Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment

Abstract

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit⁺ population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue.

Keywords: CAR-T cells; CXCR4; c-kit; hematopoietic stem cell transplantation; immunotherapy.

Figure 1

Generation and Evaluation of c-kit CAR-T Cells (A) Schematic map of c-kit CAR γ-retrovirus. (B) Timeline of CAR-T cell production and evaluation. (C) FACS analysis (day 3) of c-kit or VEGFR-2 CAR expression detected by anti-murine Fab (top row). Specific binding of Fc-conjugated murine c-kit or VEGFR-2 with c-kit CAR or VEGFR-2 CAR was detected with anti-Fc fluorescent antibody (middle and bottom rows). (D) Time course of c-kit or VEGFR-2 CAR expression (mean ± SD; n = 5; **p < 0.01 compared with mock-transduced T cells). (E) FACS analysis of c-kit expression on NIH 3T3 or E2a-PBX (CD19⁺) transduced with c-kit lentivector compared to naive. (F) Generation of IFNγ from c-kit CAR-T cells co-cultured with target lines expressing recombinant murine c-kit (mean ± SD; n = 5; **p < 0.01 comparison as indicated). (G) GFP photomicroscopy images of GFP⁺ NIH 3T3 (naive or c-kit transduced) co-cultured 24 hr with T or CAR-T cells as indicated after PBS rinse. (H) FACS analysis at 24-hr co-culture assessing cytotoxicity of c-kit CAR-T cells toward c-kit⁺ E2a-PBX that express CD19. Controls include naive T- or VEGFR-2 CAR-T cells. (I) Percentage of the c-kit⁺ E2a-PBX within the total E2a-PBX remaining at 24 hr in the cytotoxicity co-cultures, as described for (H) (n = 5; mean ± SD; **p < 0.01).

Figure 2

Congenic Thy1.1 c-kit CAR-T Cell-Mediated Depletion of HSC from Thy1.2 BM In Vitro (A) Timeline of CAR-T production and effect on Thy1.2 BM in vitro. (B) FACS analysis of c-kit⁺ cells in BM. (C) IFNγ secretion by Thy1.1 T or CAR-T cells in 24-hr co-culture at decreasing cell ratios with Thy1.2 BM (n = 5; mean ± SD; **p < 0.01). (D) FACS analysis assessment of Thy1.1 c-kit CAR-T cell-mediated depletion of c-kit⁺ Thy1.2 BM cells in 1:1 cell ratio co-culture at 24 hr. Percentge of total cell viability in the culture dishes, percentage of c-kit⁺ cells in BM population, and their mean fluorescence intensity (MFI) are shown in each panel. (E) Summary results of FACS analyses of the same cytotoxicity co-culture as (D), but at different ratios of T cells to BM (n = 5; mean ± SD; **p < 0.01 and *p < 0.05). (F) Photograph of colony formation assays following Thy1.1 c-kit CAR-T cell-mediated depletion of c-kit⁺ Thy1.2 BM cells in 1:1 cell ratio co-culture that was plated at 24 hr and read at 10 days. (G) Colony formation numbers in the same experimental conditions and analysis as (F) but at different ratios of T cells to BM (n = 5; mean ± SD; ***p <0.001 and **p < 0.01).

Figure 3

Trafficking and Expansion of Thy1.1 c-kit CAR-T Cells in Thy1.2 Mice (A) Timeline of Thy1.1 CAR-T cell production, in vivo administration, expansion, and trafficking. (B) In vivo whole-body luciferase luminescence imaging overlaid on X-ray image at day 8 following injection of luciferase⁺ T (or CAR-T) cells with or without CY pre-treatment, where white arrow indicates spleen. Additional mice are shown in Figure S3. (C) FACS analysis of CXCR4 and c-kit CAR on T cells transduced with c-kit CAR alone (upper panel) or co-transduced with CXCR4 (lower panel). (D) In vivo whole-body luciferase luminescence imaging overlaid on X-ray image at day 8 following tail vein injection of luciferase⁺ CXCR4⁺ c-kit CAR-T cells into CY-pre-treated mice (right image). White arrows show sites suggestive of BM activity. Additional mice are shown in Figure S3. (E) FACS detection in BM of c-kit CAR or CXCR4⁺ c-kit CAR-T cells without or with CY pre-treatment as indicated at days 4 and 8 after CAR-T cell administration. (F) Time course of changes in percent (left panel) and actual cell numbers (right panel) of c-kit CAR-T cells in BM using the same conditions as indicated in (E) (n = 5; mean ± SD; ***p <0.001 and *p < 0.05).

