The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug accumulation in the tumor tissue, stimulates tumor-infiltrating CD8+ T and NK cells, and leads to a dose-dependent reduction of tumor growth. These results support further characterization of the immune modulatory, anti-tumor properties of THOR-707 and represent a fundamental advance in the application of synthetic biology to medicine, leveraging engineered semi-synthetic organisms as cellular factories to facilitate discovery and production of differentiated classes of chemically modified biologics.

High-dimensional flow cytometry is proving to be valuable for the study of subtle changes in tumor-associated immune cells. As flow panels become more complex, detection of minor immune cell populations by traditional gating using biaxial plots, or identification of populations that display small changes in multiple markers, may be overlooked. Visualization of t-distributed stochastic neighbor embedding (viSNE) is an unsupervised analytical tool designed to aid the analysis of high-dimensional cytometry data. In this study we use viSNE to analyze the simultaneous binding of 15 fluorophore-conjugated Abs and one cell viability probe to immune cells isolated from syngeneic mouse MB49 bladder tumors, spleens, and tumor-draining lymph nodes to identify patterns of anti-tumor immune responses. viSNE maps identified populations in multidimensional space of known immune cells, including T cells, B cells, eosinophils, neutrophils, dendritic cells, and NK cells. Based on the expression of CD86 and programmed cell death protein 1, CD8+ T cells were divided into distinct populations. Additionally, both CD8+ T cells and CD8+ dendritic cells were identified in the tumor microenvironment. Apparent differences between splenic and tumor polymorphonuclear cells/granulocytic myeloid-derived suppressor cells are due to the loss of CD44 upon enzymatic digestion of tumors. In conclusion, viSNE is a valuable tool for high-dimensional analysis of immune cells in tumor-bearing mice, which eliminates gating biases and identifies immune cell subsets that may be missed by traditional gating.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that promotes inflammatory cytokine production by activating the MEK/ERK pathway. Tpl2 has been shown to be important for eliciting the inflammatory properties of macrophages; however, there is relatively little known about the contribution of Tpl2 to neutrophil effector functions. This is an important consideration, as neutrophils provide the first line of defense against infection in the innate immune system. We found that Tpl2 is expressed in both human and murine neutrophils, suggesting a potential function for Tpl2 in this lineage. Despite significantly higher proportions of bone marrow (BM) neutrophils in Tpl2-deficient ( ) mice compared with wild-type (WT) mice, mice have significantly reduced proportions of circulating neutrophils. neutrophils show impaired recruitment to thioglycollate, which was primarily a result of neutrophil-extrinsic factors in the host. In response to infection, neutrophils secrete inflammatory cytokines and produce reactive oxygen species (ROS), which promote bacterial killing. Tpl2 ablation impaired neutrophil TNF secretion in response to LPS stimulation, superoxide generation in response to the chemotactic peptide fMLP, and killing of the extracellular bacterium, , despite normal bacterial phagocytosis. These results implicate Tpl2 in the regulation of multiple neutrophil antimicrobial pathways, including inflammatory cytokine secretion and oxidative burst. Furthermore, they indicate that Tpl2 functions early during infection to bolster neutrophil-mediated innate immunity against extracellular bacteria.

Autoimmune diseases are approaching epidemic levels, estimated to affect 5–8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2−/− naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of Tpl2−/− T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation in vivo, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.

