Function-Preserving Single Amino Acid Substitutions Shield Hematopoietic Stem and Progenitor Cells from CD117-Targeted Immunotherapy in Vivo

Romina Marone^1,2, Rosalba Lepore^1,2,3,4, Julia Rositzka⁵, Viviane Dettmer-Monaco⁵, Alessandro Dell’ Aglio^1,2, Giuseppina Capoferri^1,2, Julie Brault⁴, Emiel Ten Buren³, Thomas Burgold³, Anna Camus³, Christopher Divsalar⁴, Laura Garcia Prat³, Anna Haydn⁴, Felix Hermann⁴, Marcel Heugel³, Andreja Knezevic³, Frank Lehmann³, Valentin Do Sacramento³, Alessandro Sinopoli⁴, Lisa C. Wellinger⁴, Amélie Wiederkehr⁴, Saniye Yumlu³, Toni Cathomen⁵, Thomas Winkler³, Tatjana I. Cornu⁵, Stefanie Urlinger^3,4, And Lukas T. Jeker^1,2,3

¹ Department of Biomedicine, Basel University Hospital and University of Basel, Basel, Switzerland; ² Transplantation Immunology & Nephrology, Basel University Hospital, Basel, Switzerland; ³ Cimeio Therapeutics AG, Basel, Switzerland; ⁴ Ridgeline Discovery GmbH, Switzerland; ⁵ Institute for Transfusion Medicine & Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany

• Current untargeted cytotoxic conditioning regimens for hematopoietic stem cell transplantation (HSCT) have been directly or indirectly associated with transplant related morbidity and mortality.
• HSPCs express the receptor tyrosine kinase c-KIT (CD117) which is activated by its ligand stem cell factor (SCF) leading to CD117 signaling that is crucial for HSPC biology.
• Blocking SCF binding prevents proliferation and leads to HSPC depletion in vivo2. Therefore, CD117 is an attractive therapeutic target as a conditioning agent prior to HSCT.
• mAb-based conditioning necessitates a washout phase of the depleting antibody before engraftment of donor HSPC and prevents post-transplant redosing.
• HSPCs engineered to be shielded from a CD117-targeted immunotherapy could overcome this limitation and enable novel HSCT approaches.

• Label-free determination of binding affinity of CD117 variants to SCF and anti-CD117 antibodies.
• In vitro mAb-mediated blocking binding and proliferation assays (TF-1, HSPC).
• Non-viral engineering of TF-1 cells and HSPCs using CRISPR-Cas9 (High fidelity Cas9 RNPs, single strand oligodeoxynucleotides (ssODN) as homology directed repair template (HDRT).
• ELISA-based p-Tyr719 signalling in TF-1 cells engineered to express CD117 variant 1.1.
• In vitro colony forming assay of HSPCs.
• In vivo: Injection of non-virally edited HSPCs into sublethally irradiated NSG mice + isotype/CIM058 injection.

1. Rational design of human CD117 protein variants and their SCF and SR-1 binding

A) 3D structure of the CD117-SCF complex (PDB ID: 2e9w)1. CD117 is shown as ribbons. SCF is shown as light grey molecular surface. CD117 residues involved in SCF binding are shown as dark grey surface. CD117 residues forming the SR-1 epitope are shown as pink surface.
B) Affinity (KD) of CD117 variants binding to SCF is shown as a ratio versus WT. Values >1 (<1) indicate lower (higher) affinity to SCF. Error bars indicate the standard deviation of three measurements. NB: No Binding observed up to 1000nM of the analyte. ND: Not determined/biphasic.
C) Binding affinity of CD117 variants to SR-1 is shown as % binding vs WT. NB: No binding observed up to 500nM of the analyte.
D) Dose titration of select CD117 variants to test residual SR-1 binding at very high analyte concentrations. CD117 variants were chosen based on SR-1 binding reduction and preserved SCF binding.

2. Generation of highly potent SCF-blocking antibodies

Antibodies used:

• Isotype control;
• SR-1 (known SCF mouse blocking mAb);
• JSP191 biosimilar (JSP191: humanized SR-1);
• CIM001 (proprietary humanized SCF blocking mAb);
• CIM058 (proprietary humanized and engineered SCF blocking mAb).

A) Incubation of the SCF-dependent cell line TF-1 with anti-CD117 antibodies results in dose-dependent inhibition of proliferation.
B) Incubation of HSPCs with anti-CD117 antibodies blocks SCF binding.
C) Incubation of mobilized CD34+ HSPCs with anti-CD117 antibodies results in reduced proliferation.
D) HSPCs were plated on methylcellulose and allowed to grow for 2 weeks in the presence of anti-CD117 antibodies. Colonies were counted. Anti-CD117 antibodies dose-dependently reduced colony formation.

3. Engineered TF-1: normal SCF binding, proliferation and c-Kit phosphorylation

A) Binding of the anti-CD117 antibody SR-1 is completely abolished in TF-1 cells expressing engineered CD117 variant1.1. Shown are results from two independent TF-1 Var1.1 clones.
B) SCF dose-dependent proliferation of TF-1 cells expressing engineered CD117 variants is comparable (EC50) to cells with wildtype receptor.
C) Anti-CD117 antibody (SR-1) blocks SCF-mediated growth of TF-1 cells expressing wildtype but not engineered variants.
D) CD117 phosphorylation at position Tyr719 upon increasing concentrations of SCF (ELISA).
E) CD117 phosphorylation at position Tyr719 upon anti-CD117 antibody treatment is not blocked in TF-1 cells expressing the engineered variant 1.1; SCF: 0.1μg/ml; antibody: 0.5μg/ml (SR-1 or CIM001).

4. In vivo CIM058-mediated HSPC depletion and engraftment + shielding of HSPC Var.1.1

• Identified CD117 variants with normal SCF binding but reduced SR-1 binding.
• Generation of potent anti-CD117 SCF-blocking antibodies which inhibit TF-1 + HSPC proliferation and colony formation.
• TF-1 cells expressing the CD117 variants display SCF-dependent growth comparable to wt cells. Moreover, SCF-dependent signaling is intact in the presence of the SCF-blocking antibodies while KO cells and wt cells are blocked.
• Edited HSPCs engraft, differentiate in vivo but are shielded from mAbs.

1. Yuzawa S et al. Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor. Cell 2007; 130: 323-334
2. Pang WW et al. Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice. Blood 2019; 133: 2069-2078

• 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)