M. Templeton

BACKGROUND: Morbidity and mortality for critically ill patients with infections remains a global healthcare problem. We aimed to determine whether β-lactam antibiotic dosing in critically ill patients achieves concentrations associated with maximal activity and whether antibiotic concentrations affect patient outcome. METHODS: This was a prospective, multinational pharmacokinetic point-prevalence study including 8 β-lactam antibiotics. Two blood samples were taken from each patient during a single dosing interval. The primary pharmacokinetic/pharmacodynamic targets were free antibiotic concentrations above the minimum inhibitory concentration (MIC) of the pathogen at both 50% (50% f T>MIC) and 100% (100% f T>MIC) of the dosing interval. We used skewed logistic regression to describe the effect of antibiotic exposure on patient outcome. RESULTS: We included 384 patients (361 evaluable patients) across 68 hospitals. The median age was 61 (interquartile range [IQR], 48-73) years, the median Acute Physiology and Chronic Health Evaluation II score was 18 (IQR, 14-24), and 65% of patients were male. Of the 248 patients treated for infection, 16% did not achieve 50% f T>MIC and these patients were 32% less likely to have a positive clinical outcome (odds ratio [OR], 0.68; P = .009). Positive clinical outcome was associated with increasing 50% f T>MIC and 100% f T>MIC ratios (OR, 1.02 and 1.56, respectively; P < .03), with significant interaction with sickness severity status. CONCLUSIONS: Infected critically ill patients may have adverse outcomes as a result of inadeqaute antibiotic exposure; a paradigm change to more personalized antibiotic dosing may be necessary to improve outcomes for these most seriously ill patients.

Emergency laparotomies have poor outcomes with variable postoperative critical care provision [1-3]. All patients requiring an emergency laparotomy with an estimated risk of death of >10% should go to critical care. Time of surgery should not affect standard of care [3,4]. In advance of the National Emergency Laparotomy Audit (NELA) results [2], our objective was to see whether the level of postoperative care and time of surgery affect outcome.

. However, concurrent inhibition of Aurk restored the TP53/CDKN1A pathway, sensitizing the tumors to Bcl2 inhibitor-mediated apoptosis. These data lay the groundwork for evaluation of this combination in the clinical setting.

Abstract Chimeric Antigen Receptor T cell (CAR-T) therapy has transformed cancer treatment. However, its application is hindered by complex manufacturing related to genetic engineering, high cost, toxicity, and resistance driven by antigen escape and tumor heterogeneity. To address these challenges, we developed a first-in-class pretargeted glycoengineered natural killer (PG-NK) cell platform that intentionally decouples the targeting moiety (ligand) from the effector cell (NK cell) using bioorthogonal click chemistry. This allows NK cells to be universally glycoengineered and paired with tumor-specific antibodies (Abs), enabling rapid, antigen-flexible targeting without genetic modification. For this, NK cells were modified with azide (NK-AZ), cyclopropene (NK-CP), or tetrazine (NK-TZ)-labeled glycans, while tumors were pretargeted with Abs functionalized with complementary click partners, dibenzocyclooctyne (Ab-DBCO) or trans-cyclooctene (Ab-TCO). The NK-TZ/Ab-TCO pair was selected as the lead configuration based on its superior affinity and efficacy. Initial glycoengineering experiments used CD16(-) NK92 cells. After labeling with azido-mannose, NK cells were modified with DBCO-TZ to display TZ on their surface. The combination of anti-CD20 (αCD20) Ab-TCO and NK-TZ resulted in a 15-fold increase in NK cell binding to CD20+ tumor cells (31.6% PG-NK vs 2.1% unmodified NK, P&amp;lt;0.0001) within 2 hours, promoting rapid and robust effector cell engagement. Cytotoxicity assays across multiple CD20+ lymphoma cell lines (Raji, DOHH2, DAUDI, and MINO) showed that the PG-NK cell platform (αCD20Ab-TCO + NK-TZ) consistently induced &amp;gt;3-fold higher cytotoxicity in a dose-dependent manner with increasing effector-to-target (E:T) ratios (e.g. 45.8% NK-TZ vs 13.1% control in MINO cells at E:T 2:1, p&amp;lt;0.01). Functional assays, including mass cytometry, revealed enhanced granzyme B and perforin expression associated with NK-TZ cells when co-cultured with target tumor cells, despite the absence of CD16 Fcɣ receptor. Glycoengineering of CD16(+) NK92 cells further enhanced therapeutic efficacy in the pretargeted setting, suggesting synergy between PG-NK and antibody-dependent cellular cytotoxicity (ADCC). To validate the off-the-shelf potential of the PG-NK platform, we treated Raji (CD20+/HER2-) cells with either αCD20 or αHER2 Ab-TCO. NK-TZ cells selectively eliminated Raji cells when pretargeted with αCD20Ab-TCO (33.5% αCD20 vs 16.3% αHER2, p&amp;lt;0.0001). Using the same batch of NK-TZ cells, BT474 (CD20-/HER2+) breast cancer cells were eliminated when pretargeted with αHER2Ab-TCO (30.3% αHER2 vs 14.0% αCD20, p&amp;lt;0.0001), demonstrating that NK-TZ cells are agnostic to tumor type or antigen profile, provided tumor cells are primed with the appropriate Ab-TCO. We next evaluated PG-NK therapy in vivo. In a subcutaneous Raji xenograft model, PG-NK treatment (αCD20Ab-TCO followed by NK-TZ 24 hours later) significantly reduced tumor volume (158 mm3 PG-NK vs 1639 mm3 unmodified NK, p&amp;lt;0.001) and improved survival after 3 weeks of therapy (80% PG-NK vs 0% all control groups at Day 50 of follow-up). A patient-derived mantle cell lymphoma model also showed superior efficacy of PG-NK therapy, with reduced bone marrow tumor burden (1.0% vs 11.6% CD20+ cells for PG-NK vs control, p&amp;lt;0.01) and extended survival (100% PG-NK vs 0% all control groups at Day 50; p&amp;lt; 0.001). Lastly, in an aggressive DOHH2 disseminated model, PG-NK treatment reduced minimal residual disease in peripheral blood (0.03% vs 0.3% CD20+ cells for PG-NK vs control, p&amp;lt;0.05) and significantly improved survival (67% PG-NK vs 0% all control groups at Day 60; p&amp;lt;0.001). Biodistribution studies confirmed selective tumor targeting and infiltration by PG-NK cells. ELISA assays showed elevated plasma levels of granzyme B, perforin, IFN-γ, and TNF-α in PG-NK-treated animals. Notably, these potent antitumor responses were achieved without discernible toxicity. Our study establishes a scalable, off-the-shelf, tumor-specific cell therapy platform that does not require genetic modification. Preclinical findings highlight Ab-TCO and NK-TZ as the optimal pairing, demonstrating rapid high-affinity binding and enhanced cytotoxicity. In vivo validation across three independent lymphoma models confirmed robust tumor killing and significant survival benefit. This PG-NK platform offers a novel framework for future cell therapy development, providing a versatile, antigen-adaptable strategy for targeted cell therapy.

