The introduction of checkpoint immunotherapeutic agents in the last decade has revolutionized cancer treatment. Although anti-PD-1, anti-PD-L1 and anti-CTLA4 are promising therapies, many patients fail to respond or relapse due to drug resistance potentially due to redundancy of immune checkpoints. One of the ways to improve the efficacy of this cancer treatment is to target two or even three immune checkpoints. To date, the benefit of combined anti-VISTA/anti-PD-L1 therapy has been confirmed, but no one has investigated the efficacy of blocking these negative immune checkpoints with a bispecific anti-VISTA/anti-PD-L1 antibody.
In this study, the bispecific antibodies (bsAbs) were produced in three formats: symmetric (IgG-HC-scFv), asymmetric (Fab-scFv-Fc(KIH)) and 2 x scFv. The binding and blocking properties of these bispecific antibodies (bsAbs) and their efficacy compared to monotherapy and combination therapy were then determined using endometrial (RL95-2), pancreatic (PANC-1) and breast (BT-20) cancer cell lines.
The bsAbs generated in this study showed weaker binding properties to PD-1 and VISTA in ELISA (EC50) than the parent antibodies (atezolizumab and onvatilimab). Blockade of VISTA/VSIG-3 binding was also weaker with bsAbs compared to onvatilimab, but the ability to block the PD-1/PD-L1 pathway was slightly better than with atezolizumab. The Fc-based bsAbs showed statistically significant higher levels of lysis of endometrial, breast and pancreatic cancer cells. The symmetric bsAbs (IgG-HC-scFv) showed the most promising therapeutic potential. Higher levels of cancer cell lysis were associated with higher levels of pro-inflammatory cytokines. Both the asymmetric and symmetric bsAbs resulted in higher secretion levels of IFN-γ, TNFα and Granzyme B than anti-VISTA, anti-PD-L1 monotherapy and anti-VISTA/anti-PD-L1 combination therapy.
The high level of tumor cell lysis and increased expression of pro-inflammatory cytokines induced by the Fc-based bsAbs suggest a novel approach for the treatment of pancreatic, endometrial and breast cancer.
Copyright © 2025 Bielski, Barczyński, Mikitiuk, Myrcha, Rykała, Boon, Gąsior, Hec-Gałązka, Holak and Sitar.

Hepatitis C virus (HCV) infection remains a leading cause of morbidity and mortality in people with human immunodeficiency virus (HIV). Liver fibrosis progression is more rapid in people with HIV/HCV coinfection compared to HCV monoinfection and the rate of resolution of liver fibrosis after HCV cure is unknown in people with HIV. Invariant natural killer T (iNKT) cells are enriched in the liver and play important roles in initiating immune responses to hepatotropic pathogens and promoting healing following injury. It was recently reported that the pro-healing CD4+ iNKT cells are preferentially infected and depleted in early HIV infection, but this effect on HCV-related liver disease outcomes is unclear.
Here we examined and compared peripheral blood iNKT cells from people with HIV/HCV coinfection and people with HIV and HCV monoinfection or no infection (controls). We evaluated the iNKT cells' expansion potential and phenotype using an unbiased Uniform Manifold Approximation and Projection (UMAP) and clustering based approach.
We observed that circulating iNKT cells from people with HIV and HIV/HCV coinfection have impaired expansion to T-cell receptor (TCR) stimulation. We also observed an enrichment of the CD8+ and CD57+ iNKT subsets, which are thought to represent terminally differentiated iNKT cells. HCV monoinfection on the other hand minimally impacted iNKT phenotypes compared to controls.
The changes observed in iNKT phenotype and proliferative ability in people with HIV/HCV coinfection suggest an impairment that may be contributing to the enhanced pathogenesis during coinfection and could inform novel therapeutic approaches.
Copyright © 2025 Nettere, White, Williams, Jha, Moody, Chan, Ferrari and Naggie.

