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While adoptive cell therapies have seen significant success in the treatment of hematological malignancies, solid tumors remain challenging for the field. A significant obstacle is the exclusion of T cells from the tumor microenvironment (TME). In contrast, monocytes/macrophages are naturally recruited to the TME. These cells then have the potential to phagocytose tumor cells, activate the TME, and prime a broad anti-tumor adaptive immune response via T cell recruitment and activation. We have previously developed CT-0508, a chimeric antigen receptor macrophage (CAR-M) targeting HER2 which showed efficacy in a variety of pre-clinical models and is currently in a Phase I clinical trial for patients with HER2+ solid tumors. Mesothelin is overexpressed in a variety of solid tumors, including mesothelioma, lung, pancreatic, and ovarian cancers. To leverage tumor biology with myeloid cells, we engineered primary human macrophages using the chimeric adenoviral vector Ad5f35 to express a CAR containing a human scFv against human mesothelin. We used both in vitro cell based assays and in vivo xenograft models to assess the activity of CT-1119. CAR-M engineered with an Ad5f35 vector demonstrated high CAR expression, high viability, upregulated M1 (anti-tumor) macrophage markers, and downregulated M2 (pro-tumor) macrophage markers. CT-1119 specifically phagocytosed multiple mesothelin expressing tumor cell lines in a CAR-dependent and antigen-dependent manner. CT-1119 demonstrated robust in vitro killing of the relevant tumor cell lines A549 and MES-OV expressing mesothelin. CAR engagement also induced the release of pro-inflammatory cytokines such as TNFα following stimulation with mesothelin in both cell-free and cell-based contexts in a dose-dependent manner. In vivo, CT-1119 significantly reduced tumor burden in a murine xenograft model of lung cancer. Similarly, human monocytes targeting mesothelin were successfully generated using the same Ad5f35 vector and demonstrated specific activity against mesothelin positive tumor cells. The presented results demonstrate that CT-1119, an autologous human anti-mesothelin CAR-M, can cause phagocytosis, tumor cell killing, and pro-inflammatory cytokine release in response to stimulation with mesothelin. These results show that CAR-M is a feasible approach for the treatment of mesothelin expressing sold tumors via the potential for induction of a systemic anti-tumor response. Citation Format: Nicholas R. Anderson, Brinda Shah, Alison Worth, Rashid Gabbasov, Brett Menchel, Kerri Ciccaglione, Daniel Blumenthal, Stefano Pierini, Sabrina Ceeraz DeLong, Sascha Abramson, Thomas Condamine, Michael Klichinsky. A mesothelin targeting chimeric antigen receptor macrophage (CAR-M) for solid tumor immunotherapy: pre-clinical development of CT-1119. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4053.

Meiosis-specific chromatin structures, guided by histone modifications, are critical mediators of a meiotic transient transcription program and progression through prophase I. Histone H3K4 can be methylated up to three times by the Set1-containing COMPASS complex and each methylation mark corresponds to a different chromatin conformation. The level of H3K4 modification is directed by the activity of additional COMPASS components. In this study, we characterized the role of the COMPASS subunits during meiosis in Saccharomyces cerevisiae. In vegetative cells, previous studies revealed a role for subunits Swd2, Sdc1, and Bre2 for H3K4me2 while Spp1 supported trimethylation. However, we found that Bre2 and Sdc1 are required for H3K4me3 as yeast prepare to enter meiosis while Spp1 is not. Interestingly, we identified distinct meiotic functions for the core COMPASS complex members that required for all H3K4me, Set1, Swd1, and Swd3. While Set1 and Swd1 are required for progression through early meiosis, Swd3 is critical for late meiosis and spore morphogenesis. Furthermore, the meiotic requirement for Set1 is independent of H3K4 methylation, suggesting the presence of nonhistone substrates. Finally, checkpoint suppression analyses indicate that Set1 and Swd1 are required for both homologous recombination and chromosome segregation. These data suggest that COMPASS has important new roles for meiosis that are independent of its well-characterized functions during mitotic divisions.

Transcriptional regulation is dependent upon the interactions between the RNA pol II holoenzyme complex and chromatin. RNA pol II is part of a highly conserved multiprotein complex that includes the core mediator and CDK8 subcomplex. In Saccharomyces cerevisiae, the CDK8 subcomplex, composed of Ssn2p, Ssn3p, Ssn8p, and Srb8p, is thought to play important roles in mediating transcriptional control of stress-responsive genes. Also central to transcriptional control are histone post-translational modifications. Lysine methylation, dynamically balanced by lysine methyltransferases and demethylases, has been intensively studied, uncovering significant functions in transcriptional control. A key question remains in understanding how these enzymes are targeted during stress response. To determine the relationship between lysine methylation, the CDK8 complex, and transcriptional control, we performed phenotype analyses of yeast lacking known lysine methyltransferases or demethylases in isolation or in tandem with SSN8 deletions. We show that the RNA pol II CDK8 submodule components SSN8/SSN3 and the histone demethylase JHD2 are required to inhibit pseudohyphal growth—a differentiation pathway induced during nutrient limitation—under rich conditions. Yeast lacking both SSN8 and JHD2 constitutively express FLO11, a major regulator of pseudohyphal growth. Interestingly, deleting known FLO11 activators including FLO8, MSS11, MFG1, TEC1, SNF1, KSS1, and GCN4 results in a range of phenotypic suppression. Using chromatin immunoprecipitation, we found that SSN8 inhibits H3 Lys4 trimethylation independently of JHD2 at the FLO11 locus, suggesting that H3 Lys4 hypermethylation is locking FLO11 into a transcriptionally active state. These studies implicate the CDK8 subcomplex in fine-tuning H3 Lys4 methylation levels during pseudohyphal differentiation.