Immunoglobulin Domain Research Articles

Orientation-dependent CD45 inhibition with viral and engineered ligands.

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as "beads on a string" that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses.

T-Cell Immunoglobulin and Mucin Domain 3 (TIM-3) Gene Expression as a Negative Biomarker of B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) accounts for 85% of all childhood ALL. Malignancies exhaust T and B cells, resulting in an increased expression of immune checkpoint receptors (ICRs), such as T-cell immunoglobulin and mucin domain 3 (TIM-3). TIM-3 has been found to be dysregulated in different types of cancer. However, there is a lack of rigorous studies on the TIM-3 expression in B-ALL. The current study aimed to measure the expression of TIM-3 at the gene and protein levels and evaluate the potential of TIM-3 as a biomarker in B-ALL. A total of 28 subjects were recruited between 2021 and 2023, comprising 18 subjects diagnosed with B-ALL and 10 non-malignant healthy controls. The B-ALL patients were divided into three groups: newly diagnosed (four patients), in remission (nine patients), and relapse/refractory (five patients). The expression levels of TIM-3 were evaluated using the real-time qPCR and ELISA techniques. The results revealed that the TIM-3 expression was significantly downregulated in the malignant B-ALL patients compared to the non-malignant healthy controls in the mRNA (FC = -1.058 ± 0.3548, p = 0.0061) and protein blood serum (p = 0.0498) levels. A significant TIM-3 gene reduction was observed in the relapse/refractory cases (FC = -1.355 ± 0.4686, p = 0.0327). TIM-3 gene expression allowed for significant differentiation between patients with malignant B-ALL and non-malignant healthy controls, with an area under the curve (AUC) of 0.706. The current study addressed the potential of reduced levels of TIM-3 as a negative biomarker for B-ALL patients.

Generation and Selection of Phage Display Antibody Libraries in Fab Format.

Monoclonal antibodies (mAbs) have exceptional utility as research reagents and pharmaceuticals. As a complement to both traditional and contemporary single-B-cell cloning technologies, the mining of antibody libraries via display technologies-which mimic and simplify B cells by physically linking phenotype (protein) to genotype (protein-encoding DNA or RNA)-has become an important method for mAb discovery. Among these display technologies, phage display has been particularly successful for the generation of mAbs that bind to a wide variety of antigens with exceptional specificities and affinities. Rather than multivalent whole antibodies, phage display typically uses monovalent antibody fragments, such as "fragment antigen binding" (Fab), as the format of choice. The ∼50-kDa Fab format consists of four immunoglobulin (Ig) domains on two polypeptide chains (light chain and shortened heavy chain), and exhibits its antigen binding site in a natural configuration found in bivalent IgG and other multivalent Ig molecules. The Fab fragment has a high melting temperature and a low tendency to aggregate, and can be readily converted to natural and nonnatural Ig formats without affecting antigen binding properties, which has made it a favored format for phage display for more than three decades. Here, I briefly summarize some of the approaches used for the generation and selection of phage display antibody libraries in Fab format, from human and nonhuman antibody repertoires.

The Putative Role of TIM-3 Variants in Polyendocrine Autoimmunity: Insights from a WES Investigation.

Autoimmune polyglandular syndrome (APS) comprises a complex association of autoimmune pathological conditions. APS Type 1 originates from loss-of-function mutations in the autoimmune regulator (AIRE) gene. APS2, APS3 and APS4 are linked to specific HLA alleles within the major histocompatibility complex, with single-nucleotide polymorphisms (SNPs) in non-HLA genes also contributing to disease. In general, variability in the AIRE locus and the presence of heterozygous loss-of-function mutations can impact self-antigen presentation in the thymus. In this study, whole-exome sequencing (WES) was performed on a sixteen-year-old female APS3A/B patient to investigate the genetic basis of her complex phenotype. The analysis identified two variants (p.Arg111Trp and p.Thr101Ile) of the hepatitis A virus cell receptor 2 gene (HAVCR2) encoding for the TIM-3 (T cell immunoglobulin and mucin domain 3) protein. These variants were predicted, through in silico analysis, to impact protein structure and stability, potentially influencing the patient's autoimmune phenotype. While confocal microscopy analysis revealed no alteration in TIM-3 fluorescence intensity between the PBMCs isolated from the patient and those of a healthy donor, RT-qPCR showed reduced TIM-3 expression in the patient's unfractionated PBMCs. A screening conducted on a cohort of thirty APS patients indicated that the p.Thr101Ile and p.Arg111Trp mutations were unique to the proband. This study opens the pathway for the search of TIM-3 variants possibly linked to complex autoimmune phenotypes, highlighting the potential of novel variant discovery in contributing to APS classification and diagnosis.

Siglec-G Suppresses CD8+ T Cells Responses through Metabolic Rewiring and Can be Targeted to Enhance Tumor Immunotherapy.

