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.