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The CD38low natural killer cell line KHYG1 transiently expressing CD16F158V in combination with daratumumab targets multiple myeloma cells with minimal effector NK cell fratricide

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Abstract

Multiple myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of the CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (MoAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells that mediate in vivo tumour immunosurveillance. NK cells also play an important role during MoAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their FcγRIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring variant of CD16 harbouring a single-point polymorphism (F158V). However, the contribution of NK cells to the efficacy of daratumumab remains debatable as clinical data clearly indicate the rapid depletion of CD38high peripheral blood NK cells in patients upon daratumumab administration. In contrast, CD38low peripheral blood NK cells have been shown to survive daratumumab mediated fratricide in vivo, while still retaining their potent anti-MM cytolytic effector functions ex vivo. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a “safe” mRNA electroporation-based approach on CD38low NK cells in combination with daratumumab could represent a novel therapeutic option for treatment of MM. In the present study, we investigate a NK cell line (KHYG-1), derived from a patient with aggressive NK cell leukemia, as a platform for generating CD38low NK cells expressing CD16F158V which can be administered as an “off-the-shelf” therapy to target both CD38high and CD38low tumour clones in patients receiving daratumumab.

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Abbreviations

ADCC:

Antibody dependent cellular cytotoxicity

ADCP:

Antibody dependent cellular phagocytosis

ADRP:

ADP-ribose pyrophosphate

CDC:

Complement-dependent cytotoxicity

E: T:

Effector: target

IMiDs:

Immunomodulatory drugs

KIR:

Killer inhibitory receptor

MM:

Multiple myeloma

MoAb:

Monoclonal antibody

NDMM:

Newly diagnosed multiple myeloma

NK:

Natural killer

PI:

Propidium iodide

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Acknowledgements

The authors would like to thank the Flowcytometry Core Facility at National University of Ireland Galway (NUIG), Ireland.

Funding

This research was supported by a Sponsored Research Agreement from ONK Therapeutics Ltd., Ireland to National University of Ireland Galway. Subhashis Sarkar received a Post-doctoral Fellowship from ONK Therapeutics Ltd. Michaela R. Reagan is supported in part by a Research Scholar Grant, RSG-19-037-01-LIB from the American Cancer Society, and the NIH, P20GM121301.

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Author notes

  1. Subhashis Sarkar and Sachin K. S. Chauhan contributed equally.

Authors and Affiliations

  1. Apoptosis Research Centre, Biomedical Sciences, National University of Ireland Galway, Newcastle Road, Galway, H91 W2TY, Ireland

    Subhashis Sarkar, Sachin K. S. Chauhan, John Daly, Alessandro Natoni, Robert Henderson, Emma Nolan, Dawn Swan, Jinsong Hu & Michael O’Dwyer

  2. ONK Therapeutics Ltd., Galway, Ireland

    Subhashis Sarkar, Sachin K. S. Chauhan & Michael O’Dwyer

  3. Maine Medical Center Research Institute, Scarborough, ME, USA

    Heather Fairfield & Michaela R. Reagan

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  1. Subhashis Sarkar
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Contributions

SS, SKSC, JD, HF and MRR performed experiments. SS, SKSC, JD and HF analysed data. AN, RH, EN, DS, JH, and MRR assisted with experiments. SS and MOD designed the project. SS and MOD wrote the manuscript.

Corresponding author

Correspondence to Michael O’Dwyer.

Ethics declarations

Conflict of interest

Subhashis Sarkar: ONK Therapeutics Ltd.: Research funding; Michael O'Dwyer: Abbvie: Membership on advisory committee; Celgene: Membership on advisory committee, and research funding; Bristol Myers Squibb (BMS): Research funding; Glycomimetics: Research funding; ONK Therapeutics Ltd.: Equity Ownership, Membership on Board of Directors, and research funding; Janssen: Membership on advisory committees, and research funding; Michaela R. Reagan: ONK Therapeutics Ltd.: Research funding. A provisional patent application was filed by Subhashis Sarkar and Michael O'Dwyer regarding the CD16 work described in the paper. All other authors declare no conflict of interest.

Ethical approval

All experiments utilizing primary cells from MM patients were conducted in accordance with the ethical standards of the Clinical Research Ethics Committee University Hospital Galway and the 1964 Declaration of Helsinki. The collection of bone marrow samples from MM patients was approved by Clinical Research Ethics Committee University Hospital Galway on June 14, 2017 (Study no. C.A.1519) All experiments utilizing blood samples from healthy donors were conducted in accordance with the ethical standards of the Clinical Research Ethics Committee University Hospital Galway and the 1964 Declaration of Helsinki. The collection of blood samples from healthy volunteers was approved by Clinical Research Ethics Committee University Hospital Galway on August 4, 2017 (Study no. C.A.1805) Animal research experiments to examine in vivo bone marrow homing were performed at the Maine Medical Center Research Institute with approval from the institutional animal care and use committee (IACUC); (Protocol #MR1508), an (Association for assessment and accrediation of laboratory animal care) AAALAC international accredited program. Approval for the experiments in this manuscript was issued by the Maine Medical Center Research Institute (MMCRI) Institutional Animal care and use committee (IACUC) on September 2, 2016.

Informed consent

NK cells were freshly isolated from peripheral blood samples of anonymous volunteer healthy donors at the University Hospital Galway, Ireland. Oral informed consent was obtained from all blood donors to the use of their blood for scientific purposes. Bone Marrow samples were provided by the Haematology Unit of University Hospital Galway, Galway from MM patients who had provided written consent for remaining samples to be used for immunology related research.

Animal source

Female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (Stock# 005557) were purchased at 4-weeks of age from Jackson Laboratory (Bar Harbor, ME) and acclimated at the Maine Medical Center Research Institute animal facility for at least two weeks prior being randomly assigned to each group for experiments.

Cell line authentication

KHYG1 was a kind gift from Dr. Armand Keating, University of Toronto (Canada) and the cell line was authenticated by STR profiling on 06/06/2017. MM1S, RPMI-8226, JJN3, H929, and U266, were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). NK-92 was purchased from Deutsche Sammlung von Mikcroorganismen and Zellkulturen  (DSMZ) (Braunschweig, Germany) The cell lines were initially grown and cryopreserved into multiple aliquots of master cell bank (MCB). All the experiments were performed with cells at low passage numbers (≤ 10). Mycoplasma was routinely tested.

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Note on previous publication: Part of this work was presented as a poster at the annual conference of the American Society of Haematology (ASH) 1–4 December 2018, San Diego, USA (Session-Myeloma: Pathophysiology and Pre-Clinical Studies, excluding Therapy: Poster II). The accompanying abstract was published in a special edition of Blood [1].

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Sarkar, S., Chauhan, S.K.S., Daly, J. et al. The CD38low natural killer cell line KHYG1 transiently expressing CD16F158V in combination with daratumumab targets multiple myeloma cells with minimal effector NK cell fratricide. Cancer Immunol Immunother 69, 421–434 (2020). https://doi.org/10.1007/s00262-019-02477-8

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