Immunotherapy for rapid bone marrow conditioning and leukemia depletion that allows efficient hematopoietic stem cell transplantation

Abstract

Hematopoietic stem cell transplantation (HSCT) is a life-saving procedure to treat hematopoietic disorders. Current bone marrow conditioning protocols create space for healthy donor stem cells by employing irradiation and/or chemotherapy, but carry severe toxicities, resulting in significant morbidity, mortality and substantial long-term complications. To develop a low-toxicity solution, we generated a bi-specific T-cell engager (BTCE) that targets CD117, an abundantly expressed receptor on hematopoietic stem and progenitor cells (HSPC) and leukemia-initiating cells (LICs). We show that the CD117×CD3 BTCE efficiently depletes in vitro and in vivo HSPCs and LICs. The CD117×CD3 BTCE was not toxic and facilitates highly efficient engraftment of human allogenic donor CD34+cells in humanized mice, thereby restoring hematopoiesis in vivo in both normal and leukemia-bearing humanized mice. We demonstrate here that a potent CD117×CD3 BTCE enables rapid HSCT in both benign and malignant conditions.

Keywords: Bispecific T cell engager - BiTE; Immunotherapy; Leukemia; Pharmacokinetics - PK; Stem cell.

Figure 1. Binding specificity of CD117×CD3 BTCE. (A) Schematic representation of CD117×CD3 and HEL×CD3 BTCEs. Heavy chains (CH and VH) are in dark colors, and light chains (Cκ, Cλ, Vκ and Vλ) are in light colors. (B) FACS plots showing binding of CD117×CD3 and control HEL×CD3 BTCEs to CD117 receptors on TF-1 cells. Negative controls U937 and RPMI-8226 do not express CD117 or CD3. Data are representative of three independent experiments. (C) EC50 of CD117×CD3 BTCE binding to TF-1 cells as determined by flow cytometry. Data are plotted as relative median fluorescent intensity and fitted to a dose-response non-linear regression model. Data are representative of three independent experiments. Error bars indicate SEM. (D) FACS plots showing the binding of CD117×CD3 and control HEL×CD3 BTCEs to CD3 receptor expressed on purified human T cells, CD4+ and CD8+ T-cell subsets in human PBMCs. PBMCs and purified T cells pretreated with blocking anti-CD3ε antibodies are also shown. Data are representative of three independent experiments. BTCE, bi-specific T-cell engager; FACS, fluorescent-activated cell sorter; PBMC, peripheral blood mononuclear cell.

Figure 2. Cytotoxic activity of CD117×CD3 BTCE in vitro. (A) Representative FACS plot showing CD117×CD3 BTCE dose-dependent T-cell proliferation, as determined by FarRed CellTrace signal dilution, when human T cells and TF-1 target cells are co-cultured. Data are representative of three independent experiments. (B) Representative FACS plots showing the percentage of T cells with diluted FarRed CellTrace signals in the indicated conditions. Data are representative of three independent experiments. (C) Percentage of CD4+ (triangles) and CD8+ (circles) T-cell subsets expressing the indicated activation markers CD69 (top) and CD25 (bottom) in the presence of TF-1 target cells and indicated reagents are shown. Data points are the average of three experiments. (D) Dose-dependent CD117×CD3 BTCE-mediated cytotoxicity of TF-1 cells after 6 hours of co-culture with human T cells at indicated E:T ratios. E:T ratio is the ratio of T cells to TF-1 cells. (E) Percentage of T cell-mediated cytotoxicity of primary human BM CD34+cells (n=4 donors) when treated with 1.1 nM of indicated BTCE and co-culture with non-HLA-matched healthy donor-derived T cells (E:T ratio of 10:1) for 24 hours is given. Colors indicate individual BM CD34+cell samples. The two-tailed Student’s t-test was used for statistical analysis. ***p<0.001. (F) FACS plots showing Annexin-V+TF-1 cells (red boxes) after 6 hours of co-culture with T cells (upper panels, E:T ratio of 10:1) or without T cells (lower panels) and indicated BTCE treatments. Error bars in C, D and E represent SEM. BM, bone marrow; BTCE, bi-specific T-cell engagers; E:T, effector to target cell ratio; FACS, fluorescent-activated cell sorter.

Figure 3. In vivo depletion of human CD117+hematopoietic stem and progenitor cells by CD117×CD3 BTCE. (A) Concentrations of CD117×CD3 BTCE in the serum of C57BL/6 mice at indicated time points post CD117×CD3 BTCE treatment are shown (n =3–5 animals per group). The calculated T_1/2 is depicted in red. (B) FACS plot showing the level of CD117×CD3 BTCEs on human T cells in the spleen of huCD34-NSG mice at indicated time points post treatment. CD117×CD3 positive control indicates ex vivo staining with the CD117×CD3 reagent. (C) Schematic overview of CD117×CD3 BTCE treatment of huCD34-NSG mice and the time points of harvesting hematopoietic tissues. (D) Representative FACS plots showing the percentage of human CD34+CD117+ cells (closed line gate) and CD34+CD117 cells (dashed line gate) of total human CD45+cells in the BM of huCD34-NSG mice after indicated treatment and at specified time points. (E) Percentages and absolute numbers of CD34+CD117+ (left panel) and CD34+CD117 cells (right panel) in the BM of huCD34-NSG mice after indicated treatment and at specified time points are plotted. For figure E, a one-way analysis of variance with Dunnett’s multiple comparisons test was used for statistical analysis. Horizontal lines in E represent average. Error bars in A and E represent SEM. **p<0.01, ***p<0.001, ****p<0.0001. FDR, False Discovery Rate; BM, bone marrow; BTCE, bi-specific T-cell engagers; FACS, fluorescent-activated cell sorter; ns, not significant.