CD8+ T cells play a critical role in cancer immune-surveillance and pathogen elimination. However, their effector function can be severely impaired by inhibitory receptors such as programmed death-1 (PD-1) and T cell immunoglobulin domain and mucin domain-3 (Tim-3). Here Siglec-G is identified as a coinhibitory receptor that limits CD8+ T cell function. Siglec-G is highly expressed on tumor-infiltrating T cells and is enriched in the exhausted T cell subset. Ablation of Siglec-G enhances the efficacy of adoptively transferred T cells and chimeric antigen receptor (CAR) T cells in suppressing solid tumors growth. Mechanistically, sialoglycan ligands, such as CD24 on tumor cells, activate the Siglec-G-SHP2 axis in CD8+ T cells, impairing metabolic reprogramming from oxidative phosphorylation to glycolysis, which dampens cytotoxic T lymphocyte (CTL) activation, expansion, and cytotoxicity. These findings discover a critical role for Siglec-G in inhibiting CD8+ T cell responses, suggesting its potential therapeutic effect in adoptive T cell therapy and tumor immunotherapy.

Identification of a seven-gene prognostic model for renal cell carcinoma associated with CD8+T lymphocyte cell.

CD8+ T lymphocytes are important elements of the tumor microenvironment, hence their involvement in the development and progression of tumors is complex. Data on the precise tumor-infiltrating lymphocytes gene signature in renal cell carcinoma (RCC) remain limited. Therefore, this study created a tumor-infiltrating lymphocytes-related predictive model for patients with RCC using data from The Cancer Genome Atlas. The most important genes associated with CD8 + T lymphocytes were identified using weighted gene co-expression network analysis. Functional categories of important genes were revealed using gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes signaling pathway analyses. A CD8 + T lymphocyte-related prognostic model with 7 important genes was simultaneously created using the least absolute shrinkage and selection operator, univariate and multivariate Cox regressions, and the 7 genes were expressed particularly in CD8 + T lymphocytes according to single-cell sequencing data obtained from the Gene Expression Omnibus. This study identified a seven-gene prognostic model associated with CD8 + T lymphocytes that may significantly influence risk stratification in patients with RCC. The genes included in the model are apolipoprotein B mRNA editing catalytic polypeptide 3G, CD3 gamma, eomesodermin, protein tyrosine phosphatase, non-receptor type 7, signal regulatory protein gamma, Fas ligand, and T-cell immunoreceptor with Ig and ITIM domains.

First contact(in): The complete structure of contactin 2

In this issue of Structure, Fan etal.1 report the structure of the full contactin 2 ectodomain, representing the first for the contactin family. The work reveals six immunoglobulin domains are essential for intercellular interactions, explores differences in proposed contactin 2 homodimerization mechanisms, and provides an updated model for contactin 2 organization on and between cells.

INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance, they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors, which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), a well-known checkpoint receptor involved in T-cell exhaustion, has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here, we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability, are rapidly and efficiently taken up by cells, and can be easily incorporated into cell product manufacturing. INTASYL PH-804, which targets TIGIT, suppresses TIGIT mRNA and protein expression in NK cells, resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition, thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy, particularly for hematological malignancies.

The Angiopoietin-Tie-2 Axis in Children and Young Adults with Dengue Virus Infection in the Philippines.

Dengue virus (DENV) infection is associated with plasma leakage, which may progress to shock. The angiopoietin (Ang)-tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 (Tie-2) axis regulates endothelial permeability. We examined the clinical utility of Ang-1, Ang-2, and the Ang-2-to-Ang-1 ratio for prediction of progression to severe DENV in a prospective cohort study of children and young adults (age 1 to <26 years) with DENV infection. Ang-1, Ang-2, Tie-2 were measured at presentation to an outpatient clinic in the Philippines from stored plasma by multiplex Luminex® assay. Patients were followed prospectively to document the clinical course (hospitalization, length of stay, intravenous fluid resuscitation, and transfer to a higher level facility). We included 244 patients (median age 9 years, 40% female). At presentation, 63 patients (26%) had uncomplicated dengue, 179 (73%) had dengue with warning signs, and 2 (0.82%) had severe dengue. One hundred eighty-one patients (74%) were hospitalized. Ang-1 levels were lower and Ang-2 higher in patients who required hospitalization. Ang-2-to-Ang-1 ratio >1 was associated with a relative risk of hospitalization of 1.20 (95% CI: 1.03-1.36, P = 0.016). A higher Ang-2-to-Ang-1 ratio was associated with longer length of hospital stay, higher frequency of transfer to a higher level facility, larger intravenous fluid requirement, hemoconcentration, and thrombocytopenia. Angiopoietin-2 was correlated with procalcitonin (Kendall's τ = 0.17, P = 0.00012), a marker of systemic inflammation, as well as soluble vascular cell adhesion molecule-1 (τ = 0.22, P <0.0001) and Endoglin (τ = 0.14, P = 0.0017), markers of endothelial activation. In conclusion, altered Ang-2-to-Ang-1 ratio can be detected early in the course of DENV infection and predicts clinically meaningful events (hospitalization, length of stay, and fluid resuscitation).

