Hematopoietic Stem Cells Expressing Engineered CD45 Enable a Near Universal Targeted Therapy for Hematologic Diseases

Romina Marone^1,2*, Simon Garaudé^1,2*, Rosalba Lepore^{1, 2, 3}, Anna Devaux^1,2, Astrid Beerlage^{1, 2}, Federico Simonetta^4,5, Anna Camus³, Izabela Durzynska⁶, Ian Kirby⁷, Patrick H. Van Berkel⁷, Christian Kunz⁶, Stefanie Urlinger³, And Lukas T. Jeker^1,2,3

* These authors contributed equally

¹Department of Biomedicine, Basel University Hospital and University of Basel, Basel, Switzerland; ²Transplantation Immunology & Nephrology, Basel University Hospital, Basel,Switzerland; ³Cimeio Therapeutics AG, Basel, Switzerland; ⁴Division of Hematology, Department of Oncology, Geneva University Hospitals, Geneva, Switzerland; ⁵Translational Research Center for Oncohematology, Department of Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland; ⁶Ridgeline Discovery GmbH, Switzerland; ⁷ADC Therapeutics (UK) Ltd,
Imperial College White City Campus, London, UK

• Current untargeted cytotoxic conditioning regimens for hematopoietic stem cell transplantation (HSCT) are directly or indirectly associated with transplant related
  morbidity and mortality..
• Antigen-specific cell depleting therapies have revolutionized clinical practice in hematology..
• The pan-hematopoietic marker CD45, a protein tyrosine phosphatase which is exclusively expressed on all nucleated hematopoietic cells, could enable targeted depletion of the entire hematopoietic system including HSCs..
• CD45 is critical for the function of immune cells (i.e, CD45 mutations can lead to severe combined immunodeficiency (SCID))..
• HSPCs engineered to be shielded from a CD45-targeted antibody-drug conjugate (ADC) could enable selective tumor ablation with preserved hematopoiesis.

Identify and characterize CD45 variants that shield from a novel, concurrently developed, highly potent CD45-antibody drug conjugate (CIM053-ADC) while preserving CD45 function.

• Biophysical characterisation of the leading variants (recombinant proteins).
• Base editing of HSPCs.
• In vitro colony forming assay of HSPCs.
• Internalisation of antibody B.
• In vitro CIM053-ADC mediated killing of tumor cells cocultured with HSPCs.
• In vivo: Injection of base edited HSPCs into NBSGW mice and application of CIM053-ADC. Secondary transplants and NGS from mouse organs.
• In vivo: Injection of MOLM-14 tumor cells (AML cell line) into humanized mice and application of CIM053-ADC.

1. Identification of base editable CD45 extracellular domain regions to achieve shielding from targeted therapies

A: Crystal structure of CD45 domains 1+2 (PDB: 5FMV); CD45 domains 1+2 were computationally identified as suitable for targeted therapy and contain antibody A and B epitopes B: Base editor screening targeting computationally selected extracellular regions of CD45 domains 1+2 predicted to be amenable for shielding. (Plasmid-based screen in K562 cells; Sanger sequencing 3 days post-electroporation). C: Flow cytometry of DF-1 cells transiently overexpressing CD45 wildtype and variants confirms reduced/abolished antibody binding to CD45 variants D: Biophysical protein characterization shows that Variant 3 retains protein stability and low aggregation propensity while Variants 1 and 2 have reduced protein integrity.

2. Base editing in CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro

A: Tiling of sgRNA49. The sgRNA was moved by 1 base pair B: Flow cytometry profile of base edited CD34+ HSPCs 12 days post-electroporation with base editor mRNA and sgRNA. Variant 3 editing efficiency was improved by repositioning sgRNAs using Cas9-SpRY’s relaxed PAM recognition capabilities. sgRNA-49.3 increased on-target editing 25-fold C: Histogram of colony forming assays. Total sgRNA-NTC colonies were set to 1, multiple experiments pooled. D: Antibody B internalization (Jurkat cells) at 37 and 4°C E: Control and edited HSPCs and Jurkat survival ratios in co-cultures in the presence of CIM053-ADC (left). Frequency of edited cells in all sorted live cells from co-culture conditions 72h after incubation with CIM053-ADC.

3. Variant 3 shields hematopoietic cells from CIM053-ADC killing in vivo

A: In vivo experimental layout: In vivo engraftment and differentiation potential of base edited HSPCs measured 18 weeks after injection in NBSGW mice (+/- CIM053-ADC) B,C: Human/mouse chimerism is comparable in mice reconstituted with unedited or Variant 3 edited HSPCs demonstrating unaltered engraftment capacity of engineered HSPCs. Variant 3 shields bone marrow hematopoietic cells from CIM053-ADC killing in vivo. Bone marrow, spleen and blood cell differentiation profiles are comparable between mice reconstituted with unedited or Variant 3 edited HSPCs D: Secondary transplant into NSG-SGM3 mice. Human chimerism in bone marrow, blood and spleen. CIM053-ADC single dose injected in the primary host. E: NGS of bone marrow and spleen cells from primary mouse hosts and from spleen of secondary mouse hosts.

4. 4. In vivo CIM053-ADC mediated selective tumor eradication with preserved hematopoiesis

A: In vivo experimental layout. In vivo engraftment of base edited HSPCs followed by MOLM-14 tumor injection in NBSGW mice B: Bioluminescence imaging of humanised NBSGW mice transplanted with MOLM-14 tumor cells (luciferase+ mCherry+). Mice were treated with CIM053-ADC or saline solution. Survival curve of tumor bearing mice C: FACS plots and D quantification of tumor cells among human cells (hCD45+ mCD45- mCherry+) and of human non-tumor cells (hCD45+ mCD45- mCherry-) in bone marrow, spleen and blood.

• Identified CD45 variants with favorable biophysical properties.
• Generation of a novel, potent anti-CD45 antibody drug conjugate (CIM053-ADC) which depletes tumor cells and HSPCs. CD45 variant 3-expressing HSPCs are shielded from CIM053-ADC while maintaining intact protein properties.
• Edited HSPCs engraft, differentiate in vivo and are shielded from CIM053-ADC.
• Selective tumor and unedited human cell depletion in vivo with preservation of edited human hematopoietic cells.

1. Palchaudhuri, R., Saez, B., Hoggatt, J. et al. Nat Biotechnol 2016
2. Walton RT. et al. Science 2020

• Research Core Facilities (animal husbandry, flow cytometry) 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))

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