaluated the efficacy of NK cells combined with tetravalent bispecific CD16A/CD19-targeting ICE® versus anti-CD19 CAR-NK cells in a preclinical proof-of-concept study using CD19-positive target cells.

Methods NK cells from PBMCs of healthy donors were expanded for three weeks in the presence of IL-2 and IL-15. Anti-CD19 CAR-NK cells were generated by transduction with baboon envelope pseudo-typed gamma-retroviral vectors. NK cell cytotoxic activation was assessed by calcein-release assays, degranulation, cytokine production, and specific CD19 positive target cell killing resolved over time using IncuCyte imaging system after co-culture of non-transduced NK cells plus CD16A/CD19-targeting ICE or anti-CD19 CAR-NK cells with CD19-positive or negative tumor target cells.

Results Combination of non-transduced NK cells with the CD16A/CD19 ICE® enhanced ADCC activity towards CD19-positive target cells and mediated elevated levels of degranulation when compared to NK cells in the absence of ICE®. In addition, this combination induced target cell dependent TNF-alpha and IFN-gamma secretion by NK cells. CD16A/CD19 ICE-induced NK cell maximal cytotoxicity as well as TNF-alpha and IFN-gamma secretion were comparable to that of a corresponding CD19 CAR NK cells at different effector to target ratios.

Conclusions No major difference in cytotoxicity and TNF-alpha and IFN-gamma release between the two approaches, NK cells in combination with CD16A/CD19 ICE vs. CD19 CAR NK cells, has been observed. Therefore, combination of allogeneic NK cells with bispecific ICE® represent a versatile innovation providing advantages over engineered CAR-NK cells such as more cost-effective manufacturing and potentially reduced safety concerns. Treatment options for ICE® molecules are more flexible since no genetic engineering of NK cells is needed and can be built basically for any target. Moreover, this approach is applicable to a variety of NK cell products of different origins.


  1. Pinto, et al. Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers. Trends in Immunology 2022;43(11):932–46.

  2. Kerbauy, et al. Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies. Clin Cancer Res 2021;27(13):3744–3756.

  3. Gong, et al. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. Hematol Oncol 2021;14:73.

  4. Liu, et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med 2020;382:545–553.

Ethics Approval This work was performed with NK cells from healthy anonymous blood donors approved by the State Chamber of Physicians of Saxony, Germany, under ethical vote number EK-BR-79/21–1.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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