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442-Q CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation
  1. Jiri Eitler1,2,
  2. Wiebke Rackwitz2,3,
  3. Natalie Wotschel3,
  4. Venugopal Gudipati4,
  5. Johannes Huppa4,
  6. Paola Ortiz Montero3,
  7. Laurent Boissel5,
  8. Hans Klingemann5,
  9. Winfried Wels6 and
  10. Torsten Tonn2,3
  1. 1Faculty of Medicine Carl Gustav Carus, Dresden, Germany
  2. 2German Red Cross Blood Donation Service North-East, Dresden, Saxony, Germany
  3. 3Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Saxony, Germany
  4. 4Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria, Austria
  5. 5ImmunityBio Inc, Culver City, CA, USA
  6. 6Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Hesse, 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 Antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or genetic engineering with chimeric antigen receptors (CARs). Nevertheless, despite continued presence of the target antigen, some tumors can escape antibody or CAR-NK cell treatment, with the underlying mechanisms only poorly understood. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells has been previously shown, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated cytotoxicity against breast cancer cells has not yet been investigated.

Methods NK-92 cells expressing high-affinity FcγRIIIa (haNK) in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR were employed to investigate the effects of ICAM-1 downregulation on breast cancer cells on NK cell cytotoxicity.

Results Blockade of the ICAM-1/LFA-1 interaction significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, while pretreatment with 5-aza-2’-deoxycytidine (5AZA) induced ICAM-1 upregulation and reversed NK cell resistance. In contrast, CAR-NK cell-mediated cytotoxicity did not rely on ICAM-1/LFA-1 interaction, and was not impaired by reduction of ICAM-1 expression on target cells or blockade of LFA-1 on NK cells (figure 1). In degranulation experiments, trastuzumab alone did not sufficiently activate NK cells but required additional LFA-1 stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. TIRF single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC but rather resulted in decreased signaling via Pyk2, which was restored by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced trastuzumab- and ICAM-1-mediated NK cell activation, CAR-NK cells were only marginally affected.

Conclusions We identified downregulation of ICAM-1 expression on breast cancer cells as a critical mechanism mediating escape from trastuzumab-triggered ADCC. Importantly, CAR-NK cells were able to overcome ICAM-1-based resistance as well as NKG2A-mediated inhibition, which may be relevant for the development of more effective NK cell-based cancer immunotherapies.

Ethics Approval Primary cells from healthy donors were obtained from the German Red Cross Blood Donation Service under an Ethics Review Board-approved protocol number EK138042014.

Abstract 442-Q Figure 1

Schematic representation of the main findings: Downregulation or loss of ICAM-1 on cancer cells leads to escape from antibody-targeted cytotoxicity of NK cells (ADCC). This can be reversed by pre-incubation of cancer cells with 5-aza-2’-deoxycytidine (5AZA) and further enhanced by production of TNF-α by activated NK cells. In contrast, CAR-targeting of NK cells is independent of ICAM-1 expression levels. Mechanistically, CAR signaling bypasses the LFA-1 signaling through the Pyk2 pathway, resulting in efficient NK cell degranulation and killing of the otherwise resistant cancer cell

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