Evaluation of the sensitizing effect of a disinfectant based on glutaraldehyde upon inhalation

When investigating new disinfectants, it is necessary to assess the degree of their danger by modeling the conditions of use. As is known, the main danger of disinfectants, especially volatiles, is posed by the inhalation route of entry into the body, exerting an irritating effect on the respiratory organs, as well as a general toxic and sensitizing effect. Despite reasonable restrictions on the use of disinfectants from the aldehyde class for decontamination of the surfaces of premises and equipment, state registration receives disinfectants based on glutaraldehyde (GA), intended for wide use in various areas, including everyday life, which is unacceptable. Experiments were carried out to confirm the danger of using GA-based disinfectants. Sensitizing effect of GA-based disinfectant under conditions of surface treatment by rubbing method is studied. Significant release of GA into the air after surface treatment (27.7±4.1 mg/m3) was detected. The concentration of GA in the air decreased by 4.5 times after 6 hours of ventilation. The sensitizing effect of the GA-based agent was determined on guinea pigs by a provocative ear swelling test and a leukocyte specific lysis reaction. The results of the study indicate a pronounced sensitizing effect of the agent. Keywords: glutaraldehyde, toxicity, disinfectants, disinfection, sensitization, inhalation, guinea pigs.

In Vitro Model to Predict Response to Daratumumab Therapy in Relapsed/Refractory Multiple Myeloma

Background Since its approval in 2015, daratumumab has become incorporated in standard-of-care regimens of antimyeloma therapy. However, response to daratumumab in patients with relapsed/refractory multiple myeloma (RRMM) is heterogeneous, and a reliable biomarker of response is lacking. Expression of CD38 was shown to be associated with response to daratumumab, but failed to predict responses consistently and is therefore not used in clinical practice. Aim of our study was to develop an in vitro method that accurately identifies patients with RRMM that will respond to daratumumab treatment. Methods We obtained CD38 mean fluorescent intensity (MFI) of plasma cells from patients with RRMM just before starting daratumumab therapy, as well as best clinical response to treatment and duration of response to daratumumab in weeks. When possible, extra material was obtained at the bone marrow biopsy performed before the start of daratumumab treatment and frozen in liquid nitrogen in our local biobank facility. All included patients provided written informed consent. Bone marrow mononuclear cells from patients with RRMM were thawed and cultured for a week in a hydrogel-based culture system, supplemented with pro-survival cytokines IL-6 and APRIL. To examine the in vitro response to daratumumab, we added 0.1 μg/ml daratumumab or 0.1 μg/ml IgG1κ isotype to the culture wells. We measured complement-dependent cytotoxicity (CDC) by adding 10% pooled human serum and antibody-dependent cellular cytotoxicity (ADCC) by adding healthy-donor peripheral blood mononuclear cells (PBMC) in a 10:1 effector-to-target ratio. After overnight incubation, hydrogel cultures were broken down mechanically and specific lysis was determined with flow cytometry, using CD138 and CD38 multi-epitope antibodies to select plasma cells, and using ToPro3 live/dead stain and Flow-Count Fluorospheres to obtain absolute numbers of surviving plasma cells. Results First, we analyzed CD38 MFI of plasma cells from 35 patients with RRMM just before starting daratumumab treatment, both as monotherapy or as part of a regimen. As was previously reported by others 1,2, we found a significant association between expression of CD38 and response (p = 0.01) (Figure 1A). However, we also observed considerable overlap between CD38 MFI values of responders and non-responders, with AUC ROC = 0.75. Therefore the discriminatory value of CD38 expression to predict response is weak. Next, we tested daratumumab in vitro on plasma cells obtained from 10 patients with RRMM. Daratumumab ADCC was associated with clinical response by the corresponding patients (p = 0.008), with a median specific lysis of 54.3% for patients who obtained a PR and 26.0% for patients who did not obtain a PR (Figure 1B). Median CDC for patients who did not obtain a PR was 3.16% versus 45.3% for patients who did obtain a PR, without a significant difference between the two groups (p = 0.18). Further evaluation of these results revealed AUC ROC of 1.00 and 0.81 for ADCC and CDC, respectively, outperforming CD38 expression. For ADCC, a threshold value of 39.6% lysis translated to a diagnostic accuracy of 100%, while for CDC a threshold value of 29.4% resulted in a diagnostic accuracy of 87.5%. Ultimately, we compared these results to CD38 expression. With an optimal CD38 MFI threshold value of 10989, diagnostic accuracy of CD38 expression was 71.4%. In addition, we examined ability of the test methods to predict the clinically experienced duration of response to daratumumab treatment. We observed no difference in duration of response when using the optimal threshold values of CD38 MFI (p = 0.13) and CDC lysis (p = 0.12), but duration of response was significantly different for patients with an ADCC lysis above and below the threshold value of 39.6% (p = 0.002) (Figure 1C). Conclusion In summary, we developed an straightforward and consistent in vitro method to predict response to daratumumab treatment in vivo. Measuring ADCC in patient-derived plasma cells using healthy-donor PBMC within our hydrogel-based culture system corresponds with the overall clinical response and duration of response to daratumumab. Our results support further clinical validation of the in vitro use of primary plasma cells to identify patients most likely to benefit from treatment. References 1. Nijhof IS, et al. Blood. 2016;128(7):959-970. 2. Kitadate A, et al. Haematologica. 2019;105(1):e37-e40. Figure 1 Figure 1. Disclosures Peperzak: Philips Healthcare: Research Funding. Minnema: Celgene: Other: Travel expenses; Alnylam: Consultancy; Janssen: Consultancy; Cilag: Consultancy; Kite/Gilead: Consultancy; BMS: Consultancy.

Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC specific T-cell bispecific antibody

Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).

The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

Soluble Expression of Fc-Fused T Cell Receptors Allows Yielding Novel Bispecific T Cell Engagers

The specific recognition of T cell receptors (TCR) and peptides presented by human leukocyte antigens (pHLAs) is the core step for T cell triggering to execute anti-tumor activity. However, TCR assembly and soluble expression are challenging, which precludes the broad use of TCR in tumor therapy. Herein, we used heterodimeric Fc to assist in the correct assembly of TCRs to achieve the stable and soluble expression of several TCRs in mammalian cells, and the soluble TCRs enable us to yield novel bispecific T cell engagers (TCR/aCD3) through pairing them with an anti-CD3 antibody. The NY-ESO-1/LAGE-1 targeted TCR/aCD3 (NY-TCR/aCD3) that we generated can redirect naïve T cells to specific lysis antigen-positive tumor cells, but the potency of the NY-TCR/aCD3 was disappointing. Furthermore, we found that the activation of T cells by NY-TCR/aCD3 was mild and unabiding, and the activity of NY-TCR/aCD3 could be significantly improved when we replaced naïve T cells with pre-activated T cells. Therefore, we employed the robust T cell activation ability of staphylococcal enterotoxin C2 (SEC2) to optimize the activity of NY-TCR/aCD3. Moreover, we found that the secretions of SEC2-activated T cells can promote HLA-I expression and thus increase target levels, which may further contribute to improving the activity of NY-TCR/aCD3. Our study described novel strategies for soluble TCR expression, and the optimization of the generation and potency of TCR/aCD3 provided a representative for us to fully exploit TCRs for the precision targeting of cancers.

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing

BackgroundWith increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function.MethodsEffectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others.ResultsOur results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30.ConclusionsThe presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

Parameters of acute and chronic toxicity of the insecticidal preparation VEIS-2

The main safety indicators of the new preparation VEIS-2, containing natural or chemically modified avermectins as an active ingredient, when exposed to the skin of mammals, intended to protect the downy feather raw material from keratophagous insects, have been studied. It has been established that the VEIS-2 compositions containing abamectin or avermectin B1a hemisuccinate as an active principle have a mild irritating effect on the conjunctival mucosa of the rabbit eye. With single and multiple applications on the skin, the above compositions do not cause such functional and structural changes in the skin as edema, erythema, cracks, desquamation. The presence of skin-resorptive and allergenic action in the compositions of the VEIS-2 preparation with abamectin or hemisuccinate avermectin B1a using the "test tube method" in rats, as well as using the reaction of specific lysis of leukocytes (RSLL), has not been established. It was shown that the VEIS-2 composition based on avermectin complex has a more pronounced local irritant effect on laboratory animals in terms of toxicity and allergenicity.

Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

Evolutionary Stabilization of Cooperative Toxin Production through a Bacterium-Plasmid-Phage Interplay

ABSTRACT Colicins are toxins produced and released by Enterobacteriaceae to kill competitors in the gut. While group A colicins employ a division of labor strategy to liberate the toxin into the environment via colicin-specific lysis, group B colicin systems lack cognate lysis genes. In Salmonella enterica serovar Typhimurium (S. Tm), the group B colicin Ib (ColIb) is released by temperate phage-mediated bacteriolysis. Phage-mediated ColIb release promotes S. Tm fitness against competing Escherichia coli. It remained unclear how prophage-mediated lysis is realized in a clonal population of ColIb producers and if prophages contribute to evolutionary stability of toxin release in S. Tm. Here, we show that prophage-mediated lysis occurs in an S. Tm subpopulation only, thereby introducing phenotypic heterogeneity to the system. We established a mathematical model to study the dynamic interplay of S. Tm, ColIb, and a temperate phage in the presence of a competing species. Using this model, we studied long-term evolution of phage lysis rates in a fluctuating infection scenario. This revealed that phage lysis evolves as bet-hedging strategy that maximizes phage spread, regardless of whether colicin is present or not. We conclude that the ColIb system, lacking its own lysis gene, is making use of the evolutionary stable phage strategy to be released. Prophage lysis genes are highly prevalent in nontyphoidal Salmonella genomes. This suggests that the release of ColIb by temperate phages is widespread. In conclusion, our findings shed new light on the evolution and ecology of group B colicin systems. IMPORTANCE Bacteria are excellent model organisms to study mechanisms of social evolution. The production of public goods, e.g., toxin release by cell lysis in clonal bacterial populations, is a frequently studied example of cooperative behavior. Here, we analyze evolutionary stabilization of toxin release by the enteric pathogen Salmonella. The release of colicin Ib (ColIb), which is used by Salmonella to gain an edge against competing microbiota following infection, is coupled to bacterial lysis mediated by temperate phages. Here, we show that phage-dependent lysis and subsequent release of colicin and phage particles occurs only in part of the ColIb-expressing Salmonella population. This phenotypic heterogeneity in lysis, which represents an essential step in the temperate phage life cycle, has evolved as a bet-hedging strategy under fluctuating environments such as the gastrointestinal tract. Our findings suggest that prophages can thereby evolutionarily stabilize costly toxin release in bacterial populations.

Targeting Intracellular WT1 in AML Utilizing a T Cell Bispecific Antibody Construct: Augmenting Efficacy through Combination with Lenalidomide

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.