Background: In the current era of acute myeloid leukemia (AML) management, hypomethylating agents (HMAs) remain as the backbone of combination regimens for front-line treatment of elderly or unfit patients (pts) and/or as salvage therapy in relapsed/refractory pts. Data on biological predictors of HMA response are limited. Our previous work explored cytidine deaminase (CDA), known to inactivate HMAs, and nucleophosmin 1 (NPM1), determined by core pathway analysis to indirectly influence CDA expression. No clear correlation was identified in pt samples between CDA protein expression and NPM1 status or response, and pharmacogenomic analysis showed no CDA single nucleotide polymorphisms were predictive of response to HMAs. We aimed to expand prior findings using RNA sequencing (RNA seq) and gene set enrichment analysis (GSEA) to identify gene signatures predictive of HMA response and propose potential therapeutic targets. Methods: AML pts with banked samples who received frontline, bridging, or salvage HMA-based therapy between January 2014 to December 2018 were reviewed. Responses following at least 2 cycles of HMA were categorized as complete response (CR), CRi (CR with incomplete hematologic recovery), morphologic leukemia-free state (MLFS), complete hematologic response (CHR), or refractory. Pts were categorized as responders (CR, CRi, MLFS, CHR) or non-responders (refractory). Tumor cells were purified using immunomagnetic selection from bone marrow aspirates collected at diagnosis (dx). RNA seq was performed on 20 available pt tumor samples. Unsupervised clustering was performed and GSEA was completed to compare identified clusters using a false discovery rate (FDR) cut-off of less than 25%. GSEA-derived gene pathway scores were assessed by Wilcoxon rank-sum test. To evaluate AML cell lines to explore the non-responder phenotype, viability of AML cell lines was assessed following treatment with HMA azacitidine (Aza) and MS177 (a proteolysis targeting chimeric EZH2 degrader, also known to degrade MYC). Immunoblotting was used to assay proteolysis targeting chimeric (PROTAC)-induced MYC degradation. Results: Unsupervised clustering of RNA seq from 20 tumor samples at dx, labeled for response and NPM1 status, revealed 3 distinct groups: a non-responder group, a responder group, and a mixed response group ( Figure 1A). GSEA comparing the non-responder group to the responder group revealed several enriched genes in the non-responder group, including MYC targets (FDR &amp;lt;0.001), E2F targets (FDR &amp;lt;0.001), and G2M checkpoint (FDR=0.244). Upregulation of MYC targets was confirmed by enrichment profiling. Application of enrichment pathway scores revealed significant differences between non-responders and responders for E2F targets (p=0.026), G2M checkpoint (p=0.026), and MYC targets (p=0.0022). To explore MYC as a therapeutic target, we assayed MYC degradation in leukemia cells treated with two PROTAC degraders. The PROTAC MS177 induced marked MYC degradation in HL-60 cells ( Figure 1B). Cytotoxicity assays showed that in Kasumi-1 cells, the 50% inhibitory concentrations (IC50) for Aza and MS177 were 2.9 µM (responder phenotype) and 1.1 µM, respectively ( Figure 1C); in HL-60 cells the IC50 for Aza and MS177 were 10.9 µM (non-responder phenotype) and 0.06 µM, respectively ( Figure 1D). MS177 also showed efficacy in cytotoxicity assays using pt derived tumor samples. Conclusions: Using tumor samples from AML pts receiving HMAs, we identified a non-responder gene phenotype enriched with MYC upregulation. GSEA-derived pathway scoring differentiated non-responders from responders in our dataset. To further evaluate whether the enrichment pathway score is predictive for response across other AML cohorts, we are applying enrichment scores to publicly available AML datasets. With MYC upregulation identified as a potential driver of HMA failure, MYC degradation or inhibitory targets are recognized as potential therapeutic targets for pts with a non-responder gene signature. We established the HL-60 AML cell line, found to be less sensitive to Aza than the Kasumi-1 cell line, demonstrated greater sensitivity to MYC degradation compared to Kasumi-1 cells and may reflect the non-responder phenotype. Further in vitro efforts are ongoing to evaluate MYC targets for the HMA non-responder phenotype.