Targeted hematopoietic stem cell depletion through SCF-blockade

Abstract

Background: Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However, HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections, graft vs. host disease, infertility, and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models, which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches, though none are optimal and how any of these mAbs fully function is unknown.

Methods: To improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings, it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here, we compare the antagonistic properties of αCD117 mAb clones including ACK2, 2B8, and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further, to augment efficacy, combination regimens were also explored.

Results: We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further, we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings, but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings, highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice.

Conclusion: These findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further, they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide.

Fig. 1

αCD117 ACK2 mAb has the unique property of inhibiting SCF binding which is maintained with Fab, F(ab’)2 and deglycosylated mAb derivatives. Representative flow cytometry plots showing the binding of αCD117 mAb to mouse HSCs (Lin⁻Sca-1⁺CD150⁺CD48⁻CD244⁻) detected by goat-α-Rat-FITC (left), fluorophore labeled mSCF-A647 (center), and various αCD117 mAbs followed by mSCF-AF647 (right) with overlayed graphs of A untreated control (Ctl) and αCD117 mAbs (clone ACK2, 2B8, and 3C11). B αCD117 ACK2 mAb compared to ACK2 fragments Fab, F(ab’)2, and mAb with deglycosylated Fc were subsequently similarly tested for SCF-AF467 binding antagonistic capacities (n = 5–7)

Fig. 2

Only αCD117 ACK2 mAb and its derivatives robustly inhibit HSC growth and differentiation in vitro. FACS sorted mouse HSCs (Lin⁻Sca-1⁺CD150⁺CD48⁻CD244⁻) were cultured for 4 days with A different αCD117 mAb clones and B ACK2 mAb derivatives (Fab, F(ab’)2, and mAb with deglycosylated Fc) to determine effects on HSPC growth kinetics as assessed by light microscopy (n = 5–7). Statistics calculated using unpaired t-test compared with untreated controls (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 3

αCD117 ACK2 mAb and its fragment derivates uniquely deplete HSCs in SCID settings. A Experimental outline to assess the time of clearance of each αCD117 mAb (clone ACK2, 2B8, 3C11) or each αCD117 ACK2 mAb derivatives (Fab, F(ab’)2, and mAb with deglycosylated Fc) in the SCID mouse model with subsequent assessment of HSC depletion at time of mAb or fragment clearance. B BM HSC depletion was measured when αCD117 mAbs were cleared in peripheral blood (PB) serum and compared to untreated control mice. Profound BM HSC depletion was only observed post treatment with αCD117 ACK2 mAb as determined by phenotypic assessment with flow cytometry (Lin⁻Sca-1⁺CD117⁺CD150⁺CD48⁻) and functional assessment of HSPCs by in vitro colony forming capacity. (C) Subsequently, a parallel experiment was performed assessing αCD117 ACK2 mAb fragment derivatives’ depletion capacity with similarly robust HSC depletion observed in all treatment groups as determined by phenotypic assessment by flow cytometry (Lin⁻Sca-1⁺CD117⁺CD150⁺CD48⁻) and functional assessment by in vitro colony forming capacity. (n = 5). Statistics calculated using unpaired t-test compared with untreated controls (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 4

αCD117 ACK2 mAb and its fragment derivates uniquely enhance donor engraftment post HSCT in SCID settings. A Experimental outline to assess the conditioning efficacy of each αCD117 mAb (clone ACK2, 2B8, 3C11) or each αCD117 ACK2 mAb derivatives (Fab, F(ab’)2, and mAb with deglycosylated Fc) in the SCID mouse model with subsequent assessment of donor chimerism post HSCT. B PB donor chimerism and BM HSC donor chimerism were measured 20 weeks post HSCT of αCD117 mAb conditioned animals with 4 × 10⁶ CD45.1 donor cKIT⁺ enriched cells. Enhanced donor engraftment was only observed post conditioning with αCD117 ACK2 mAb, whereas αCD117 2B8 and 3C11 mAbs decreased donor engraftment (n = 5). C Subsequently, a parallel experiment was performed assessing αCD117 ACK2 mAb fragment derivatives’ conditioning capacity with similarly robust donor engraftment observed with full intact αCD117 ACK2 mAb and its Fab, F(ab’)2, and deglycosylated Fc derivative (n = 5). Statistics calculated using unpaired t-test compared with unconditioned controls (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Fig. 5

Only αCD117 ACK2 mAb augmented with αCD47 mAb enables depletion of HSCs and enhances donor engraftment post HSCT in WT settings. A Experimental outline to showcase mAb treatment and assess HSC depletion and HSCT transplantation efficacy with combination αCD117 mAbs and αCD47 mAb treatment in WT mice. B BM HSC depletion was measured 7 days post treatment with profound depletion in αCD117 ACK2 and 3C11 mAb augmented with αCD47 mAb treated groups as determined by phenotypic assessment by flow cytometry (Lin^-Sca-1⁺CD117⁺CD150⁺CD48⁻) and functional assessment by in vitro colony forming capacity. C PB donor chimerism and D BM HSC donor chimerism were measured 20 weeks post HSCT of conditioned animals with 10 × 10⁶ CD45.1 donor WBM cells. Only αCD117 ACK2 mAb augmented with αCD47 mAb was observed to result in robust donor chimerism in peripheral blood and HSCs (n = 5). Statistics calculated using unpaired t-test compared with unconditioned controls (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 6

Functional Fc is required for αCD117 ACK2 mAb and αCD47 mAb combination treatment to results in HSC depletion and enhanced donor engraftment post HSCT in WT settings. A Experimental outline to assess mAb treatment, HSC depletion and HSCT transplantation efficacy with combination αCD117 ACK2 mAb, deglycosylated Fc derivative and αCD47 mAb treatment in WT mice. B BM HSCs depletion was measured 7 days post treatment with profound depletion only observed post intact αCD117 ACK2 mAb and αCD47 mAb treatment as determined by phenotypic assessment by flow cytometry (Lin^-Sca-1⁺CD117⁺CD150⁺CD48^-) and functional assessment by in vitro colony forming capacity. C PB donor chimerism and D BM HSC donor chimerism were measured 20 weeks post HSCT of conditioned animals with 10 × 10⁶ CD45.1 donor WBM cells with robust donor engraftment only observed with conditioning with the full αCD117 ACK2 augmented with αCD47 mAb (n = 5). Statistics calculated using unpaired t-test compared with unconditioned controls (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).