Hematopoiesis after anti-CD117 monoclonal antibody treatment in the settings of wild-type and Fanconi anemia mice.

Anti-CD117 monoclonal antibody (mAb) agents have emerged as exciting alternative conditioning strategies to traditional genotoxic irradiation or chemotherapy for both allogeneic and autologous gene-modified hematopoietic stem cell transplantation. Furthermore, these agents are concurrently being explored in the treatment of mast cell disorders. Despite promising results in animal models and more recently in patients, the short- and long-term effects of these treatments have not been fully explored. We conducted rigorous assessments to evaluate the effects of an antagonistic anti-mCD117 mAb, ACK2, on hematopoiesis in wild-type and Fanconi anemia (FA) mice. Importantly, we found no evidence of short-term DNA damage in either setting following this treatment, suggesting that ACK2 does not induce immediate genotoxicity, providing crucial insights into its safety profile. Surprisingly, FA mice exhibited an increase in colony formation after ACK2 treatment, indicating a potential targeting of hematopoietic stem cells and expansion of hematopoietic progenitor cells. Moreover, the long-term phenotypic and functional changes in hematopoietic stem and progenitor cells did not differ significantly between the ACK2-treated and control groups, in either setting, suggesting that ACK2 does not adversely affect hematopoietic capacity. These findings underscore the safety of these agents when utilized as a short-course treatment in the context of conditioning, as they did not induce significant DNA damage in hematopoietic stem or progenitor cells. However, single-cell RNA sequencing, used to compare gene expression between untreated and treated mice, revealed that the ACK2 mAb, via c-Kit downregulation, effectively modulated the MAPK pathway with Fos downregulation in wild-type and FA mice. Importantly, this modulation was achieved without causing prolonged disruptions. These findings validate the safety of anti-CD117 mAb treatment and also enhance our understanding of its intricate mode of action at the molecular level.

Targeted hematopoietic stem cell depletion through SCF-blockade.

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.

Non-genotoxic Restoration of the Hematolymphoid System in Fanconi Anemia.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients with many different blood and immune diseases; however, current treatment regimens contain non-specific chemotherapy and/or irradiation conditioning, which carry both short-term and long-term toxicities. The use of such agents may be particularly harmful for patients with Fanconi anemia (FA), who have genetic mutations resulting in deficiencies in DNA repair, leading to increased sensitivity to genotoxic agents. mAb-based conditioning has been proposed as an alternative conditioning strategy for HSCT that minimizes these toxicities by eliminating collateral tissue damage. Given the high need for improved treatments for FA patients, we aimed to evaluate the efficacy of different αCD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion and explored their ability to safely establish therapeutic donor hematopoiesis post-HSCT in FA disease models. We evaluated the effects of different concentrations of αCD117 mAbs in vitro and in vivo on HSC growth and depletion. To further assess the efficacy of mAb-based conditioning, Fancd2-/- animals were treated with αCD117 mAb and combination agents with αCD47 mAb and antibody-drug-conjugates (ADCs) for syngeneic HSCT. Immunosuppression αCD4 mAb was added to all in vivo experiments due to a slightly mismatched background between the donor grafts and recipients. Immunosuppressant cocktails were also given to Fancd2-/- animals to evaluate the efficacy of mAb-based conditioning in the haploidentical setting. Statistical analyses were done using the unpaired t-test. We found that antagonistic αCD117 mAbs alone do not deplete host HSCs or enhance HSCT effectively in FA mouse models; however, the potency of αCD117 mAbs can be safely augmented through combination with αCD47 mAbs and with ADCs, both of which lead to profound HSC depletion and establishment of long-term donor engraftment post-syngeneic HSCT. This is the first time these approaches have been tested in parallel in any disease setting, with the greatest donor engraftment observed after CD117-ADC conditioning. Interestingly, our data also suggest that HSC-targeted conditioning is not necessary in HSCT for FA, as high donor HSC engraftment was observed with mAb-based immune suppression alone with immunologically matched and mismatched haploidentical grafts. These results demonstrate the safety and efficacy of several different non-genotoxic mAb-based conditioning strategies in the FA setting. In addition, they show that if sufficient immunosuppression is given to obtain initial donor HSC engraftment, turnover of a majority of the hematolymphoid system can result, likely owing to the survival advantage of wild-type HSCs over FA HSCs. Such non-toxic all-mAb-based conditioning strategies could be transformative for FA patients and those with other hematolymphoid diseases.