Base-Edited HSPCs Are Shielded from Targeted CD33 Therapy but Preserve CD33 Expression

¹Department of Biomedicine, University of Basel, Basel, Switzerland ²Transplantation Immunology & Nephrology, Basel University Hospital, Basel, Switzerland ³Cimeio Therapeutics AG, Basel, Switzerland

• CD33 is expressed in >90% of patients with acute myeloid leukemia (AML)
• Due to higher expression on leukemic blasts compared to their healthy counterparts, CD33 is an attractive target and already used in clinical routine (antibody drug
  conjugate gemtuzumab ozogamicin)
• Additionally, there are clinical trials evaluating allogeneic hematopoietic cell transplantation (HCT) with CD33 knock-out cells to avoid hematotoxicity. However,
  longterm outcome of a CD33 deficient hematopoiesis remains unclear
• Our group and others recently demonstrated that single amino acid changes can protect hematopoietic stem and progenitor cells (HSPCs) from targeted therapies while maintaining their function^1,2,3
• The adenine base editor ABE8e allows for targeted introduction of A > G changes

Identification and characterization of a base-editable CD33 variant, that maintains function while loosing binding to a therapeutic CD33 antibody. Thereby, we create a tumor-specific antigen allowing targeted therapy for AML after allogeneic HCT without depletion of HSPCs.

• Alanine scan epitope mapping to identify single amino acid substitutions within the extracellular domain of CD33 maintaining protein structure
• Affinity screening by bio-layer interferometry (BLI) and biophysical characterization of variants by assessing melting temperature and monomer content
• Base editing screen using ABE8e_SpRY and 21 tiled sgRNAs
• Readout after editing by assessing binding to therapeutic CD33 antibody and CD33 control antibody via flow cytometry and Sanger sequencing of bulk cells
• NGS of sorted cell populations after base editing
• In vitro differentiation and colony forming assay of baseedited HSPCs

1. Biophysical characterization of CD33 protein variants harboring single amino acid substitutions

A: Schematic domains of CD33, containing one C2 and one V domain B: 3D Structure of CD33 extracellular domain (PDB ID: 5IHB). Residues involved in the binding to antibody 1 are shown in sphere based on a full ECD alanine scan C: Binding affinity of CD33 variants to antibody 1 as % binding versus WT measured by BLI. Blue circles show no binding observed up to 500nM D: Monomeric content in % of purified CD33 extracellular domain E: Melting temperature of purified CD33 extracellular domain histidine tagged proteins.

2. Base editing of human CD34+ HSPCs in vitro

A: Timeline of base editing in HSPCs B: Base editing screen by tiling of 21 sgRNAs around the intended edit C: Histograms of CD33 target epitope antibody binding after editing of human CD34+ HSPCs with different sgRNAs in comparison to non-target control (NTC) D: Flow cytometry of edited human CD34+ HSPCs, staining with therapeutic and control CD33 antibody E: Sequencing of sgRNA4 edited bulk (Sanger sequencing) and sorted (NGS) cells, % edited and unedited cells.

3. Differentiation of sgRNA4 edited HSPCs in vitro

A: Colony-forming unit assay (CFU) with NTC and sgRNA4 edited human CD34+ HSPCs, image after 14 days B: Quantification of colony forming assays (two technical replicates) C: Sanger Sequencing of DNA extracted from single colonies from sgRNA4 edited cells in CFU, assignment to hetero- or homocygously edited cells by % of edited cells using EditR D: In vitro differentiation assay with NTC and sgRNA4 edited human HSPCs, readout with FACS after 14 days E: Quantification of three technical replicates of in vitro differentiation assay comparing myeloid and erythroid differentiation of cells.

• We identified a base-editable CD33 variant showing loss of antibody 1 binding while maintaining binding to a control CD33 antibody and unaltered biophysical properties compared to wildtype CD33
• HSPCs expressing this variant show a differentiation potential comparable to NTC edited HSPCs in CFU assay and in vitro differentiation assay
• Studies to further characterize the differentiation of edited cells in vivo as well as tumor models to investigate selective killing of tumor cells while preserving edited HSPCs are ongoing

1. Marone R, Landmann E, Devaux A, et al. Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy. J Exp Med. 2023;220(12):e20231235. doi:10.1084/jem.20231235
2. Wellhausen N et al. Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy. Sci Transl Med. 2023;15(714):eadi1145. doi:10.1126/scitranslmed.adi1145
3. Casirati G et al. Epitope editing enables targeted immunotherapy of acute myeloid leukaemia. Nature. 2023;621(7978):404-414. doi:10.1038/s41586-023-06496-5

Department of Biomedicine, Basel University Hospital and University of Basel, Basel, Switzerland lukas.jeker@unibas.ch

• Flow cytometry facility at the University of Basel and Department of Biomedicine
• Funding from European Research Council (ERC) European Union’s Horizon 2020 research and innovation programme (grant agreement No. 818806 (LTJ))
• Sponsored Research Collaboration Agreement (University of Basel/Cimeio Therapeutics AG (LTJ))