Mgta-456 Contains Large Numbers of Expanded Cord Blood (CB) CD34+CD90+ Hematopoietic Stem Cells (HSC) Which Confer All Engraftment Activity and Correlate with Accelerated Neutrophil Recovery after Myeloablative Conditioning in Patients with Hematologic Malignancy

Background: Patient access to well-matched CB containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and a risk of graft failure relative to other hematopoietic stem cell (HSC) sources. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. Thus far, 40 patients with hematological malignancy (n=36) and non-malignant diseases (n=4) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (20 to 143 days) respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p<0.01). The aim of the current study was to fully characterize the expanded CD34+ cell fraction of MGTA-456 phenotypically and functionally and identify the cell population that correlates with time to neutrophil recovery. We found that the expanded CD34+CD90+ population of MGTA-456 were the cells responsible for engraftment in NOD-scid IL2Rgammanull (NSG) mice. We hypothesized that the dose of CD34+CD90+ cells/kg would have the strongest correlation with time to neutrophil recovery.

Methods: The expansion culture consisted of StemSpan SFEM supplemented with SCF, FLT- 3L, TPO and IL-6 (all at 50 ng/mL) and the AHRa without the addition of antibiotics. After a 10 or 15 day culture, the CD34 expanded product was characterized by cell surface markers (CD34, CD90, CD133, CD41, CD71, CD235a, CD3, CD4, CD8, CD14, CD15, CD16, CD11b, CD33, CD19, CD56, and CD10), as well as colony-forming unit (CFU) capacity and engraftment potential in NOD-scid IL2Rgammanull (NSG) mice in a subset of products.

Results: The expanded CD34+ cell fraction of MGTA-456 consisted of CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded fraction contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present (<1%) in the CD34 expanded fraction of MGTA-456. To identify which of these cell populations contain the NSG engraftment activity, we sorted CD34-, CD34+CD90- and CD34+CD90+ cells from MGTA-456 and transplanted the cell fractions into NSG mice. These studies clearly demonstrated that all NSG engraftment activity resided in the CD34+CD90+ cell subpopulation (Figure 2). Based on this result, we correlated the dose of CD34-, CD34+CD90-, CD34+CD90+ cells, and CFU cells/kg infused with time to neutrophil recovery of patients treated with MGTA-456. The dose of TNC (r²=0.49, P<0.05), CD34+CD90- (r²=0.45, p<0.05), and CD34+CD90+ (r²=0.52, p<0.05) cells/kg all significantly correlated with time to neutrophil recovery. The dose of CD34+CD90+ cells/kg had the strongest correlation with time to neutrophil recovery consistent with the NSG engraftment result.

Conclusion: In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large doses of CD34+CD90+ HSC and progenitors, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically.

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