BackgroundTHOR-707 (SAR444245) is a recombinant human IL-2 molecule irreversibly bound to a PEG chain to block alpha-binding while retaining near-native affinity for beta/gamma IL-2 receptor subunits. We report updated results from the ongoing HAMMER phase 1/2 trial.MethodsSAR444245 was given via IV infusion as monotherapy Q2W [A] or Q3W [B], with pembrolizumab 200mg IV Q3W [C], or Q3W with cetuximab 400mg/m2 IV on D1 then 250mg/m2 IV QW [D] after pre-medication and peri-infusion hydration. A 3+3 design was used to identify the MTD/RP2D in pts with advanced solid tumors. Key objectives included assessments of safety, efficacy, pharmacokinetics (PK) and pharmacodynamics (PD).Results68 pts, median age 61.5 (37–78) yrs with median 3 (1–10) prior therapies enrolled. Most common tumors: melanoma (n=10), colorectal (n=11). Doses tested by cohort: [A]: 8–16 µg/kg (n=9); [B]: 8–40 µg/kg (n=29); [C]: 8–32 µg/kg (n=20); [D]: 16–24 µg/kg (n=10). The most common (>30%) AEs included pyrexia (52.5%), nausea (50.0%), flu-like symptoms (44.1%), vomiting (36.8%), chills (32.4%), fatigue (32.4%), AST elevation (30.9%). AEs generally resolved promptly with supportive care. Grade(G) 3/4 (>5%) related AEs included ALT/AST elevation (5.9%), and decreased lymphocyte count (26.5% within first 24 hrs, recovering by 48–72 hrs, this lymphocyte migration is mechanistically consistent with immune cell margination). G3/4 CRS was observed in 2 pts. Two DLTs occurred: G3 infusion reaction (32 µg/kg [B]) and G3 AST/ALT/G2 bilirubin elevation with G2 CRS (24 µg/kg [C]). No vascular leak syndrome, QTc prolongation, cardiac, or end organ toxicity was observed. Half-life was ~10 h. Sustained increases in CD8 T and NK cells were observed (fold relative to baseline): monotherapy (1–9.4x and 2–43.3x); with pembrolizumab (0.5–5.78x and 1.5–26.9x); with cetuximab (1.3–7.57x and 3.6–45.4x). Max CD4 and eosinophils increased to 136 cell/µL and 1078 cell/µL. No IL-5 elevation or ADAs were observed. Transient IL-6 increases in 4 pts (500, 627, 1000, 1100 pg/mL) were not associated with AEs. Four pts had confirmed PRs (1 PD1-treated SCC, unknown primary [B]; 2 PD1-naïve BCC and 1 PD1-treated HNSCC [C]); 3 pts had minor responses -- prostate (-24%) and PD1-treated melanoma (-17%) [B]; PD1-treated NSCLC (¬-29%) [C] -- after ≥2 scans. 23 pts completed ≥5 cycles.ConclusionsSAR444245 was well tolerated and demonstrated antitumor activity in heavily pretreated patients, including prior checkpoint inhibitor therapy. Clinical safety, efficacy and PD suggest a wide therapeutic window. Combination with pembrolizumab and cetuximab leveraged SAR44245’s effects on CD8 T and NK cells.Trial RegistrationNCT04009681Ethics ApprovalThe clinical trial was approved by each institutions ethics’ and review board prior to beginning study enrollment.

The serine/threonine kinase tumor progression locus 2 (Tpl2, also known as Map3k8/Cot) is a potent inflammatory mediator that drives the production of TNF␣, IL-1␤, and IFN␥. We previously demonstrated that Tpl2 regulates T cell receptor (

Tumor progression locus 2 (Tpl2) is a serine-threonine kinase that regulates Th1 differentiation, secretion of the inflammatory cytokine gamma interferon (IFN-␥), and host defense against the intracellular pathogens Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis. However, relatively little is known about the contribution of Tpl2 to Th17 differentiation and immune cell function during infection with an extracellular pathogen. The goal of this study was to determine whether Tpl2 influences the immune response generated to the extracellular bacterium Citrobacter rodentium, which induces a mixed Th1 and Th17 response. During peak infection with C. rodentium, Tpl2 Ϫ/Ϫ mice experienced greater bacterial burdens with evidence of dissemination to the liver and spleen but ultimately cleared the bacteria within 3 weeks postinfection, similar to the findings for wild-type mice. Tpl2 Ϫ/Ϫ mice also recruited fewer neutrophils and monocytes to the colon during peak infection, which correlated with increased bacterial burdens. In mixed bone marrow chimeras, Tpl2 was shown to play a T cell-intrinsic role in promoting both IFN-␥ and interleukin-17A production during infection with C. rodentium. However, upon CD4 T cell transfer into Rag Ϫ/Ϫ mice, Tpl2 Ϫ/Ϫ CD4 T cells were as protective as wild-type CD4 T cells against the dissemination of bacteria and mortality. These data indicate that the enhanced bacterial burdens in Tpl2 Ϫ/Ϫ mice are not caused primarily by impairments in CD4 T cell function but result from defects in innate immune cell recruitment and function. KEYWORDS Citrobacter, T helper cells, gastrointestinal infection, intestinal immunity, neutrophilsC itrobacter rodentium is a nonmotile Gram-negative rod that is a natural mouse and gerbil pathogen (1, 2). Upon infection, C. rodentium colonizes the large intestine, primarily the cecum and distal portion of the colon (3), and forms a close association with the epithelium and lamina propria that results in attaching and effacing lesions in the large intestine (4, 5). However, C. rodentium can disseminate out of the intestines and be found in the nasopharynx, lung, heart, liver, and spleen (6). Early innate responses to C. rodentium are associated with recruitment and the antimicrobial functions of neutrophils, macrophages, NK cells, and innate lymphoid cells (7-12). Neutrophils secrete interleukin-17A (IL-17A) and IL-22, promote the production of antimicrobial defensins by epithelial cells, and protect against the development of diarrhea (11,13). The bacterial association with the lamina propria of the large intestine subsequently induces a mixed Th1 and Th17 response associated with IL-12, gamma interferon (IFN-␥), tumor necrosis factor (TNF), . Clearance of the bacteria occurs within 3 weeks in a wild-type host and is dependent upon both CD4 T cell and B cell functions (18, 19).Tumor progression locus 2 (Tpl2; also known as MAP3K8) is a serine-threonine Citation Acuff NV, Li X, Latha K, Nagy T, Watford WT. 2017. Tpl2 promotes innat...

