Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice

Abstract

Inherited hematologic defects that lack an in vivo selective advantage following gene correction may benefit from effective yet minimally toxic cytoreduction of endogenous hematopoietic stem cells (HSCs) prior to transplantation of gene-modified HSCs. We studied the efficacy of administering a novel sequential treatment of parenteral ACK2, an antibody that blocks KIT, followed by low-dose irradiation (LD-IR) for conditioning of wild-type and X-linked chronic granulomatous disease (X-CGD) mice. In wild-type mice, combining ACK2 and LD-IR profoundly decreased endogenous competitive long-term HSC repopulating activity, and permitted efficient and durable donor-derived HSC engraftment after congenic transplantation. ACK2 alone was ineffective. The combination of ACK2 and LD-IR was also effective conditioning in X-CGD mice for engraftment of X-CGD donor HSCs transduced ex vivo with a lentiviral vector. We conclude that combining ACK2 with LD-IR is a promising approach to effectively deplete endogenous HSCs and facilitate engraftment of transplanted donor HSCs.

Figure 1

Effect of ACK2 with low-dose irradiation treatment on marrow HSPC. (A) Schematic of treatment schedule using 2 mg ACK2 (IP) ± LD-IR at 300 cGy. Control mice received 0.5 cc saline on day 1. (B) Marrow HSCs (lin⁻ Sca-1⁺ KIT⁺ CD135⁻CD150⁺), HPCs (common myeloid progenitor lin⁻ Sca-1⁻KIT⁺ CD34^lowFcγR^low or, for the ACK2 treatment group at day 7, colony forming unit-granulocyte, monocyte) and cellularity at different times following treatment of F1 C57/BoyJ mice with ACK2, LD-IR, or ACK + LD-IR compared with control mice treated with saline. Data are either the number per 2 femurs or the percentage of the saline-treated cohort on the day of study; mean ± standard deviation (n = 3 to 4). *P < .05; **P < .01 versus control. (C) Relative LTRA was determined by a competitive repopulation assay. Marrow obtained at day 7 (Figure 1A) was studied from either control mice or mice conditioned with ACK2 (n = 4), LD-IR (n = 4), or ACK2 + LD-IR (n = 8). **P < .01.

Figure 2

ACK2 treatment plus LD-IR allows long-term engraftment of syngeneic wild-type bone marrow cells and gene-modified donor HSCs. (A) Posttransplantation donor chimerism of F1 C57/BoyJ mice conditioned with LD-IR (300 cGy) or ACK2 + LD-IR prior to transplantation with 1 × 10⁶ freshly isolated C57 marrow cells. Recipients received 2 mg ACK2 or saline on day 1 and LD-IR on day 4, followed by transplantation on day 7. Peripheral blood was sampled at different times posttransplantation. Solid squares, ACK2 + LD-IR cohort; gray diamonds, LD-IR cohort. Donor cell chimerism at 24 weeks in ACK2 + LD-IR cohort was 79.3 ± 8.3% versus 15.3 ± 4.6% in LD-IR cohort (**P < .01, n = 4). (B) Posttransplant donor chimerism of F1 C57/BoyJ mice conditioned with saline, ACK2 alone, LD-IR (300 cGy), or ACK2 + LD-IR prior to transplantation with 20 × 10⁶ (saline or ACK2) or 1 × 10⁶ (LD-IR or ACK2 + IR) freshly isolated C57 marrow cells. PB leukocyte donor chimerism was determined 24 weeks posttransplantation. Bars represent the mean donor cell CD45.2 chimerism. **P < .01 (n = 4 in each group). (C) Secondarily transplanted donor HSCs from ACK2-treated mice give rise to long-term engraftment in secondary recipients. Marrow from 2 primary recipients in each cohort shown in Figure 2A was harvested at 24 weeks posttransplantation and for each primary recipient, 1 × 10⁶ cells were transplanted into each of 4 lethally irradiated F1 C57/BoyJ mice. Donor chimerism in primary recipients was 3.4% and 5.2% for the 300 cGy mice and 71.2% and 69.3% for the ACK2 + LD-IR mice. Secondary recipients were analyzed for PB leukocyte donor chimerism 18 weeks after transplantation (mean ± standard deviation is shown). **P < .01 compared with 300 cGy cohort. One of 2 independent experiments is shown. (D) Conditioning with ACK2 + LD-IR prior to transplantation of lentivirus-tranduced cells in murine X-CGD. Lineage-negative BM cells from C57 X-CGD mice were purified using a MACS Separation System, then transduced in vitro with vesicular stomatitis virus–glycoprotein–pseudotyped CL20i4r-EF1a-gp91-OPT lentiviral vector overnight in the presence of interleukin-6 and SCF. Recipient F1C57/BoyJ X-CGD mice were treated with LD-IR (300 cGy) alone or ACK2 + LD-IR as described in panel A prior to transplantation with 5 × 10⁵ transduced X-CGD lin- BM cells. n = 4 in each group. PB leukocyte donor chimerism and neutrophil NADPH oxidase activity was assayed at various times after transplantation. Different colored symbols represent individual mice. *P < .02 compared with LD-IR cohort. One of 2 independent experiments is shown.