Figure 4

In Vivo Depletion of BM Cells after CY Only or after CY + CXCR4⁺ c-kit CAR-T Cell Treatment (A) Time course of PB hemoglobin (Hgb), platelets (Plt), total leukocyte (WBC), and absolute neutrophil (Neut) count following CY alone (blue), CY + CXCR4⁻ c-kit CAR-T (orange), or CY + CXCR4⁺ c-kit CAR-T cell (red) compared with the naive (purple) (n = 5; mean ± SD; ***p < 0.001, **p < 0.01, and *p < 0.05). (B) Photomicrographs of H&E-stained tibia sections from naive mouse or mice following CY only, CY + CXCR4⁻ c-kit CAR-T, or CY + CXCR4⁺ c-kit CAR-T cell injection. (C) Photographs of in vitro colony formation assay of BM cells from naive mice or mice on day 12 following CY only, CY + CXCR4⁻ c-kit CAR-T, or CY + CXCR4⁺ c-kit CAR-T cell injection. Numbers of colonies per plate are shown in the graph (n = 5; mean ± SD; ***p < 0.001; NS, not significant). (D) FACS analysis of BM obtained from naive mice or from mice at days 4 and 8 after treatment with CY only, CY + CXCR4⁻ c-kit CAR-T, or CY + CXCR4⁺ c-kit CAR-T cells, where boxes show percentage of KSL (c-kit⁺sca1⁺Lineage⁻) cells. (E) Time course of the same FACS analyses of mice as indicated in (D), but calculating and plotting absolute number of BM, c-kit⁺, KSL, or LT-HSC cells per 2 femurs per mouse (n = 3 [5 mice/experiment; 15 mice total]; mean ± SD; ***p < 0.001, **p < 0.01, and *p < 0.05).

Figure 5

CY Plus Congenic Thy1.1⁺ CD45.2⁺ CXCR4⁺ c-kit CAR-T Cell Facilitated Engraftment of Congenic Donor Thy1.2⁺ CD45.1⁺ BM into Thy1.2⁺ CD45.2⁺ C57BL/6J Mice following the Depletion of CAR-T Cells with Anti-Thy1.1 Antibody (A) Timeline of c-kit CAR-T cell production and in vivo injection followed by Thy1.1 antibody depletion of c-kit CAR-T cells, transplantation of donor BM, and subsequent evaluation of donor engraftment. (B) FACS analysis of congenic Thy1.1⁺ CD45.2⁺ CXCR4⁺ c-kit CAR-T cells in recipient BM at day 8 comparing before and 24 hr after (day 9) i.p. injection of anti-Thy1.1 antibody (upper versus lower panels). (C) Percentage of all LT-HSCs in recipient BM that are donor phenotype by FACS analysis at 12 weeks after transplant without conditioning, with CY alone, or after CY plus c-kit CAR-T cell conditioning and Thy1.1 antibody depletion of the c-kit CAR-T cells (n = 5; mean ± SD; ***p < 0.001). (D) Time course of percentage of B cell, T cell, and granulocyte lineages in the PB that are donor phenotype by FACS analysis at 0, 4, 8, and 12 weeks and 6 and 9 months after transplant mediated by CY plus c-kit CAR-T cell conditioning and Thy1.1 antibody depletion of the c-kit CAR-T cells (n = 3 [5 mice/experiment; 15 mice total]; mean ± SD; ***p < 0.001 and **p < 0.01).

Figure 6

Treatment of gp91-phox-Deficient CGD Mice with Normal Wild-Type BM Transplantation Using Sequential CY + CAR-T + Thy1.1 Ab Conditioning (A) Timeline of c-kit CAR-T cell production and in vivo injection into CGD mice followed by Thy1.1 antibody depletion of c-kit CAR-T cells, transplantation of donor normal wild-type BM, and subsequent evaluation of donor engraftment and assessment of the appearance of DHR⁺ oxidase normal granulocyte in the PB of transplanted CGD mice. (B) DHR flow cytometry analysis of ROS production by PB granulocytes without or with PMA stimulation from a normal wild-type mouse (middle and left panels) or CGD mouse (right panel) (gated on granulocyte Gr1⁺Mac1⁺ population). (C) FACS analysis of BM obtained from untreated and CY + CAR-T-treated CGD mice at day 8, where boxes show percentage of KSL (c-kit⁺sca1⁺Lineage⁻) cells. (D) Normal wild-type donor PB granulocyte chimerism (CD45.1) in c-kit CAR-T cell-conditioned CGD mice (CD45.2) was assessed together with measurement of DHR oxidase activity (PMA stimulation) before transplantation (left panel) and 12 weeks after transplantation of normal wild-type BM into CGD mice conditioned with c-kit CAR-T cells (right panel). Time course of percentage of DHR⁺ cells (in granulocyte fraction) is shown in right panel at 0, 4, 8, and 12 weeks after transplantation (n = 5; mean ± SD; ***p < 0.001).