Multiple myeloma (MM) is a plasma cell malignancy characterized by clonal accumulation of malignant plasma cells in the bone marrow. CD38 is highly expressed on MM cells, and antibodies targeting CD38 (such as Isatuximab and Daratumumab) induce MM cell killing though several mechanisms, particularly by NK cell-mediated antibody dependent cellular cytotoxicity (ADCC). However, NK cells also express CD38 and treatment with anti-CD38 antibodies results in a rapid decrease of NK cells presumably due to fratricide, which may potentially limit NK cell mediated ADCC and reduce the efficacy of anti-CD38 antibody treatment. NK cells lacking CD38 in combination with Daratumumab have been shown to be resistant to NK cell fratricide and have improved ADCC. Here, we investigated the effect of knocking out CD38 in K-NK cells (CD38KO K-NK) in combination with Isatuximab. Peripheral blood NK cells were isolated from healthy donors and expanded using Kiadis PM21 particle technology (Oyer et al, Cytotherapy 2016) to produce highly activated K-NK cells. To produce CD38KO K-NK cells, CRISPR gene editing was applied during NK cell expansion by electroporating with Cas9/RNP complexes targeting CD38, and successful deletion of CD38 was confirmed by flow cytometry analysis. Analysis of key NK cell receptors by flow cytometry evidenced very similar receptor profiles between WT and CD38KO K-NK cells, suggesting that CD38 deletion does not affect the potent activation state of the K-NK cells. Importantly, CD38KO K-NK cells were found to be resistant to Isatuximab-induced fratricide. The cytotoxic activity of CD38KO K-NK cells in combination with Isatuximab against LP-1 and H929 MM cell lines was also measured. Calcein release assay and Incucyte based analysis revealed that cytotoxicity of CD38KO K-NK cells is enhanced in presence of Isatuximab, and that CD38KO K-NK cells are more cytotoxic than WT when combined with Isatuximab. Furthermore, the effect of Isatuximab/CD38KO K-NK cells combination also in association with SAR444245 (also termed THOR-707; Ptacin et al, Nat Commun 2021), an engineered non-α binding IL-2 that promotes NK cell activation and proliferation, was tested. Cytotoxicity of WT or CD38KO NK cells is enhanced in the presence of SAR444245. Indeed, addition of SAR444245 was found to further enhance the cytotoxic activity of CD38KO K-NK cells against LP-1 MM cells when combined with Isatuximab, resulting in an overall superior and sustained cytotoxicity. These data suggest that deletion of CD38 mitigates NK cell fratricide and improves Isatuximab-mediated ADCC against MM cells, and provide evidence for the therapeutic potential of the triple combination CD38KO NK cells, Isatuximab and SAR444245 in the setting of MM. Citation Format: Marco Meloni, Pauline Perrin, Erik Slinger, Chrissta Maracle, Alain Fournier, Nicole Acuff, Jill Mooney, Robert Y. Igarashi, Angela Virone-Oddos, Marielle Chiron. CD38KO K-NK cells prevent NK cell fratricide effect and improve isatuximab-mediated cytotoxicity against multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4209.