Novel Anti-CD117 Antibodies for Rapid and Efficient Hematopoietic Stem Cell Depletion and Safe Bone Marrow Conditioning

Giada Dal Collo, Antoinette Beijen, Yu He, YM Mueller, Yuandong Wang, Joe Zhao, Peter Katsikis, Stefan Erkeland

Research output: Contribution to journalMeeting AbstractAcademicpeer-review

Abstract

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) has proven to be effective for the treatment of hematopoietic disorders, including immune deficiencies, bone marrow failure syndromes (BMFS) and leukemia. However, current preparation for HSCT carries significant toxicity due to radiation and/or chemotherapy that put the patients at high risk of life-threatening infections and toxicity. Severe infections frequently present in the immediate post-HSCT period, while complications caused by chemotoxicity and irradiation damage affect multiple organs at the long term. The high mortality and morbidity rates, although acceptable for malignant diseases, make the risk-benefit ratio too high to apply HSCT for young patients with for instance BMFS and immune deficiencies. The low-risk solution that we propose in this report is the engineering and pre-clinically testing of novel antibodies that rapidly and transiently deplete hematopoietic stem cells (HSCs) to create the desired bone marrow (BM) space that allows for sufficient HSC engraftment. Highly effective HSC depleting antibodies that have a controllable activity in the body would be ideal for rapid and efficient treatment of patients. Such antibodies would facilitate BM conditioning without the risk of infections and toxicity. In this project, we developed and tested novel antibodies that target the c-KIT/CD117 receptor, which is a characteristic molecule on HSCs.

METHODS: We developed novel anti-CD117 reagents including anti-CD117 IgG Fc-engineered (anti-CD117) antibody and bispecific T-cell engager (BiTE, CD117xCD3). For our experiments we used an anti-CD117 reference antibody, which is currently tested in clinical trials. We first determined the binding capacity and affinity of these novel reagents to human CD117 expressing TF-1 cell line. The in vitro depletion capacity of our anti-CD117 antibody was tested with an antibody-dependent cytotoxicity reporter Bioassay and NK-cell-mediated cell depletion assays using TF-1 cells and human primary BM-CD34+ cells as targets. The cytotoxicity of the BiTE was shown in vitro in a T-cell-mediated cell depletion assay with the same type of target cells. In addition, we determined T-cell activation, T-cell proliferation and inflammatory cytokine levels. Next, we tested our novel reagents in a CD34+ transplanted humanized mouse model (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) for their ability to deplete human CD117+ HSCs. Mice were treated with our BiTE reagents for three days by multiple injections. The percentage of depleted human HSCs in the BM was determined at various time points by flow cytometry. Finally, the levels of the inflammatory cytokines were tested in the serum with ELLA Simple Plex assays.

RESULTS: We demonstrated that our novel anti-CD117 reagents bind specifically to CD117 expressed on TF-1 and human CD34+ cells. Our novel anti-CD117 antibody showed an enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), with a more than 2.5 improved killing of TF-1 cells compared to the reference antibody in vitro. Furthermore, we observed a greater than 4-fold improved killing of human CD34+ cells. We demonstrated that our CD117xCD3 BiTE also binds specifically to human T cells leading to a strong T-cell activation, T-cell proliferation, and upregulation of cytokines, in the presence of human CD117+ target cells. In in vitro cytotoxicity assays using our CD117xCD3 BiTE, we observed an 80% killing of TF-1 within 6 hours and 60% depletion of human CD34+ cells at 24 hours, whereas control BiTE did not induce cell death. We observed up to 80% depletion of human CD117+ HSCs in the BM of humanized mice already at 1 day post-treatment, whereas control-treated mice did not show depletion of CD117+ cells. This depletion was even more pronounced and on average 95% at 5 days post-treatment. Notably, no inflammatory cytokines in the serum or obvious toxicities were found in the treated mice.

CONCLUSION: We showed that our novel anti-CD117 antibody and CD117xCD3 BiTE exhibit improved cell depletion in vitro. Furthermore, we showed that the CD117xCD3 BiTE rapidly depletes human HSCs in the BM of humanized mice. Our novel anti-CD117 reagents are promising for further development of safe BM conditioning protocols prior to HSCT.
Original languageEnglish
Pages (from-to)4496–4497
JournalBlood
Volume140
Issue numberSuppl 1
DOIs
Publication statusPublished - 15 Nov 2022

Bibliographical note

© 2022 by The American Society of Hematology

Fingerprint

Dive into the research topics of 'Novel Anti-CD117 Antibodies for Rapid and Efficient Hematopoietic Stem Cell Depletion and Safe Bone Marrow Conditioning'. Together they form a unique fingerprint.

Cite this