Effect and Kinetics of Depleting ACK-2 Anti C-Kit Monoclonal Antibody on Hematopoeisis and Hematopoetic Progenitors and Ability To Condition for Bone Marrow Transplantation.

Abstract

c-kit is expressed on hematopoetic stem and progenitor cell populations. ACK-2 is an anti-mouse c-Kit monoclonal antibody which has been shown (by Nishikawa et al) to antagonize the function of c-kit and deplete the bone marrow of treated mice. We wished to characterize the effect of the anti c-kit mAb ACK-2 on the peripheral blood cell counts and marrow hematopoetic stem cell and progenitor populations. We also tested the hypothesis that treatment with ACK-2 may serve as a novel means of non-myeloablative conditioning for hematopoeitic stem cell transplantation. METHODS: Adult C57 black mice were injected either intravenouslyl or intraperitonealy with 1mg of ACK2 mAb every other day on Day 0, Day 2 and Day 4. Peripheral blood cell counts, including white cell differentials were followed over time in recipient mice. Peripheral blood and bone marrow was analyzed at Day 7, and marrow was analyzed for fraction and proportions of hematopoetic stem cells (HSC), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and macrophage erythrocyte progenitors (MEP). A subset of Ly5.1 mice which had received ACK2 mAb on Days 0,2 and 4 received a bone marrow transplant on Day 7 with 1 Million bone marrow cells from a Ly5.2 donor. Peripheral blood from transplanted recipient mice was analyzed three weeks post transplant for presence of Ly5.2 donor derived cell engraftment. RESULTS: Both intravenous and intraperitoneal administration of ACK-2 resulted in rapid development of anemia, neutropenia and thrombocytopenia. In mice treated with intravenous ACK-2 on Days 0,2,4, the WBC dropped from a mean (3 mice) of 12.12 at Day 0 in untreated controls, to 6.6 at Day 2 to 4.2 at Day 4, and to 2.56 at day 7, recovering to a WBC of 9.5 by Day 11. The mean neutrophil dropped rapidly from a control mean of 796 prior to treatment, to 180 at Day 2, to 32 on Day 4, 26 on Day 7, recovering to a mean of 320 by Day 11. Hemoglobin dropped from a pretreatment mean of 15.1 to 14.5, 13.2, 6.2 and 5.3 on Days 2,4,7,11 respectively. Platelet counts fell from 1107 pre ACK-2 to 763 at Day 7 and 220 and Day 11, recovering to 1226 by Day 16. Analysis of Day 7 peripheral blood revealed an decrease in Mac-1+ cells from 12.2 of circulating white cells in untreated controls to 3.1%, while the proportion of B220+ B cells increased from 49.7 to 72.7%. The fraction of circulating T cells decreased from 24.5% to 11.1% at Day 7. The bone marrow fraction of KTLS HSC decreased from.07% in controls to.003% in Day 7 treated mice. Marrow fraction CMP decreased from 12% in untreated to 4.7% in treated, wheras the GMP population dropped from 66 to 53%. In ACK-2 treated mice which received 1 Million Ly5.2 syngeneic bone marrow cells analyzed at Day 21 post transplant, there was no evidence of Ly5.2+ donor derived peripheral blood white cells, as compared to 80% Ly5.2 donor derived cells in control mice which had received 9 Gray of radiation for conditioning. CONCLUSIONS: The anti-kit Monocolonal antibody rapidly induces anemia, neutropenia and thrombocytopenia with decreases in the marrow HSC and other progenitor populations. ACK-2 does not appear to facilitate non-myelablative conditioning for a syngeneic graft when given alone.