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Acute myeloid leukemia

Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2

Leukemia volume 33, pages 762–808 (2019)Cite this article

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CD33 has long been pursued as immunotherapeutic target in acute myeloid leukemia (AML) [1, 2]. Improved survival with gemtuzumab ozogamicin (GO) validates this approach [3]. Partly stimulated by GO’s success, several investigational CD33-directed therapeutics are currently in clinical testing [4]. However, CD33 expression on normal hematopoietic cells leads to “on-target, off-leukemia” toxicity with significant morbidity/mortality from profound cytopenias, limiting the use of CD33-directed immunotherapies [4]. This toxicity should be minimal if normal blood cells did not express the epitope targeted by these antibodies. Supporting the feasibility of CD33-engineering the hematopoietic system are the findings that CD33-deficient mice have a very mild phenotype and show no difference in cellular response to pro-inflammatory stimuli compared to wild-type animals, indicating functional degeneracy between CD33 and other proteins [5]. Moreover, recent studies have shown that CRISPR/Cas9-mediated disruption of the CD33 coding region in CD34+ hematopoietic stem and progenitor cells (HSPCs) may not affect engraftment [6], suggesting that the generation of CD33-manipulated hematopoiesis is a clinically viable strategy to protect from “off-leukemia” cell toxicity of CD33-directed immunotherapy. Here we have investigated an alternative, precise CD33 genome-editing approach that would only eliminate exon 2 and therefore the V-set immunoglobulin-like domain, which is the target of all current clinical CD33-directed approaches. Our editing strategy is expected to result in expression of a naturally occurring shorter isoform of CD33 (CD33∆E2) but not full-length CD33 (CD33FL), which may minimize potential adverse effects associated with disruption of the entire CD33 locus. We used CRISPR/Cas9 [7,8,9,10] to accomplish this goal and functionally assessed genome-edited human hematopoietic cells in vitro and in immunodeficient mice.

Human myeloid ML-1 cells and human fetal liver CD34+ HSPCs were used for our studies. ML-1 cells were maintained as described [11]. Human fetal liver CD34+ cells were enriched by immunomagnetic separation from tissue obtained from Advance Bioscience Resources Inc. (ABR, Alameda, CA). Cells were cultured in StemSpan SFEMII media (StemCell Technologies, Cambridge, WA) supplemented with penicillin/streptomycin (Life Technologies, Carlsbad, CA), Stem cell factor , Thrombopoietin  (both PeproTech, Rocky Hill, NJ), and FLT3-L (Miltenyi Biotec, Auburn, CA). CRISPR/Cas9-editing was carried out by electroporation of purified Cas9 protein (TrueCut Cas9 V2; ThermoFisher Scientific, Waltham, MA) complexed with synthetic guide RNAs (sgRNAs; Supplementary Table 1), which were modified at the 5′ and 3′ ends with 2′-O-methyl-3′-phosphorothiate (Synthego, Redwood City, CA) using the ECM 380 Square Wave Electroporation system (Harvard Apparatus, Cambridge, MA) [12]. For evaluation of colony-forming units (CFUs), 1500 CD34+ cells were seeded in 3.5 mL ColonyGEL 1402 (ReachBio, Seattle, WA) and scored after 12–14 days. CFU DNA was extracted in QuickExtract (Epicentre, Madison, WI).

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Acknowledgements

We thank Dr. Colin E. Correnti and the Molecular Design and Therapeutics core facility team (Fred Hutchinson Cancer Research Center) for the generation of AMG 330. We also thank Helen Crawford for help preparing and formatting this manuscript, and Jerry Chen for general mouse colony maintenance. Research reported in this publication was supported by the Leukemia & Lymphoma Society (Translational Research Program, grant 6489-16). RBW is a Leukemia & Lymphoma Society Scholar in Clinical Research. H-PK is a Markey Molecular Medicine Investigator and received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Fred Hutchinson Cancer Research Center Endowed Chair for Cell and Gene Therapy.

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  1. These authors contributed equally: Olivier Humbert, George S. Laszlo and Hans-Peter Kiem, Roland B. Walter

Authors and Affiliations

  1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Olivier Humbert, George S. Laszlo, Sophie Sichel, Christina Ironside, Kevin G. Haworth, Olivia M. Bates, Mary E. Beddoe, Ray R. Carrillo, Hans-Peter Kiem & Roland B. Walter

  2. Division of Medical Oncology, Department of Medicine, University of Washington, Seattle, WA, USA

    Hans-Peter Kiem

  3. Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA

    Roland B. Walter

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  1. Olivier Humbert
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Correspondence to Olivier Humbert.

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Conflict of interest

RBW has received laboratory research grants and/or clinical trial support from Actinium Pharmaceuticals, Inc, Amgen Inc., Amphivena Therapeutics, Inc., Covagen AG, and Seattle Genetics, Inc.; has ownership interests with Amphivena Therapeutics, Inc.; and is (or has been) a consultant to Amphivena Therapeutics, Inc., Boehringer Ingelheim Pharma GmbH & Co. KG, Covagen AG, Pfizer, Inc., and Seattle Genetics, Inc. H-PK is a consultant for Rocket Pharma and Homology Medicines. The other authors declare that they have no conflict of interest.

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Humbert, O., Laszlo, G.S., Sichel, S. et al. Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2. Leukemia 33, 762–808 (2019). https://doi.org/10.1038/s41375-018-0277-8

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