Alica Joana Emhardt

-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components.

Background: The myeloid associated antigen CD33 is overexpressed in over 88% of Acute myeloid leukemia (AML) patients, making it a suitable target antigen for CAR T cell therapy across different genetic subtypes. Despite the development of clinical trial data, there remains a concern about on-target off-leukemia toxicity. To enhance specificity, we generated dual targeting CAR T cells that target CD33 and/or T-cell immunoglobulin and mucin-domain containing-3 (TIM3). The latter is known to be expressed on LSCs, but not on healthy hematopoietic precursor or stem cells. Notably, TIM3 is recognized for its inhibitory immunomodulatory role further enhancing the suitability of this target antigen. The objective of this research was to explore various CD33 and TIM3 dual-targeting CAR T cells approaches that enhance AML specificity and maintain high anti-AML activity. Methods: The hybridoma technology was used to generate antibodies against TIM3 immunized mice. Antibodies were screened for TIM3 specificity via ELISA and FACS. The anti-TIM3 single-chain variable fragment (scFv) DNA sequence was sequenced from hybridoma cells. Furthermore, the binding ability to TIM3 antigen (PDB ID: 5F71) was evaluated using AlphaFold. The CD33 scFv was derived from Gemtuzumab ozogamicin (clone: hP67.6). ScFv sequences and co-stimulation domains (CD28 or 4-1BB) were cloned into a pMP71 vector. Retrovirus for transduction was produced using the 293Vec-GALV and RD114 retroviral production system. In vitro co-culture assays of CAR T cells and target cells were performed to study the efficacy of CAR T cells. The cytotoxicity against wild-type and TIM3 transduced AML cell lines (THP-1 and OCI-AML3) was assessed by multiparameter flow cytometry (MPFC). The secretion of effector cytokines (IFN-γ, TNF, IL-2) was analyzed via CBA assays. In addition, avidity between CAR T cells and target cells was determined by Z-Movie analyzer. The different CAR constructs were screened for on-target off-tumor toxicity in colony forming unit assays (CFU) using isolated CD34 + hematopoietic stem and progenitor cells (HSPCs) from healthy doners after 14 days. In order to compare CAR constructs regarding long-term efficacy in antigen restimulation assays, CAR T cells were co-cultured with irradiated TIM3 transduced OCI-AML3 every 4 days at an E: T ratio of 1:1 for 24 days. Moreover, CAR T-cell proliferation, checkpoint marker expression and T-cell subset differentiation were analyzed via MPFC. Results: All dual CAR T cells (compound, split, tandem, pooled) were generated with high and robust transduction efficacy (Figure 1A). TIM3-dependent fratricide was not observed during CAR T cell manufacturing. Compared to single antigen targeting CAR T cells, dual CAR T cells showed enhanced cytotoxicity against AML cell lines and primary AML cells (Figure 1B). Moreover, we also observed a strong increase in the secretion of proinflammatory cytokines (IFN-γ and IL-2) and higher avidity of dual CAR T cells. Notably, split CAR T cells demonstrated greater specificity in cocultures directed against mono vs. dual-target antigen expression cell lines. In accordance with these observations, split CAR T cells did not exert on-target off-leukemia toxicity against healthy HSC in CFU assays. In the antigen restimulation assay, we observed that compound CAR T cells exhibited diminished expanding capacity and heightened expression of exhaustion markers in comparison to the other CAR T constructs. Conclusion: We developed multiple CD33 and TIM3 dual CAR T cells using both “AND” and “OR” gating strategies in this study. Our findings revealed that dual CAR T cells provided higher avidity and cytotoxicity compared to single targeting CAR T cells against dual antigen expressing target cells in vitro. Importantly, only the split CAR T constructs demonstrated specific killing of CD33 +TIM3 + cell lines and primary AML cells while sparing HSPCs. Prospectively, compound, tandem, pooled CD33 TIM3 specific CAR T cells might be helpful in a bridge to transplant setting, whereas split CAR T cells might provide a higher safety profile, thereby allowing a transplant independent approach. To advance our concepts, we are currently conducting in vivo experiments in an NSG mouse model.

