All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.
INTRODUCTION: Hematopoietic stem and progenitor cells (HSPCs) support life-long hematopoiesis. A single HSPC can also be at the origin of hematological malignancies, such as Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). Allogeneic HSCT with the intent to eliminate recipient AML or MDS and at the same time replace recipient HSPC with donor-HSPC and immune cells is a life-saving therapeutic option for many patients. However, chemotherapy (and sometimes in addition gamma-irradiation based conditioning regiments) prior to HSCT are associated with substantial toxicity. Thus, due to benefit-outweighing treatment-related toxicity and mortality, frail, multi-morbid and elderly patients are usually excluded from potentially curative allo-HSCT approaches. For these reasons, more selective preconditioning strategies, leading to residual AML/MDS elimination and creating "space" for incoming HSPCs, are required. Selective targeting of CD117 with monoclonal antibodies has been proposed as a strategy to remove endogenous HSPCs, enabling an effective but mild preconditioning. However, specific conditioning of AML and MDS patients, prior to HSCT, might require a more potent effector cell type. We hypothesized that a CD117 and CD3 binding, T cell engaging and activating antibody construct (CD117xCD3 TEA) with a short half-life might be an ideal means to selectively eliminate CD117-expressing healthy HSPCs and residual CD117-expressing AML or MDS cells prior to allo-HSCT. METHODS: We cloned and expressed CD117xCD3 TEA in tandem scFv format and produced it by transient gene expression in Chinese hamster ovary cells (CHO-S). The fusion proteins were purified to homogeneity by protein A affinity chromatography. We derived target cell lines with varying surface levels of CD117 (high, medium and low) from CD117 negative parental cell lines HL-60 and MOLM-14 (Myburgh et al., Leukemia, 2020). To assess T cell mediated killing of target cells, we mixed them with human T cells (purified and enriched after negative selection) at varying Effector-to-Target (E:T) cell ratios and added CD117xCD3 TEA at different concentrations. The mixture was incubated and specific killing was quantified via flow cytometry at different time-points. RESULTS: In order to characterize the biocidal properties of CD117xCD3 TEA, we performed in vitro killing experiments against cell lines, HSCPs from healthy donors and blast cells from AML patients. A dose-dependent in vitro killing of the cell lines was observed in the presence of various concentrations of CD117xCD3 TEA and of human T cells at an E:T cell ratio of 10:1 after 24h. The HL60 CD117 high cell line was efficiently lysed (~90%) at 100 ng/ml of CD117xCD3 TEA, corresponding to ~1.8 nM. In similar experiments with different E:T cell ratios, we observed that both HL60 CD117 high and CD117 medium cells could be quantitatively killed at E:T ratios as low as 1:1, while the killing of CD117 low cells required a higher density of T cells. The biocidal effect on non-transduced HL60 cells was negligibly low, confirming the requirement of a simultaneous engagement of CD117 and CD3 for specific killing. We repeated the same experiment with an engineered MOLM14 cell line, which also expressed CD117 at comparable high levels, incubating the target cell line with human T cells at an E:T of 1:1 for 24, 48 or 72, 120 or 192 hours. Complete killing of the target cell line was achieved at 120 and 192 hours and after supplemental addition of T cells and CD117xCD3 TEA at 72 hours (see example figure). Experiments with primary cells (HSPCs from healthy donors or blast cells from AML patients) at an E:T of 1:1 confirmed specific killing of target cells in an antigen-density- and concentration-dependent manner after 48h. CONCLUSIONS: We have generated a novel bispecific antibody, which binds to human CD117 (expressed on HSCPs and AML/MDS blast cells) and to CD3 (expressed on T cells), which we term CD117xCD3 TEA. The antibody induces selective T cell-mediated killing of cell lines with different surface levels of CD117, as well as of healthy HSPCs and primary human AML cells. Thus, the newly generated CD117xCD3 TEA might be developed clinically in order to erradicate residual AML/MDS and at the same time serve as a milder preconditioning approach prior to allo-HSCT in frail AML/MDS patients. Figure 1 Figure 1. Disclosures Kiefer: ETH Zurich: Current Employment, Patents & Royalties: CD117xCD3 TEA. Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Guggisberg: F. Hoffmann-La Roche AG: Current Employment. Abdelmotaleb: F. Hoffmann-La Roche AG: Current Employment. Mock: Philogen S.p.A.: Current Employment. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: University of Zurich: Patents & Royalties: CD117xCD3 TEA; CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company.
