BN-CD38 spares HSCs and noncancer immune cells. (A) Healthy donor (HD)-derived total BM MNCs were treated with 1.0 ng/mL control human IgG (n = 7), CD38 NB (n = 3), BN-CD38Mut (n = 7), and BN-CD38 (n = 7) and plated in CFC assay for 14 days. Representative images of CFC assay for 2 representative HDs’ BM MNCs showing effect of 4 treatments. Images were acquired in tiles by the City of Hope microscopy core facility using ZEN 3.1 (blue edition, Carl Zeiss Microscopy GmbH). (B) Violin plot comparing CFU of each treatment group. CFU of each treatment group was normalized to control human IgG for each HD and shown as F.C. over IgG. One-way ANOVA with multiple comparisons was used to calculate statistical significance. (C-E) Three HD-derived total BM MNCs were treated with 1.0 ng/mL control human IgG, CD38 NB, BN-CD38Mut, or BN-CD38. BM MNCs were collected at 48 and 120 hours after treatment and subjected to surface staining with CD45, CD34, and CD38. (C) Representative dot plot of 2 HDs’ BM MNCs showing effect of control human IgG, CD38NB, BN-CD38Mut, and BN-CD38 treatment on CD34^posCD38^pos healthy progenitors and CD34^posCD38^neg HSCs. (D-E) Violin plots compare the effect of different treatment groups on CD34^posCD38^pos healthy progenitors and CD34^posCD38^neg HSCs after treatment. The percent CD34^posCD38^pos and percent CD34^posCD38^neg of each treatment group was normalized to control human IgG and shown as F.C. over IgG. One-way ANOVA with multiple comparisons was used to calculate statistical significance. (F-H) HDs’ total BM MNCs (n = 3) treated in panel B were gated in total CD4 or CD8 T cells and each population was assessed for CD69 expression. (F) Violin plots compare the percent CD4^posCD69^pos and percent CD8^posCD69^pos activated T cells at 48 hours. One-way ANOVA with multiple comparisons was used to calculate statistical significance for T-cell activation. (G-H) Violin plots compare percent CD4^pos and percent CD8^pos T cells over the total cellular population between different treatment groups at 48 and 120 hours. Paired Student t test was used to compare percent frequencies of T cells between treatment groups. (I) HD-derived total PB MNCs were treated with 1.0 ng/mL control human IgG, CD38 NB, BN-CD38Mut, or BN-CD38 for 72 hours and gated for monocytes (n = 5), NK cells (n = 4), B cells (n = 4), and CD4^pos and CD8^pos T cells (n = 4), followed by gating with DAPI to assess percent killing. Violin plots compare percent killing (DAPI positivity) between different treatment groups. One-way ANOVA with multiple comparisons was used to calculate statistical significance. (J) Representative dot plot showing CD38 expression in CD45^Dim population after treatment of bulk MNCs with 1.0 ng/mL of BN-CD38, BN-CD38Mut, and control human IgG. (K) Violin plot comparing CD38 surface expression in CD45^Dim population of AML (n = 7: 5 PB, 1 BM, and 1 leukapheresis [LP]) and HD (n = 3 BM) MNCs treated with 1.0 ng/mL of control human IgG, BN-CD38Mut, or BN-CD38 for 48 hours. BN-CD38 and BN-CD38Mut CD38 surface expression measured by flow cytometry in MFI were normalized to control human IgG. Ordinary 1-way ANOVA with Dunnett multiple comparisons test was used to calculate significance; ∗P < .05. (L) Total CD34^pos cells (HSCs and CD38⁺ progenitors) were isolated from HDs and cocultured with THP1 GFP^pos cells (CD34^neg; supplemental Figure 6J-K) at a HSCs:THP1 ratio of 1:10, and T cells were added at an E:T (T cells:THP1) of 1:1 overnight in the presence of increasing doses of BN-CD38. The experiment was repeated using n = 2 donors for a total of 4 independent replicates for each point. DAPI^neg THP1 GFP and CD34^pos alive cell frequencies were determined by gating in the GFP or CD34^pos populations, respectively. Each dose frequency was normalized to vehicle control, and simple linear regression analyses were used to determine the BN-CD38 dose effect on THP1 and total CD34^pos cells. (M) Schematic representation of the generation and treatment of CD34^pos humanized NSG mouse model. Specifically, 5 × 10⁵ CD34^pos selected cells from HDs were IV injected into irradiated NSG mice. Once engraftment of human CD45^pos cells was confirmed on day 150, mice were randomized into 3 groups: control human IgG (n = 4), BN-CD38Mut (n = 4), and BN-CD38 (n = 5). Each mouse was IV treated with 2.5 mg/kg and 3 million autologous T cells, as indicated. Following 3 treatments, mice were euthanized on day 173, and total BM cells were isolated and subjected to flow cytometry analysis for human immune cell engraftment (hCD45⁺). (N) Representative contour plots of hCD34 in hCD45^pos selected cells. One representative mouse is shown for each treatment group. (O) Bar graph comparing percent of different immune subsets in human CD45^pos selected cells in BM, as indicated. Ordinary 1-way ANOVA with multiple comparisons was used as statistical test. ns, not significant.