Motixafortide (CXCR4 Inhibition) Alone and in Combination with Natalizumab (VLA-4 Inhibition) As a Novel Regimen to Mobilize Hematopoietic Stem Cells for Gene Therapies in Sickle Cell Disease: A First-in-Human, Proof-of-Principle Safety and Feasibility Study

Autologous hematopoietic stem cell (HSC) based gene-modified therapies for sickle cell disease (SCD) offer potential cure and decreased toxicity relative to allogeneic hematopoietic cell transplant (HCT). HSC-based gene therapy requires collecting sufficient HSCs via peripheral blood (PB) mobilization to manufacture a gene-modified product. G-CSF mobilization is unsafe in SCD, while CXCR4 inhibition (CXCR4i) with plerixafor (P) alone requires multiple mobilization attempts and often yields suboptimal HSC numbers. Thus, developing novel, rapid, G-CSF-free HSC mobilization for gene therapy in SCD is needed. Motixafortide (M) is an optimized, high-affinity, long-acting CXCR4i approved for HSC mobilization for autologous HCT in myeloma. M mobilizes higher CD34+ HSC numbers relative to P, while preferentially mobilizing immunophenotypically and transcriptionally primitive HSCs. Natalizumab (N), a monoclonal antibody VLA-4 inhibitor (VLA4i), is a relatively weak HSC mobilizer in prior studies. Our pre-clinical data demonstrate combination CXCR4i + VLA4i may further enhance HSC mobilization.

Participants 18-40 yrs with SCD (SS or Sβ⁰) on automated RBC exchange were enrolled. RBC exchange occurred within 72h prior to mobilization. Participants received M (1.25mg/kg, subcutaneous injection) followed by 1 blood volume (BV) leukapheresis procedure (LP) ~14h post-M. After 8wks, a 2nd HSC mobilization with N (300mg, IV infusion) + M (~32h post-N) followed by 1 BV LP ~14h post-M (~46h post-N) was completed. Primary endpoint was safety/tolerability. Secondary endpoints were PB CD34+ cell mobilization kinetics, CD34+ cells/kg collected in 1 BV LP and predicted CD34+ cells/kg collected if 4 BV LP was performed. Immunophenotypic and transcriptional profiling of CD34+ HSCs was performed by flow cytometry (FACS) and single-cell RNA sequencing (scRNA seq).

Five completed mobilization and LP with M and 4 of 5 with N+M. Median age = 32 yrs, 40% female. M and N+M were safe and well-tolerated. Common adverse events (AEs) were transient, Grade 1-2 injection site (pruritis, 100%; tingling/pain, 22%) and systemic reactions (pruritis, 100%; hives, 33%). No Grade 4 AEs or vaso-occlusive events occurred. M mobilized median of 198 CD34+ cells/μl (range 77-690) to PB at 10-14h post-M, with median 3.49x10⁶ CD34+ cells/kg collected in 1 BV LP and 13.9x10⁶ CD34+ cells/kg if 4 BV LP was performed. N mobilized median 44 CD34+ cells/μl at 24-32h post-N. Whereas N+M mobilized median of 231 CD34+ cells/μl (range 117-408) at 14h post-M (~46h post-N), with median 4.64x10⁶ CD34+ cells/kg collected in 1 BV LP and 18.6x10⁶ CD34+ cells/kg if 4 BV LP was performed. Relative to M, N+M mobilized median of 1.55-fold higher PB CD34+ cells/μl (range 0.59-1.77). PB CD34+ cells/μl peaked at ~10-14h post-M but had not peaked at time of LP post-N+M. Two participants had mobilized with P alone on a prior HSC-based gene therapy study but did not receive gene therapy. Relative to P, M led to 2.7-fold higher PB CD34+ cells/μl and 2.8-fold higher CD34+ cells/kg; while N+M led to 2.8-fold higher PB CD34+ cells/μl and 3.2-fold higher CD34+ cells/kg. FACS immunophenotyping of N+M products vs M alone revealed increased absolute numbers of primitive HSCs (RA^-123^lo38^-90⁺49f⁺), multipotent and common myeloid progenitors (MPP/CMP) and high CXCR4 expressing lymphoid progenitors (CLP, B, NK); as well as decreased granulocyte/monocyte progenitors (GMP) and plasmacytoid dendritic cell progenitors (pDCP) (all p≤0.003). Transcriptional profiling by scRNA seq revealed marked upregulation of >100 genes with N+M vs M, including CXCR4 and multiple CLP-associated genes.

Here we present the first proof-of-principle report of optimized CXCR4i with M alone and combination VLA4i + CXCR4i with N+M to mobilize HSCs for potential gene therapy in SCD. M and N+M were safe, well-tolerated and resulted in robust CD34+ HSC mobilization to PB (198 and 231 CD34+ cells/μl) sufficient to enable collection of 13.9x10⁶ and 18.6x10⁶ CD34+ cells/kg in a single 4 BV LP, respectively. In those with prior P mobilization, M and N+M enabled 2.8- and 3.2-fold greater HSC mobilization, respectively. Immunophenotypic and transcriptional profiling of CD34+ HSCs mobilized with P, M and N+M is ongoing but preliminary data suggest increased mobilization of primitive HSCs, MPP/CMPs and CLPs; decreased pDCPs; and upregulated expression of CXCR4 and CLP-associated genes with N+M vs CXCR4i alone.

Crees:BioLineRx, Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sorani:BioLineRx, Ltd.: Current Employment. King:UpToDate: Patents & Royalties; Cigna: Consultancy. DiPersio:RiverVest: Consultancy, Other: consulting fees; Incyte: Other: grant; BioLineRx: Consultancy, Other: grant, consulting fees; Wugen: Other: grant, Patents & Royalties: royalties/licenses ; MacroGenics: Other: grant; Magenta Therapeutics: Patents & Royalties: royalties/licenses ; Vertex Pharmaceuticals: Consultancy, Other: consulting fees.

Motixafortide is a CXCR4 inhibitor that is US FDA approved, in combination with G-CSF, to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous hematopoietic cell transplantation in patients with multiple myeloma. Natalizumab is a monoclonal antibody VLA-4 inhibitor that is US FDA approved for the treatment of multiple sclerosis and Crohn's disease. In this study, we evaluate Motixafortide alone and in combination with Natalizumab to mobilize hematopoietic stem cells to the peripheral blood for collection and potential use in hematopoietic stem cell-based gene therapies.