Background:Chimeric Antigen Receptor (CAR) T-cells have been clinically implemented to eradicate B-cell malignancies by targeting lineage specific cell-of-origin antigens, eliminating both tumor cells as well as healthy B-and plasma-cell counterparts, which can subsequently regenerate again from hematopoietic stem and progenitor cells (HSPCs). Similar on-target, off-tumor side effects would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), where the heterogeneous overlapping antigen expression on tumor and HSPC would lead to terminal ablation of hematopoiesis. Thus, higher tumor-cell selectivity and/or tunable effector activity is required.
Mastocytosis is a malignant disease resulting from oncogenic transformed mast cells. Up to 80% of malignant cells harbor a D816V mutation in the KIT-receptor (CD117), leading to constitutive kinase activation and proliferation and survival of mast cells. Advanced forms of mastocytosis (aggressive systemic mastocytosis: ASM, systemic mastocytosis with associated hematological disease: SM-AHN, mast cell leukemia: MCL) present as a therapeutic challenge. Although the recently approved poly tyrosine kinase inhibitor Midostaurin provides some improvement, the median overall survival ranges from 3.5 years (ASM) to less than six months (MCL). The reduced life expectancy is frequently due to mast cell infiltration resulting in multi organ failure. Additionally, there are patients who do not benefit from the treatment with Midostaurin (overall response 60%) or suffer from side effects, which lead to reduction or termination of therapy. Currently, the only available curative approach is conditioning poly-chemotherapy followed by allogenic stem cell transplantation (allo-HSCT). However, allo-HSCT is associated with substantial side-effects and, also due to high rates of relapse, only leads to an overall survival of 43% for ASM and 17% for MCL after three years. Thus, better therapeutic options are needed. Recently, we demonstrated that CD117 (KIT-receptor) positive human AML can be efficiently eradicated by anti-CD117 CAR T-cells in vitro and in vivo (Myburgh et al., Leukemia 2020). As mast cells, and also transformed mast cells, highly express CD117, we here tested if anti-CD117 CAR T-cells would equally efficiently eliminate this malignant cell population. We thus co-cultured various established mast cell lines (partly harboring the oncogenic driver mutation KIT D816V) with anti-CD117-CAR T-cells in a 1:1 effector to target ratio in vitro. After 24 hours of co-culturing, the tumor cells were effectively killed, and this was still observed despite increasing the effector to target ratio to 1:4. Also, within 28 days of co-culture, the longest time followed in vitro, tumor cells were controlled and did not outgrow. Increased proliferation of anti-CD117-CAR T-cells in the presence of mast cells was observed and tracked throughout the 28-day experiment. In conclusion, we demonstrate that the human mast cell lines HMC-1.1 KIT V560G, HMC-1.2 KIT V560G, KIT D816V, ROSA KIT WT, ROSA KIT D816V, LAD2 and MCPV-1 can be efficiently targeted and killed in vitro by allogeneic anti-CD117-CAR T-cells. Given that CD117 is expressed on healthy hematopoietic stem and progenitor cells (HSPCs) on a substantially lower level, there might be a therapeutic window for anti-CD117 immunotherapy in advanced forms of mastocytosis. However, as CAR T-cells are highly efficient, collateral damage on healthy HSPCs will likely need to be compensated by subsequent HSC transplantation. We are currently translating these promising in vitro immunotherapeutic settings into surrogate xenogeneic in vivo models. Disclosures Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.
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