Figure 4. In-depth immunophenotype of the major hematopoietic cell subsets in the BM of huCD34-NSG mice after CD117×CD3 BTCE treatment. (A) Population cluster identification in high-dimensional 41-color flow cytometry data using Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction. Cell populations in the BM of five CD117×CD3 BTCE and five HEL×CD3 BTCE-treated huCD34-NSG mice at 5 days post-treatment are shown in an overlay plot. (B) Overlay UMAP plots of BM cells of five representative huCD34-NSG mice treated with indicated BTCE and isolated at 5 days post-treatment are shown. cDC-2 are indicated by red circles. (C) Volcano plot showing the fold change of five individual BM cell populations of CD117×CD3 BTCE-treated huCD34-NSG compared with HEL×CD3-treated controls. A significant positive fold change indicates that the cellular population was decreased in CD117×CD3 BTCE-treated huCD34-NSG mice. (D) Percentage of cDCs-2 in the BM of huCD34-NSG mice treated with indicated BTCE (n=5 per group) at 5 days post-treatment. The two-tailed Student’s t-test was used for statistical analysis. Lines represent average. *p<0.05. (E) Concentrations of indicated cytokines in the serum of CD117xCD3 BTCE-treated and HEL×CD3 BTCE-treated huCD34-NSG mice at specified time points are shown. Results are representative of two independent experiments. (F) Absolute numbers of human T cells in the BM of huCD34-NSG mice (n=5 or 6 animals per group) after indicated treatment and at the specified time points are shown. A one-way analysis of variance with Dunnett’s multiple comparisons test was used for statistical analysis. ***p<0.001, ns=not significant. Horizontal lines represent average. Error bars in D, E and F represent SEM. BM, bone marrow; BTCE, bi-specific T-cell engagers; cDC2, conventional dendritic cell type 2; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Figure 5. CD117×CD3 BTCE treatment allows rapid and highly efficient allogenic HSCT in humanized NSG mice. (A) Schematic overview of the treatments for CD117×CD3 BTCE-mediated HSPC depletion and allogenic HSCT of huCD34-NSG mice. Control mice received HEL×CD3 BTCE. ACK2 anti-mouse CD117 blocking antibody. (B) STR genotyping of DNA isolated from human BM cells at 6 weeks post transplantation of huCD34-NSG mice. Recipient BM cells (top), CD34+donor cells (middle) and BM cells from transplanted mice (bottom) are shown. Peaks indicate amplicons, and the chromosomal localization of the amplicons is indicated by the green boxes at the x-axis. (C, D) Representative FACS plots showing the percentage of huCD45+HLA-A2+ donor and huCD45+HLA-A3+ recipient cells in the BM and PB of BTCE-treated huCD34-NSG mice at 6 weeks post-HSCT. FACS plots are representative of four mice per group. (E) Absolute numbers of human CD45+HLA-A2+ in BM (left) and percentage of CD45+HLA-A2+ cells in PB (right) in huCD34-NSG mice (n=4) treated with indicated BTCE at 6 weeks post-HSCT are shown. The two-tailed Student’s t-test was used for statistical analysis. Lines represent average. Error bars in E and F represent SEM. *p<0.05, **p<0.01. (F) Absolute numbers of huCD45+HLA-A2+ and huCD45+HLA-A3+ in immune cell populations in the BM of huCD34-NSG mice (n=4) treated with indicated BTCE at 6 weeks post-HSCT are shown. BM, bone marrow; BTCE, bi-specific T-cell engagers; DPH, diphenhydramine hydrochloride; HSCT, hematopoietic stem cell transplantation; PB, peripheral blood.

Figure 6. CD117×CD3 BTCE efficiently depletes AML and LICs. (A) Percentage of T cell-mediated depletion of indicated CD117+cells, when treated with 1.1 nM of indicated BTCE and co-cultured with human T cells (effector to target cell ratio of 4:1) for 24 hours (Kasumi-1) or 48 hours (primary AML samples and AML-PDX-CD117^ASP(816)-VAL in vitro) are shown. AML1 expresses the highest level of CD117, followed by AML2. AML3 and AML 4 express low CD117 levels. Data are representative of three experiments. (B) Schematic overview of the CD117×CD3 BTCE-mediated AML depletion and secondary recipient transplantation protocol of AML-PDX transplanted mice. (C) Absolute numbers of huCD117+AML PDX cells (left) and huCD33+AML PDX cells (right) in the BM of AML-PDX-transplanted mice (n=4 or 5 animals per group) at 36 hours post indicated BTCE treatments are shown. (D) NSG mice receiving BM from BTCE-treated AML-PDX animals. Percentages of huCD45+AML PDX cells in the PB of secondary transplanted NSG mice (n=6 animals per group) after indicated treatments and at specified time points are shown. (E) Absolute numbers of human CD45+cells in the BM (left) and spleen (right) of NSG mice (n=6 animals per group) at 6 months post-secondary BM transplantation depicted. (F) Schematic overview of AML-PDX depletion and allogenic HSCT protocol of AML-PDX mice. (G) Percentage of donor HLA-A3- HLA-B7+cells within the BM (left), huCD34+ (central) and huCD117+ (right) in the BM of AML-PDX mice (n=4 or 5 animals per group) after indicated treatments (F) and at 6 weeks post-HSCT. For figures A, C, E and G, the two-tailed Student’s t-test was used for statistical analysis. Lines (A, C, E, G) represent average, error bars in A, C, D, E, and G represent SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. AML, acute myeloid leukemia; BM, bone marrow; BTCE, bi-specific T-cell engagers; DPH, diphenhydramine hydrochloride; HSCT, hematopoietic stem cell transplantation; PB, peripheral blood; PDX, patient-derived xenograft.