Colorectal cancer with chromosomal instability (CIN+) phenotype is immunosuppressive and refractory to immune checkpoint blockade (ICB) therapy. Recently, KIF18A is found to be a mitotic vulnerability in chromosomally unstable cancers, but whether targeting KIF18A affects antitumor immunity in CIN+ colorectal cancer is unknown. In our study, western blot, cell viability assay, transwell migration and invasion assays, flow cytometry, animal model, immunohistochemistry (IHC) staining, reverse transcription-quantitative PCR (RT-qPCR) and ELISA assay were conducted to evaluate the potential function of KIF18A in CIN+ colorectal cancer. We found that KIF18A inhibition by short hairpin RNAs (ShRNAs) or small inhibitor AM-1882 suppressed proliferation, migration, invasion and tumor growth and metastasis of CIN+ colorectal cancer cells in vitro and in vivo. Moreover, targeting KIF18A disrupted cell-cycle progression and induced G2/M arrest in CIN+ colorectal cancer cells. In addition, KIF18A inhibition promoted immune infiltration and activation in CIN+ colorectal tumors. KIF18A inhibition suppressed proliferation of Tregs and increased infiltration and activation of cytotoxic CD8+ T cells in CIN+ colorectal tumors. Mechanically, KIF18A inhibition stimulated type I IFN signaling and cGAS-STING activation in CIN+ colorectal tumors. Finally, targeting KIF18A enhanced PD-1 blockade efficiency in CIN+ colorectal tumors through T cells. Our data elucidated a novel role of KIF18A in antitumor immunity of CIN+ colorectal cancer.
© 2025. The Author(s).

NK cells are endowed with immunological memory to a range of pathogens but the development of NK cell memory in bacterial infections remains elusive. Here, we establish an assay inducing memory-like NK cell response to Burkholderia pseudomallei, the causative agent of the severe bacterial disease called melioidosis, and explore NK cell memory in a melioidosis patient cohort. We show that NK cells require bacteria-primed monocytes to acquire memory-like properties, demonstrated by bacteria-specific responses, features that strongly associate with CD160 expression. Induction of this memory-like NK cell is partly dependent on CD160 and IL-12R. Importantly, CD160 expression identifies memory-like NK cells in a cohort of recovered melioidosis patients with heightened responses maintained at least 3 months post hospital admission and reduced numbers of this cell population independently correlate with recurrent melioidosis. These newly identified memory-like NK cells are a promising target for future vaccine design and for monitoring protection against infection.
© 2023 The Authors.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.
Copyright: © 2023 Shepherd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Lung cancer remains a devastating disease with a poor clinical outcome. A biomarker signature which could distinguish lung cancer from metastatic disease and detect therapeutic failure would significantly improve patient management and allow for individualized, risk-adjusted therapeutic decisions. In this study, circulating Hsp70 levels were measured using ELISA, and the immunophenotype of the peripheral blood lymphocytes were measured using multiparameter flow cytometry, to identify a predictive biomarker signature for lung cancer patients pre- and post-operatively, in patients with lung metastases and in patients with COPD as an inflammatory lung disease. The lowest Hsp70 concentrations were found in the healthy controls followed by the patients with advanced COPD. Hsp70 levels sequentially increased with an advancing tumor stage and metastatic disease. In the early-recurrence patients, Hsp70 levels started to increase within the first three months after surgery, but remained unaltered in the recurrence-free patients. An early recurrence was associated with a significant drop in B cells and an increase in Tregs, whereas the recurrence-free patients had elevated T and NK cell levels. We conclude that circulating Hsp70 concentrations might have the potential to distinguish lung cancer from metastatic disease, and might be able to predict an advanced tumor stage and early recurrence in lung cancer patients. Further studies with larger patient cohorts and longer follow-up periods are needed to validate Hsp70 and immunophenotypic profiles as predictive biomarker signatures.

Chimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand-based CAR targeting the cancer antigen IL13Rα2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13Rα2 versus the more ubiquitous unintended target IL13Rα1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3ζ-containing CARs (IL13ζ), second-generation 4-1BB (CD137)-containing or CD28-containing CARs (IL13-BBζ or IL13-28ζ), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BBζ). In vitro coculture assays at high tumor burden establish that second-generation IL13-BBζ or IL13-28ζ outperform first-generation IL13ζ and third-generation IL13-28BBζ CAR designs, with IL13-BBζ providing superior CAR proliferation and in vivo antitumor potency in human xenograft mouse models. IL13-28ζ displayed a lower threshold for antigen recognition, resulting in higher off-target IL13Rα1 reactivity both in vitro and in vivo. Syngeneic mouse models of GBM also demonstrate safety and antitumor potency of murine IL13-BBζ CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BBζ CARs for greater selective recognition of IL13Rα2 over IL13Rα1, higher proliferative potential, and superior antitumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition.
This study reveals how modulating CAR design outside the antigen targeting domain improves selective tumor recognition. Specifically, this work shows improved specificity, persistence, and efficacy of 4-1BB-based IL13-ligand CARs. Human clinical trials evaluating IL13-41BB-CAR T cells are ongoing, supporting the clinical significance of these findings.
© 2023 The Authors; Published by the American Association for Cancer Research.