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Development of Conditioning Regimens Based on HSC-Targeted Antibody ACK2
Background: Marrow conditioning prior to HSC transplantation incorporates chemotherapy and/or radiotherapy to eradicate malignant disease, eliminate immunologic barriers to allogeneic cell engraftment, and to ‘make space’ for donor HSC within the HSC niche. The sequelae of such treatments can be substantial, including direct organ toxicity. Selective targeting of HSC with monoclonal antibodies presents an alternative approach to induce stem cell depletion with fewer off target effects. Previous studies in a congenic mouse transplant model established that antibody-mediated blockade of c-Kit, the receptor for stem cell factor, given in combination with low dose irradiation (LD-IR) efficiently depletes HSC and substantially enhances engraftment of donor HSC in immunocompetent mice (Xue et al, Blood 2010). However, since even low doses of irradiation have long-term risks, particularly in children, an important goal is to develop improved regimens utilizing c-Kit blockade.
Objective: To determine if replacing low dose irradiation (LD-IR) with chemotherapy will synergize with c-Kit blockade and improve engraftment in a congenic mouse transplant model.
Methods: Mice were transplanted according to standard protocol. C57B6 (CD45.2) mice were used as donors, and F1 progeny of C57B6 and BoyJ mice (CD45.1) were used as recipients. Recipient mice were treated with 2 mg of the KIT-blocking monoclonal antibody ACK2 (injected intraperitoneally) combined with various schedules of Fludarabine 100mg/kg IP / Cyclophosphamide 200mg/kg IP (FluCy); or 5-flurouracil (5-FU )150 mg/kg IP; or Busulfan (Bu) 20mg/kg IP. Control mice were treated with the ACK2 and 300 cGy. This was followed by transplantation of 1- 2 million freshly isolated congenic marrow cells and subsequent long-term engraftment analysis by flow cytometry.
Results:In comparison to ACK2/LD-IR, the level of engraftment post-transplant was much lower in all of the other treatment arms. ACK2/FluCy had < 5% engraftment at 13 weeks compared to 80% engraftment in the ACK2/LD-IR arm. ACK2/Bu had < 5% engraftment compared to 70% in the ACK2/LD-IR arm at 20 weeks and ACK2/5-FU had < 5% compared to 80% engraftment at 13 weeks in the ACK2/LDR arm.
Conclusion: Our study demonstrates that combination of antibody-mediated c-Kit blockade and chemotherapy did not enhance congenic donor cell engraftment, at least in the regimens tested. Further experiments to elucidate how LD-IR and c-Kit blockade synergize to deplete HSCs and enhance engraftment are ongoing.