Abstract 3177: Advancing CAR T-cell therapy in AML: Enhancing specificity and bone marrow homing via chemokine receptor engineering
Authors: Lis Winter, Katharina Konnerth, Anne-Sophie Neumann, Luisa Schmid, Stefanos Michaelides, Yingshuai Wang, Louisa von Baumgarten, Sebastian Kobold, Veit L. Bücklein, Marion Subklewe
Journal: Cancer Research (2025)

Abstract To date, the success of CD19-directed CAR T cells in B-cell malignancies has not been replicated in other hematological or solid malignancies. Key hurdles include the absence of suitable target antigens and limited tumor site homing. CAR T-cell efficacy can be enhanced by engineering T cells to express receptors specific to chemokines released by cancer or cancer-associated cells1. Thus, we aimed to identify receptors that promote directed migration to the bone marrow niche, thereby enhancing CAR T-cell efficacy in AML. Köhnke et al.2 identified upregulated expression of CCR2 and CXCR2 on the surface of AML cells via mass spectrometry, which we confirmed on primary AML cells using MPFC. Next, we used a retroviral-based transduction to engineer αCD33-28ζ-CAR T cells to co-express CCR2 or CXCR2. We analyzed CCR2high- or CXCR2high-CD33 CAR T-cell mediated cytotoxicity against the AML cell lines MV4-11 and MOLM-13 by MPFC. Next, we evaluated the specific migration towards the ligands CCL2 and CXCL8, conditioned AML cell line medium as well as towards bone marrow serum from primary AML samples using transwell assays and 3D migration chambers. The engineered CCR2high- and CXCR2high- CD33 CAR T cells showed no significant phenotypic differences compared to CD33 CAR T cells, as shown by activation or exhaustion marker expression or T-cell subset formation. To our surprise, we observed a reduced target cell killing together with reduced inflammatory cytokine secretion at high E:T ratios (1:20 &amp; 1:30) with the CCR2high- or CXCR2high- CD33 CAR T compared to CD33 CAR T cells. These effects were more prominent in MOLM-13 than MV4-11 cells and we are currently investigating the underlying mechanism. Next, we tested for the migratory capacity towards the specific ligands CCL2 and CXCL8. Enhanced migration, cell velocity and speed of CCR2high- and CXCR2high-CD33 CAR T cells were observed compared to CD33 CAR T cells. As expected by the surfacome data of primary AML cells, we identified CCL2 and CXCL8 to be highly abundant in the bone marrow serum of AML patients. Accordingly, transwell assays using bone marrow serum of primary AML patients demonstrated a higher targeted migration of CCR2high- and CXCR2high CD33 CAR T cells compared to CD33 CAR T-cell controls. Taken together, we demonstrated that CCR2high- and CXCR2high- CD33 CAR T cells show enhanced specific migration towards their ligands but also towards AML bone marrow serum. Surprisingly, we detected a reduction in specific lysis after transduction of the chemokine receptors into CD33 CAR T cells. Further studies are needed to understand the influence of the chemokine receptors on killing capacity. Next, we aim to validate these findings in a model system permitting in vivo live-cell imaging. 1. Biondi M et al.; Blood Journal 2023:blood. 20220183302. 2. Köhnke T et al.; Blood 2017;130:3968 Citation Format: Lis Winter, Katharina Konnerth, Anne-Sophie Neumann, Luisa Schmid, Stefanos Michaelides, Yingshuai Wang, Louisa von Baumgarten, Sebastian Kobold, Veit L. Bücklein, Marion Subklewe. Advancing CAR T-cell therapy in AML: Enhancing specificity and bone marrow homing via chemokine receptor engineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3177.