P-04.01 CSF1R-targeting T cell engaging bispecific antibodies for Acute Myeloid Leukemia treatment

Abstract

<h3>Background</h3> The potential of T-cell engaging bispecific antibodies (TCEs) is illustrated by the success of targeting CD19 (Blinatumomab) and CD20 (Epcoritamab, Glofitamab, Mosuenetuzumab) in relapsed and refractory B-cell non-Hodgkin lymphoma. However, in AML, TCEs targeting CD33 (JNJ-67571244, AMG330) and CD123 (Vibecotamab) have shown low efficacy and high toxicity. This is paralleled by preclinical studies addressing these targets evidencing on-target off-tumor toxicity against hematopoietic stem and progenitor cells (HSPCs). To address the need for safer, effective immunotherapeutic targets in AML, we previously identified the colony-stimulating factor 1 receptor (CSF1R) using an unbiased single-cell RNA sequencing approach. To further evaluate its potential efficacy and safety profile, we developed a CSF1R-targeting TCE structurally based on the CrossMAb^® technology in the 2+1 format shared with the FDA-approved CD20-targeting TCE Glofitamab. <h3>Materials and Methods</h3> In co-culture experiments with T cells or peripheral blood mononuclear cells and human AML cell lines (Mv4-11, THP-1, OCI-AML3, PL-21) as well as primary AML blasts, the binding of CSF1R-TCE was characterized using flow cytometry, while luciferase bioluminescence and live cell imaging readouts investigated the killing capacity.The on-target off-tumor toxicity against HSPCs <i>in vitro</i> was analyzed using multicolor flow cytometry or classical colony-forming unit assays. On-target-off-tumor-toxicity <i>in vivo</i> was analyzed in CD34⁺ cord blood (CB)-stem cell-humanized as well as using fully syngeneic, immunocompetent mouse models. Xenograft-derived AML cell line models were used to evaluate efficacy of CSF1R-TCE in humans. <h3>Results</h3> First, we confirmed dose-dependent binding of our CSF1R-TCE and specific lysis of AML cell lines (effector-to-target (E:T) ratio 1:2, p &lt; 0.0001 for both Mv4-11 and THP-1) and primary AML blasts (E:T 1:1, p &lt; 0.0001). The CSF1R-TCE did not cause relevant HSPC lysis <i>in vitro</i>, whereas the CD33-TCE led to a near complete depletion of HSPCs (E:T 2:1, p &lt; 0.0001). These results were confirmed in a CD34⁺ CB-stem cell-humanized mouse model, in which CSF1R-TCE showed less evidence of cytokine release and minimal HSPC count reduction compared to CD33-TCE. Similarly, in fully syngeneic mouse models, we observed no signs of on-target-off-tumor toxicity in mice treated with a CSF1R-TCE targeting the murine receptor. In the Mv4-11 AML xenograft model, the CSF1R-TCE reduced tumor outgrowth and progression compared to a control TCE (p &lt; 0.0001). <h3>Conclusions</h3> We demonstrated the safety and efficacy of our CSF1R-TCE <i>in vitro</i> and <i>in vivo</i>. Consistent with our single-cell RNA sequencing-based target analysis, the CSF1R-TCE exhibited a superior safety profile than the CD33-TCE while maintaining anti-tumor activity. CSF1R-targeting TCEs may represent a novel immunotherapeutic approach for AML with high translative potential. <b>A. Gottschlich:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Tabby Therapeutics, Nanogami. <b>T. Janert:</b> None. <b>G. Hoffmann:</b> None. <b>J. Sam:</b> A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. <b>S. Gebhardt:</b> A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Roche. <b>L. Rohrbacher:</b> None. <b>S. Nandi:</b> None. <b>E. Carlini:</b> None. <b>T. Herold:</b> D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; Jazz Pharmaceuticals, Servier Deutschland. <b>M. von Bergwelt-Balidon:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TABBY, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, KITE/Gilead Mologen, Miltenyi, MSD Sharp + Dohme, Novartis, Priothera, Roche. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; TABBY, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, KITE/Gilead Mologen, Miltenyi, MSD Sharp + Dohme, Novartis, Priothera, Roche. F. Consultant/Advisory Board; Significant; TABBY, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, KITE/Gilead Mologen, Miltenyi, MSD Sharp + Dohme, Novartis, Priothera, Roche. <b>S. Endres:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Arcus Bioscience, Plectonic GmbH, TCR2 Inc, Catalym GmbH. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2 Inc, Carina Biotech. <b>M. Subklewe:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Amgen, BMS/Celgene, Gilead/Kite, Janssen, Miltenyi Biotec, Molecular Partners, Novartis, Roche, Seagen, Takeda. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; AstraZeneca, BMS, Gilead/Kite, GSK, Janssen, LAWG, Novartis, Pfizer, Roche, Springer Healthcare, AbbVie, Amgen, Autolus, AvenCell, CanCell Therapeutics, Genmab US, Ichnos Sciences, Incyte Biosciences, Interius BioTherapeutics, Miltenyi Biomedicine, Molecular Partners, Nektar Therapeutics, Orbital Therapeutics, Pfizer. <b>C. Klein:</b> A. Employment (full or part-time); Significant; F. Hoffmann-La Roche Ltd. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; F. Hoffmann-La Roche Ltd. <b>S. Kobold:</b> B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; CR2 Inc., Tabby Therapeutics, Catalym GmBH, Plectonic GmBH, Arcus Bioscience. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; CR2 Inc., Miltenyi, Galapagos, Novartis, BMS, GS. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; CR2 Inc., Carina Biotech.