Bispecific T-cell recruiting antibody constructs (BsAb) have shown clinical efficacy with Blinatumomab, a CD3xCD19 BiTE ® construct used for the treatment of relapsed or refractory B-cell precursor ALL. Despite the success of BsAbs in B-cell malignancies, the translation to a myeloid setting has been unsuccessful, independently of the chosen target antigen. For example, targeting CD33 has failed to provide long-term benefits. Several reports have demonstrated that the diverse genetic makeup of AML confers resistance to T-cell based immunotherapy. Mutations of TP53 occur in 5-10% of patients with de novo AML and up to 50% in older patients with therapy related AML, thus highlighting its role as a potential resistance mechanism. We hypothesize, that genetic aberrations of TP53 in AML contribute to cell intrinsic and cell extrinsic resistance against T-cell based immunotherapy approaches. To study the impact of TP53 aberrations on T-cell based immunotherapy, MV4-11 p53 WT or MV4-11 p53 KD were cocultured with healthy donor T cells at an effector to target ratio of 1:6 with or without BsAbs. BsAb-mediated cytotoxicity was analyzed by specific lysis of the CD33 + target cells and T-cell proliferation was assessed by FarRed ® staining or CD2 + T-cell fold change calculated to day 0. Cytokine secretion was monitored in the coculture supernatants by cytometric beads arrays. To differentiate between secretome vs surfacome mediated differences, transwell assays were performed. To identify the T-cell immunosuppressive factor, secretome analysis were performed using the Olink® proteomics platform. To further dissect the effect of the p53 KD MV-411 on T cells, we performed RNA-Seq analysis of T cells after coculture with MV4-11 p53 KD vs p53 WT. As expected, BsAb-mediated cytotoxicity was reduced against p53 KD vs p53 WT MV4-11 (% specific lysis: p53 KD=47.86±7.37 vs p53 WT=76.42±3.82; p=0,0001). However, BsAb-mediated T-cell proliferation was also significantly decreased in cocultures with p53 KD vs p53 WT (% proliferated: 30.26±2.92 vs 44.25±4.08; p=0.002). In line with these findings, we observed a significant decrease in secretion of the proinflammatory cytokines IFNγ (p53 KD=2985,01±896,69 vs p53 WT=4009.15±764.97 pg/ml; p=0.068), TNF (29,38±8.16 vs 110.21±37.61 pg/ml; p=0.044) and IL-2 (1391.64±375.93 vs 2253.88±604.76 pg/ml; p=0.018). RNA-Seq analysis of T cells after coculture with MV4-11 p53 KD vs p53 WT revealed a decrease in the expression of genes associated with mitosis (such as E2F targets and mitotic spindle related genes). A lower mitotic rate provides a potential explanation for the reduction in T-cell proliferation after coculture with p53 KD. To dissect if the observed effects were due to the AML surfacome or the secretome, we performed transwell assays. Indeed, the negative impact of p53 KD AML cells on T-cell function was also observed without direct cell-cell contact. To gain further insights in the secretome of the AML cells, coculture supernatant was analysed using the Olink ® platform and demonstrated significantly higher secretion of IL-18 and LAP TGF-β1 (TGF-β1 latency associated protein) in the coculture with MV4-11 p53 KD vs MV4-11 p53 WT (normalized protein expression for IL-18: p53 KD=1.86±0.22 vs p53 WT=0.96±0.052; p=0.0153; for LAP TGF-β1: 6.52±0.18 vs 5.84±0.14; p=0.0394). The relevance of TGF-β on T-cell fitness was confirmed by the analysis of BsAb-mediated cytotoxicity with or without the addition of TGF-β. Addition of 3 ng/µl TGF-β to the coculture with p53 WT decreased the specific lysis significantly and was now comparable to the lysis of p53 KD. Confirmatory studies in primary AML samples with or without p53 aberrations further validated our findings with a clear reduction of BsAb-mediated cytotoxicity (% specific lysis: p53 KD=35.82±5.51 vs p53 WT=61.32±8.27; p=0.015), T-cell proliferation (CD2 + fold change: 3.17±0,61 vs 7.1±2.8; p=0.111) and secretion of proinflammatory cytokines. Taken together, we demonstrated that AML cells carrying a p53 aberration are less susceptible to BsAb-mediated cytotoxicity. Surprisingly, immune evasion was mediated by the AML secretome leading to a decrease in T-cell proliferation and cytokine secretion. Secretome analyses indicated TGF-β as a prominent cytokine leading to an impairment of T-cell function. Further studies are needed to understand the influence of p53 on the TGF-β pathway in AML cells.

Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 presents with a rather favorable expression profile due to broad AML expression and limited expression within the normal hematopoietic compartment (Brauchle et al., 2020). Importantly, expression on AML bulk cells and leukemic stem cells is observed independent of FLT3 mutational status. Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR-mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. On-target off-leukemia toxicity was evaluated in mixing assays of pAML cells and healthy donor bone marrow (hBM). The impact of positive costimulation on both approaches was tested using our previously established Ba/F3 model system (Marcinek et al., 2023), which is devoid of any human costimulatory molecules. As a surrogate for synapse formation, conjugate formation as well as consecutive T-cell degranulation were studied. Further, we studied the cell death pathway mediating cytotoxicity against AML target cells, e.g. FAS/FASL vs perforin/granzyme B. The impact of continuous stimulation by BiTE®-and CART on evolving T-cell exhaustion was tested using our previously established 28-day long-term culture assay (Philipp et al., 2022). Finally, we compared BiTE® and CAR in an in vivo xenograft AML model. In a second in vivo experiment, all mice were harvested 18 days after T-cell injection, followed by T-cell isolation from the bone marrow and subsequent bulk RNA sequencing. BiTE®- and CAR-redirected T cells led to comparable effector-to-target (E:T) ratio-dependent specific lysis of various AML cell lines and pAML cells. Using our in vitro mixing experiments, we could show that BiTE®- and CAR-redirected T cells efficiently killed pAML cells while mostly sparing the hBM. Using the Ba/F3 model system, we observed that the expression of the costimulatory molecule CD86 significantly increased the cytotoxicity of BiTE®-redirected T cells while CAR-mediated lysis was unaltered. Furthermore, we were able to show that the percentage of T cell-AML conjugates formed as well as degranulation of CD107a was significantly higher when using CART compared to BiTE® molecules. Interestingly, when we added a FAS-blocking antibody to our cocultures, we could show that CART-mediated lysis of the target cells was significantly reduced. This was not observed when using the BiTE® construct. Using our long-term culture system, we found that T-cell proliferation and cytokine secretion decreased with both constructs over time. The cytotoxic capacity of BiTE®-redirected T cells seemed to decrease faster compared to the CART construct. Moving to an in vivo AML xenograft model, we observed that CART-treated mice had an improved OS compared to the BiTE®-treated mice (Fig.1). This was accompanied by enhanced T-cell proliferation and splenic homing of the CAR T cells. Bulk RNA sequencing of T cells isolated from the murine bone marrow 18 days post T-cell injection revealed signs of exhaustion in BiTE®-redirected T cells compared to CAR T cells. We found an upregulation of several inhibitory immune receptors and transcription factors (Fig.2) that have been well described to promote T-cell exhaustion. Furthermore, gene set enrichment analysis revealed that genes upregulated in effector vs exhausted were also upregulated in CAR T cells. This was accompanied by an upregulated glycolysis and fatty acid metabolism in CAR T cells. We conclude, that FLT3 is a promising target antigen with limited on-target off-leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR construct provided an advantage in T-cell activation and might lead to better T-cell homing, resulting in improved efficacy and delayed T-cell exhaustion in vivo. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell-based immunotherapy platforms and identify suitable combination partners.