The application of programmed cell death protein 1 (PD-1) antibodies has brought significant benefits to patients with non-small cell lung cancer (NSCLC). However, not all patients respond to PD-1 immune therapy. The aim of this study was to identify response biomarkers to predict the efficacy of chemotherapy combined with anti-PD-1 therapy in NSCLC patients.
Thirty-two NSCLC patients receiving chemotherapy combined with anti-PD-1 therapy were recruited, and peripheral blood samples were collected before and after treatment. Flow cytometry was used to detect the proportions of circulating T-cell subsets, and cytokines in the blood serum were detected via ELISA.
The results revealed that, among the CR/PR group (CR, complete response; PR, partial response; n = 22), the proportions of CD3+TIM-3+PD-1+, CD3+CD4+TIM-3+PD-1+, and CD3+CD8+TIM-3+PD-1+, CD3+γδT+PD-1+, CD3+γδT+Vδ1+PD-1+, and CD3+γδT+Vδ2+PD-1+T cells were lower after treatment, with no significant differences found between the stable disease (SD) and progressive disease (PD) groups (n = 10). Some proinflammatory cytokines are highly expressed in patients with NSCLC.
This study suggests that monitoring changes in immune biomarkers in the circulating cells of NSCLC patients may help differentiate CR/PR patients from SD/PD patients, providing a potential new approach for assessing the efficacy of chemotherapy combined with anti-PD-1 therapy.
Copyright © 2025 Cao, Zhang, Guo, Wu, Guo, Zhang, Zhang, Liu, Li, Yang, He, Bai, Lv, Xie, Huang, Xiao, Deng, Li, Zhu, Jia, Yin and Wang.

Cystic fibrosis (CF) is caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, resulting in multi-organ dysfunction and decreased lifespan and quality of life. A durable cure for CF will likely require a gene therapy approach to correct CFTR. Rapid advancements in genome editing technologies such as CRISPR/Cas9 have already resulted in successful FDA approval for cell-based gene editing therapies, providing new therapeutic avenues for many rare diseases. However, immune responses to gene therapy delivery vectors and editing tools remain a challenge, especially for strategies targeting complex in vivo tissues such as the lung. Previous findings in non-CF healthy individuals reported pre-existing antibody and T cell dependent immune responses to recombinant Cas9 proteins, suggesting potential additional obstacles for incorporation of CRISPR/Cas9 technologies in gene therapies. To determine if pre-existing immunity to Cas9 from S. aureus or S. pyogenes was present or augmented in people with CF (PwCF), anti-Cas9 IgG levels and Cas9-specific T cell responses were determined from peripheral blood samples of PwCF and non-CF healthy controls. Overall, non-CF controls and PwCF displayed evidence of pre-existing antibody and T cell responses to both S. aureus and S. pyogenes Cas9, although there were no significant differences between the two populations. However, we observed global changes in activation of Th1 and CD8 T cell responses as measured by IFNγ and TNF that warrant further investigation and mechanistic understanding as this finding has implications not only for CRISPR/Cas9 gene therapy for PwCF, but also for protection against infectious disease.

Glioblastoma (GBM) stem cells (GSCs) contribute to poor prognosis in patients with GBM. Identifying molecular markers is crucial for developing targeted therapies. Here, we identify cluster of differentiation 97 (CD97) as an optimal GSC surface antigen for potential targeting by chimeric antigen receptor (CAR) T cell therapy through in vitro antibody screening. CD97 is consistently expressed in all validated patient-derived GSCs and positively correlated with known intracellular GSC markers. Silencing CD97 reduces GSC tumorigenicity-related activities, including self-renewal, proliferation, and tumor progression. Transcriptome analysis reveals that CD97 activates mTORC2, leading to AKT S473 phosphorylation and enhanced expression of the downstream genes ARHGAP1, BZW1, and BZW2. Inhibiting mTORC2 with JR-AB2-011 suppresses GSC tumorigenicity and downstream gene expression. We develop CD97-CAR T helper (Th) 9 cells, which exhibit potent cytotoxic effects in vitro and extend survival in mice. These findings suggest that CD97 is a promising GSC-enriched antigen and that targeting it with CAR Th9 cells offers a potential therapeutic strategy for GBM.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.