Engineered Single Amino Acid Substitutions Protect Hematopoietic Stem and Progenitor Cells From CD123 Targeted Immunotherapy

E. Landmann1,2*, A. Devaux1,2*, R. Lepore1,2,3,4*, R. Marone1,2*, C. Engdahl1,2, M. Hasiuk1,2, G. Capoferri1,2, A. Wiederkehr4, L. Wellinger4, A. Sinopoli4, V. Do Sacramento3, A. Haydn4, L. Garcia Prat3, C. Divsalar4, A. Camus3, J. Brault4, L. Xu5, T. Schwede6, M.H. Porteus7, S. Urlinger3,4, L.T. Jeker1,2,3

*These authors contributed equally
1 Transplantation Immunology & Nephrology, University Hospital Basel, Basel, Switzerland; 2 Department of Biomedicine, University of Basel, Basel, Switzerland; 3 CimeioTherapeutics AG, Basel, Switzerland; 4 Ridgeline Discovery GmbH, Basel, Switzerland; 5 Department of Pathology, Stanford University, Palo Alto, CA, USA; 6 Biozentrum, University of Basel, Basel, Switzerland; 7 Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA


  • Targeted immunotherapy using monoclonal antibodies (mAb), T cell engager (TCE), or chimeric antigen receptor (CAR)) cells is standard of care in hematologic malignancies.
  • CD123 (IL-3 receptor α-chain) is expressed in hematologic malignancies (e.g. acute myeloid leukaemia AML, blastic plasmacytoid dendritic cell neoplasm BPDCN), but also on healthy hematopoietic stem and progenitor cells (HSPCs)1.
  • Shared antigen expression leads to on-target off-tumor effect with risk of extensive myelotoxicity following targeted cell depletion2.
  • Proposed solution includes HSC transplantation with antigen-deleted HSPCs prior to immunotherapy3-5. This is, however, limited to dispensable targets and bears risk for antigen negative relapses.


We aimed to genetically engineer the CD123 epitope on HSPCs to shield the cells from targeted immunotherapy while preserving the antigen’s structure and function.


  • HEK cell lines stably expressing wildtype CD123 (HEK-CD123) and 28 CD123 variants with individual amino acid substitutions.
  • Targeted immunotherapy:
    • CSL362 biosimilar mAbMIRG123
    • CSL362/OKT3 TCE
    • Non-viral CRISPR/Cas9-based second generation CSL362-derived CAR T cells
  • Non-viral engineering of HSPCs using CRISPR-Cas9 (High fidelity Cas9 RNPs and single strand oligodeoxynucleotides(ssODN) as homology directed repair template (HDRT).
  • AAV6-mediated HDR editing of HSPCs (p-Stat5).
  • In vivo: Injection of non-virally edited HSPCs into sublethally irradiated NSG mice.


In silico design of human CD123 protein variants

In silico design of human CD123 protein variants

a. Crystal structure of CD123 and the fragment antigen binding (Fab) of the CSL362 mAb(talacotuzumab). CSL362 epitope at the N-terminal domain (NTD) is indicated in light blue(red arrow). b. Per residue relative solvent accessibility computed on the CSL362-free (solid line) and -bound (dashed line) states based on the crystal structure. c.E51, S59 and R84 were identified as key amino acid (AA) residues for CSL362 binding and a total of 28 AA substitutions were chosen (not shown) that potentially disrupt binding but preserve function.

2. Abolished binding to theCSL362 biosimilar despite preserved expression of CD123

Abolished binding to theCSL362 biosimilar despite preserved expression of CD123
Stable expression of CD123 wildtype (HEK-CD123) and all 28 variants in HEK cells. a. Variants were categorised as non binding (blue), weak binding (orange) and strong binding (red) using flow cytometry staining with the CSL362 biosimilar MIRG123 and the expression control mAb6H6. b. 13 non-binding, 12 weak-binding and 3 strong-binding variants were identified.

3. Biophysical characterization of selected CD123 variants

Biophysical characterization of selected CD123 variants
a. Biolayer interferometry confirms non-binding of soluble CD123 variant proteins to CSL362 even at high concentrations. b.All variants but two maintained binding to increasing concentrations of IL-3. c. Thermostability measured by differential scanning fluorimetry (DSF) indicates preserved protein stability for most variants, except R84E, that also showed reduced binding to IL-3.

4. Engineered CD123 variants are shielded from TCE-and CAR-mediated cytotoxicity in vitro

Engineered CD123 variants are shielded from TCE-and CAR-mediated cytotoxicity in vitro
HEK cells stably expressing non-binding CD123 variants were protected from MIRG123-mediated cytotoxicity induced by a.CSL362/OKT3 TCE and b. second generation CSL362-derived CD123 specificCAR T cells in vitro.

5. Engineered human HSPCs expressing CD123 variants are functional, engraft and differentiate in vivo

Engineered human HSPCs expressing CD123 variants are functional, engraft and differentiate in vivo
Mobilised peripheral blood CD34+HSPCs were genetically engineered to express the non-binding variants E51K and E51T using a non-viral and close to GMP-grade CRISPR/Cas9 protocol. a. Representative flow cytometry dot plots and b. summary of KI (MIRG123-6H6+) frequency for multiple donors 5 days post-electroporation with RNPs and ssODN HDRT. c. Representative histogram indicating regular pSTAT5 signaling upon IL-3 exposure in engineered HSPCs. d.Multi-lineage differentiation of engineered HSPCs in blood 15 weeks after injection in NSG mice.

6. Engineered HSPCs enable tumor-selective CD123 immunotherapy

Engineered HSPCs enable tumor-selective CD123 immunotherapy
a. Selective CSL362/OKT3 TCE-mediated killing of non-edited HSPCs in vitro. E51K/E51T expressing HSPCs are spared. b. CD123 expressing MOLM-14 (CTV labelled AML cell line) cells are selectively killed in co-culture with engineered CD34+HSPCs and the CSL362/OKT3 TCE.


  • Single amino acid substitutions abolish CSL362 binding but preserve function
  • In vitro shielding of epitope engineered CD123 variants from TCE-and CAR-mediated killing
  • Edited HSPCs are functional, engraft and differentiate regularly in vivo
  • Tumor-selective killing of edited HSPCs in vitro
  • Epitope engineering to shield cells may enable targeting of essential proteins


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  2. Gill, S. et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood 123, 2343-2354, doi:10.1182/blood-2013-09-529537 (2014).
  3. Kim, M. Y. et al. Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia. Cell 173, 1439-1453 e1419, doi:10.1016/j.cell.2018.05.013 (2018).
  4. Borot, F. et al. Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies. Proc Natl AcadSciU S A 116, 11978-11987, doi:10.1073/pnas.1819992116 (2019)
  5. Humbert, O. et al. Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33exon 2. Leukemia 33, 762-808, doi:10.1038/s41375-018-0277-8 (2019)


  • Research Core Facilities (animal husbandry, flow cytometry) at the University of Basel and Department of Biomedicine.
  • Dario Neriand MihaelaZavolanfor sharing reagents.
  • Funding from European Research Council (ERC) European Union’s Horizon 2020 research and innovation programme (grant agreement No. 818806).