Engineered Stem Cell Antibody Paired Evasion-2 (ESCAPE-2): Paired HSC Epitope Engineering and Direct Editing of Sickle Allele for Antibody-Mediated Autologous Hematopoietic Stem Cell Therapy Conditioning for the Treatment of Sickle Cell Disease

Introduction of the naturally-occurring Hb G-Makassar variation in the human betaglobin gene (HBB) through base editing to eliminate the polymerizing sickle protein, HbS, the major molecular driver for sickle cell disease (SCD), represents a promising new paradigm for the potential treatment of individuals with this disease. While several ex vivo delivery of gene editing technologies for SCD are advancing in the clinic, several challenges remain for this potentially transformative cell therapy, namely the requirement of myeloablative conditioning necessary for autologous hematopoietic stem cell transplant (HSCT) to be fully effective. Aiming to address this, we have developed a strategy whereby a monoclonal antibody (mAb) that binds to CD117, a critical receptor on HSPCs essential for viability, is coupled with multiplexed engineered HSCs (eHSCs). Our eHSCs are designed to both evade mAb binding and harbor the Makassar therapeutic edit. Our engineered stem cell antibody paired evasion (ESCAPE) strategy is designed to provide a non-genotoxic alternative to current conditioning regimens and a potential curative therapy for the treatment of SCD.

In our ESCAPE-1 approach, we demonstrated that we could successfully engineer epitopes of the stem cell marker and receptor, CD117, by introducing missense mutations in the ligand binding domain that preserved normal CD117 function and prevented the binding of a high affinity antibody against wildtype (WT) CD117. We leveraged this selective advantage and screened guides that could install missense mutations that covered the entirety of the CD117 ectodomain and were compatible in a multiplexed edited HSC with a next-generation adenine base editor that could install the therapeutic Hb G-Makassar edit.

We generated a library of CD117 variants via highly-efficient base editing of the CD117 gene, in mobilized peripheral blood CD34⁺ HSPCs and then counter-screened these variants against a library of mAbs that bound to wild-type CD117. We identified an antagonist mAb that interacts in the ligand binding domain of CD117, blocking normal SCF ligand binding to the WT receptor, but was unable to bind eHSCs. In vitro biological de-risking of this engineered epitope showed no significant changes in receptor function in response to SCF, or during in vitro differentiation and colony forming unit (CFU) assays when compared to WT CD117. Furthermore, we could demonstrate that this engineered epitope allowed for evasion of cytotoxicity in vitro when edited HSCs were exposed to increasing concentrations of the mAb.

We next optimized the multiplex editing conditions in HSCs and achieved outcomes where all cells harboring a CD117 edit, also included at least one Makassar edit. We previously demonstrated that sickle cells harboring even a single Makassar edit exhibited reduced sickling when exposed to hypoxia in vitro. Subsequent F_c receptor silencing modifications were also optimized to reduce any mast cell degranulation that could be the result of antibody-based binding to F_c-receptor on mast cells.

Altogether, our ESCAPE-2 strategy is compatible with the installation of the therapeutic Makassar edit that, when combined with a mAb as a conditioning agent, represents a promising potential alternative to busulfan-based myeloablative regimens in an autologous HSCT setting for the treatment of SCD.

Chu:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Budak:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mondal:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhang:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Harmon:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lam:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lucini:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Coisman:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Qiao:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bai:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sen:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barrera:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cafferty:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kopesky:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonagh:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.