Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Xu:Roche: Employment, Equity Ownership, Patents & Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents & Royalties. Sam:Roche: Employment, Equity Ownership, Patents & Royalties. Pulko:Roche: Employment, Equity Ownership, Patents & Royalties. Schönle:Roche: Employment, Equity Ownership, Patents & Royalties. Challier:Roche: Employment, Equity Ownership, Patents & Royalties. Carpy:Roche: Employment, Equity Ownership, Patents & Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.
Introduction TAK-981 is a first-in-class small molecule inhibitor of the SUMO activating enzyme currently in Phase I/II clinical trials. TAK-981 has been shown to increase NK cell activation and M1 macrophage polarization via upregulation of Type I interferon (IFN) signaling, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in combination with rituximab (Nakamura 2019, AACR). Tafasitamab (MOR208) is a CD19-targeting antibody with enhanced Fc effector function mediating ADCC, ADCP and direct cytotoxic activities against B-lymphoma cells. Based on the Phase II clinical study L-MIND (Salles et al., 2020 and Duell et al., 2021), tafasitamab in combination with lenalidomide received accelerated approval by the Food and Drug Administration for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Due to the potential for TAK-981 to enhance the activity of tafasitamab via activation of innate effector cells, we aimed to investigate the effects of this drug combination on ADCC, ADCP and tumor cell viability in vitro. Additionally, combinatorial activity of TAK-981 plus tafasitamab was evaluated in lymphoma xenograft models. Methods A panel of 9 aggressive lymphoma cell lines was analyzed (7 DLBCL and 2 Burkitt lymphoma). For ADCC, PBMC effector cells from healthy human donors were pre-treated with 0.1 or 1 µM TAK-981 or dimethyl sulfoxide (DMSO) control for 24 hours. Tumor cells were incubated with/without 1 nM tafasitamab in the presence of TAK-981 pretreated PBMCs at effector-to-target (E:T) ratios of 5:1 to 10:1 for 2 hours. Degranulation of NK cells was determined via CD107a surface expression after co-incubation of TAK-981 pre-treated PBMCs with tumor cells and 0.1 or 10 nM tafasitamab for 3 hours. Cytokine levels in the supernatant were investigated upon incubation of PBMCs with lymphoma cells, 1 µM TAK-981 and/or 10 nM tafasitamab for 24 hours. For the ADCP assays, in vitro differentiated macrophages were treated with 1 µM TAK-981 for 24 hours. Next, macrophages were incubated with lymphoma cells and 1 or 10 nM tafasitamab at an E:T ratio of 2:1 for 3 hours. For cell viability assays, tumor cells were treated with 1-1000 nM TAK-981 and/or 5 nM tafasitamab for 24 hours in the absence of effector cells. Cytotoxicity, phagocytosis, degranulation and cytokine release were analyzed by flow cytometry. Cell viability was assessed by determination of ATP levels. For in vivo analysis, effects of TAK-981 (7.5 mg/kg IV twice weekly) in combination with tafasitamab (3, 10 or 20 mg/kg IP twice weekly) on tumor growth were evaluated in Daudi and WSU-DLCL2 xenograft models of Burkitt lymphoma and DLBCL grown in SCID mice. Results In ADCC experiments, increased cytotoxicity was observed upon combination treatment with TAK-981 and tafasitamab compared to the respective mono treatments in 5/8 tested lymphoma cell lines (Daudi, SU-DHL-2, SU-DHL-6, TMD8, OCI-LY10). Moreover, TAK-981 plus tafasitamab enhanced degranulation of NK cells and cytokine release compared to mono treatments. In ADCP assays, combination of TAK-981 and tafasitamab resulted in increased phagocytosis rates in comparison to mono treatments in 2/2 tested cell lines (Daudi, Ramos). Cell viability analysis revealed a combination benefit by increased direct cytotoxic effects against SU-DHL-6 cells. Finally, TAK-981 and tafasitamab were investigated in Daudi and WSU-DLCL2 xenograft models with 3 weeks of dosing. In the Daudi model, the combination treatments of TAK-981 with 10 or 20 mg/kg tafasitamab performed better than either treatment alone, and in the WSU-DLCL2 model, the combination treatments of TAK-981 with 3, 10 or 20 mg/kg tafasitamab performed better than the single agent treatments. Conclusions The combination of TAK-981 with tafasitamab significantly enhanced anti-tumor effects compared to the respective monotherapies in vitro and in vivo. These preclinical data support a clinical evaluation of this drug combination in patients with lymphoma including aggressive subtypes such as Burkitt lymphoma and DLBCL. The study was funded by MorphoSys AG and Takeda Development Center Americas, Inc. Disclosures Patra-Kneuer: MorphoSys AG: Current Employment. Nakamura: Takeda Development Center Americas, Inc.: Current Employment. Song: Takeda Pharmaceuticals International Co.: Current Employment. Grossman: Takeda Development Center, Cambridge MA: Current Employment. Polzer: MorphoSys: Current Employment. Ginzel: MorphoSys: Current Employment. Steidl: MorphoSys AG: Current Employment. Berger: Takeda Development Center Americas, Inc.: Current Employment. Proscurshim: Takeda Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company. Heitmüller: MorphoSys AG: Current Employment.
Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that targets CD19 and has shown promising clinical activity in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). CD19 is homogeneously expressed among different B-cell malignancies, and the binding of tafasitamab to CD19 directly mediates cell death, induces antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Aiming to potentiate the tafasitamab-mediated "eat me" signal, we tested a combination with a CD47-directed monoclonal antibody (mAb) to inhibit the CD47/SIRPα "don't eat me" signal and further enhance macrophage-mediated phagocytosis. Preclinical studies demonstrated that blocking the CD47/SIRPα checkpoint in combination with antibodies, such as rituximab, increased phagocytosis by macrophages, resulting in effective anti-tumor effects in non-Hodgkin lymphoma (NHL) (Chao, et al. 2010). Additionally, the combination of the anti-CD47, magrolimab, and the anti-CD20, rituximab, demonstrated beneficial outcomes for patients with refractory NHL (Advani, et al. 2019). Here, we present in vitro and in vivo data on the combinatory effect of tafasitamab and an anti-CD47 mAb in preclinical models of Burkitt's lymphoma (BL). Methods During in vitro studies, CD14+ monocytes were isolated from the whole blood of healthy volunteers and differentiated with 50 ng/mL M-CSF for 6 days. ADCP was analyzed by flow cytometry in co-culture experiments with Ramos cells (BL) after 3 hours of treatment with tafasitamab and anti-CD47 mAb (clone B6H12). In vivo, the combination of tafasitamab with an anti-CD47 mAb was tested in a Ramos disseminated survival and subcutaneous tumor model in SCID and NOD-SCID mice, respectively. In both models, tafasitamab was administered therapeutically twice a week either at 3 mg/kg (disseminated) or 10 mg/kg (subcutaneous) for max. 4 weeks. The anti-CD47 mAb was administered at 4 mg/kg three times per week. Main study readouts were to assess animal survival and any delays in tumor growth. Results The combination of tafasitamab + CD47/SIRPα checkpoint blockade enhanced ADCP activity of primary M2 macrophages on BL-derived Ramos cells, in comparison with the anti-CD47 mAb or tafasitamab monotherapies (Figure 1A). In vivo, a significant increase in anti-tumor activity was observed with the combination of tafasitamab + anti-CD47 mAb. In the Ramos disseminated survival model, the combination showed an increased life span (ILS) of >182% compared with tafasitamab monotherapy control, with an overall survival of all animals treated with the combination (15/15) until the end of the study (Day 99 post-cell injection). Additionally, pronounced anti-tumor efficacies were detected in the Ramos subcutaneous tumor model. Here, the combination resulted in a significant delay in tumor growth compared with the tafasitamab or anti-CD47 mAb monotherapies (ILS >175% tafasitamab and ILS >72% anti-CD47 mAb vs tafasitamab + B6H12) (Figure 1B). Conclusions The ADCP activity of primary macrophages was increased by combining tafasitamab with an anti-CD47 mAb in vitro, resulting in enhanced anti-tumor activity compared with tafasitamab or anti-CD47 mAb monotherapies in vivo. Overall, results indicate the combination of tafasitamab with a CD47/SIRPα checkpoint blockade may be a promising novel combination approach for lymphoma therapy. Disclosures Mangelberger: MorphoSys AG: Current Employment. Augsberger:MorphoSys AG: Current Employment. Landgraf:MorphoSys AG: Current Employment. Heitmüller:MorphoSys AG: Current Employment. Steidl:MorphoSys AG: Current Employment.
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