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For adoptive therapy with T cell receptor engineered T (TCR-T) cells, the quantity and quality of the final cell product directly affect their anti-tumor efficacy. The post-transfer efficacy window of TCR-T cells is keen to optimizing attempts during the manufacturing process. Cbl-b is a E3 ubiquitin ligase previously shown with critical negative impact in T cell functions. This study investigated whether strategic inclusion of a commercially available small inhibitor targeting Cbl-b (Cbl-b-IN-1) prior to T cell activation could enhance the quality of the final TCR-T cell product. Examination with both PBMCs and TCR-T cells revealed that Cbl-b-IN-1 treatment promoted TCR expression efficiency, T cell proliferation potential and, specifically, cell survival capability post antigenic stimulation. Cbl-b-IN-1 exposure facilitated T cells in maintaining less differentiated states with enhanced cytokine production. Further, we found that Cbl-b-IN-1 effectively augmented the activation of TCR signaling, shown by increased phosphorylation levels of Zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase c-γ1 (PLCγ1). In conclusion, our results evidence that the inclusion of Cbl-b inhibitor immediately prior to TCR-T cell activation may enhance their proliferation, survival, and function potentials, presenting an applicable optimization strategy for immunotherapy with adoptive cell transfer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Continuous stimulation of tumor neoantigens and various cytokines in the tumor microenvironment leads to T cell dysfunction, but the specific mechanisms by which these key factors are distributed among different cell subpopulations and how they affect patient outcomes and treatment response are incompletely characterized. By integrating single-cell and bulk sequencing data of non-small cell lung cancer patients, we constructed a clinical outcome-associated T cell exhaustion signature. We discovered a significant association between the T cell exhaustion state and tumor cell hypoxia. Hypoxic malignant cells were significantly correlated with the proportion of exhausted T cells, and they co-occurred in patients at advanced stage. By analyzing the ligand-receptor interactions between these two cell states, we observed that T cells were recruited towards tumor cells through production of chemokines such as CXCL16-CXCR6 axis and CCL3/CCL4/CCL5-CCR5 axis. Based on 15 immune checkpoint blockade (ICB)-treatment cohorts, we constructed an interaction signature that can be used to predict the response to immune checkpoint blockade therapy. Among genes composed of the signature, CXCR6 alone has similarly high prediction efficacy (Area Under Curve (AUC) = 1, 0.89 and 0.73 for GSE126044, GSE135222 and GSE93157, respectively) with the signature and thus could serve as a potential biomarker for predicting immunotherapy response. Together, we have discovered and validated a significant association between exhausted T cells and hypoxic malignant cells, elucidating key interaction factors that significantly associated with response to immunotherapy., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Short peptides are poor immunogens. One way to increase their immune responses is by arraying immunogens in multivalency. Simple and efficient scaffolds for spatial controlling the inter-antigen distance and enhancing immune activation are required. Here, we report a molecular vaccine design principle that maximally drives potent SARS-CoV-2 RBD subunit vaccine on DNA duplex to induce robust and efficacious immune responses in vivo. We expect that the DNA-peptide epitope platform represents a facile and generalizable strategy to enhance the immune response. STATEMENT OF SIGNIFICANCE: DNA scaffolds offer a biocompatible and convenient platform for arraying immunogens in multivalency antigenic peptides, and spatially control the inter-antigen distance. This can effectively enhance immune response. Peptide (instead of entire protein) vaccines are highly attractive. However, short peptides are poor immunogens. Our DNA scaffolded multivalent peptide immunogen system induced robust and efficacious immune response in vivo as demonstrated by the antigenic peptide against SARS-CoV-2. The present strategy could be readily generalized and adapted to prepare multivalent vaccines against other viruses or disease. Particularly, the different antigens could be integrated into one single vaccine and lead to super-vaccines that can protect the host from multiple different viruses or multiple variants of the same virus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Potent neutralizing antibodies (Abs) have been proven with therapeutic efficacy for the intervention against SARS-CoV-2. Majority of these Abs function by directly interfering with the virus entry to host cells. Here, we identified a receptor binding domain (RBD) specific monoclonal Ab (mAb) 82A6 with efficient neutralizing potency against authentic SARS-CoV-2 virus. As most Abs targeting the non-receptor binding motif (RBM) region, 82A6 was incapable to block the RBD-ACE2 interaction. In particular, it actively promoted the S1 subunit shedding from the S protein, which may lead to effective reduction of intact SARS-CoV-2 viruses. Importantly, it could block potential syncytia formation associated with post-infectious cell surface expression of S proteins. Our study evidenced a RBD specific Ab with unique beneficial efficacy against SARS-CoV-2 infection, which might bring informative significance to understand the collective effects of neutralizing Abs elicited in COVID-19 patients., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or agonistic antibodies targeting TRAIL-receptors (TRAIL-Rs) can selectively induce apoptosis in cancer cells. However, they have limited antitumor efficacy in clinical trials. We previously generated ten fully human monoclonal Abs to TRAIL-receptor type 1 (TR1-mAbs) using immunospot array assay on a chip (ISAAC technology). We found that the TR1-mAbs exhibited different effects on TRAIL-induced apoptosis (enhanced or blocked apoptosis). Here, we further demonstrated that some mAbs competed with TRAIL for binding to TRAIL-R1 expressed on tumor cells that blocked TRAIL-induced apoptosis (B-TR1-Ab), whereas others did not compete with TRAIL that enhanced TRAIL-induced apoptosis (E-TR1-Ab). Combination of E-TR1-Ab (TR1-419) with TRAIL leads to enhanced antitumor activity in various tumor cells in vitro. E-TR1-419 and TRAIL could cooperate to upregulate the mRNA expression and protein levels of TRAIL-R1 and to promote caspase-8 cleavage and increased JNK phosphorylation. Our results suggest that combining E-TR1 Ab with TRAIL could provide a new therapeutic strategy for tumor immunotherapies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cβ antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10(-5)M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cβ antibody, its binding affinity for p/MHC increased by 5-fold (2.2 × 10(-6)M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-Cβ antibody, which is probably due to the stabilization of the Vα/Vβ region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing functional sTCR as a diagnostic and therapeutic tool for cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10(-10)M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration., (2010 Elsevier B.V. All rights reserved.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.