Alpha-1-B glycoprotein (A1BG) inhibits sterol-binding and export by CRISP2

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in various processes, including sperm maturation and cancer progression. They are mostly secreted glycoproteins and share a unique conserved CAP domain. The precise mode of action of these proteins, however, has remained elusive. Saccharomyces cerevisiae expresses three members of this protein family, which bind sterols in vitro and promote sterol secretion from cells. This sterol-binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with alpha-1-B glycoprotein (A1BG), a human plasma glycoprotein that is upregulated in different types of cancers. Here we examined whether the interaction between CRISP proteins and A1BG affects the sterol-binding function of CAP family members. Co-expression of A1BG with CAP proteins abolished their sterol export function in yeast and their interaction inhibits sterol-binding in vitro. We map the interaction between A1BG and CRISP2 to the third of five repeated immunoglobulin-like (Ig) domains within A1BG. Interestingly, the interaction between A1BG and CRISP2 requires magnesium, suggesting that coordination of Mg2+ by the highly conserved tetrad residues within the CAP domain is essential for a stable interaction between the two proteins. The observation that A1BG modulates the sterol-binding function of CRISP2, has potential implications for the role of A1BG and related Ig domain containing proteins in cancer progression and the toxicity of reptile venoms containing CRISP proteins.

Tim-1-mediated extracellular matrix promotes the development of hepatocellular carcinoma.

Tim-1 (T-cell immunoglobulin and mucin domain 1), also known as Kim-1 (kidney injury molecule 1) or hepatitis A virus cellular receptor 1 (HAVCR1), is a transmembrane protein expressed on various immune and epithelial cells. It plays a role in modulating inflammatory and immune responses. In this study, we find that Tim-1 is overexpressed in hepatocellular carcinoma (HCC) samples and that its expression is significantly correlated with postoperative survival. Bulk RNA sequencing reveals a general upregulation of extracellular matrix-related genes in HCC tissues with Tim-1 overexpression. The results of the cell and in vivo experiments reveal that Tim-1 in HCC not only affects biological processes such as the proliferation, migration, and invasion of HCC cells but also broadly promotes extracellular matrix processes by influencing cytokine secretion. Further studies demonstrate that Tim-1 mediates the activation of hepatic stellate cells and upregulates Th1 and Th2 cytokines, thereby promoting HCC progression. Thus, Tim-1 may represent a novel target for future interventions in HCC and liver fibrosis.

A bispecific antibody targeting the Ig domains of Siglec-E displays enhanced antitumor effects

Siglec-9 is a promising immune checkpoint molecule, and therapeutics targeting Siglec-9 have the potential to augment anti-tumor immunity. Here, we generated a bispecific antibody, named as aSE4-1-Fc, by fusing two distinct alpaca derived nanobodies, which can simultaneously target the extracellular Ig variable (V)-set domain and C2-set domains of murine Siglec-9 (also known as Siglec-E) with high affinity. In vivo studies showed that aSE4-1-Fc was better than its component antibodies in inhibiting tumor growth/metastasis, and Siglec-E blockade using aSE4-1-Fc generated protective anti-tumor T cell memory. Furthermore, the combination of aSE4-1-Fc with anti-PD-L1 therapy greatly improved the antitumor effects by augmenting both T and NK cells. Taken together, this study emphasizes the importance of Siglec-9 as a potential cancer therapeutic target, demonstrates the synergistic effect of co-inhibition of Siglec-9 and PD-L1, and may have implications for developing engineered antibodies targeting Siglec-9 with enhanced therapeutic efficacy.

The Functional and Prognostic Impact of TIGIT Expression on Bone Marrow NK Cells in Core Binding Factor-Acute Myeloid Leukemia Patients at Diagnosis.

Background: The effect of the expression of the newly identified immune checkpoint, T cell immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) on NK cells in core binding factor-acute myeloid leukemia (CBF-AML) remains to be investigated. Methods: Fresh bone marrow samples from a total of 39 newly diagnosed CBF-AML patients and 25 healthy donors (HDs) were collected for testing the phenotype and function state of total NK, CD56bright, and CD56dim NK cell subsets after in vitro stimulation. Results: The frequencies of TIGIT+ cells in total NK, CD56bright, and CD56dim NK cell subsets had no significant difference between patients and HDs. TNF-α and INF-γ levels were uniformly lower in TIGIT+ cells than the corresponding TIGIT- cells in all HDs, whereas those for TIGIT+ to TIGIT- cells in patients were highly heterogenous; TIGIT expression was not related to PFP and GZMB expression in HDs, whereas it was related to higher intracellular PFP and GZMB levels in patients. Patients' TIGIT+ NK cells displayed lower K562 cell-killing activity than their TIGIT- NK cells. In addition, high frequencies of TIGIT+ cells in total NK and CD56dim NK cells were associated with poor RFS. Conclusions: TIGIT expression affected the diagnostic bone marrow-sited NK cell function and had prognostic significance in CBF-AML patients.

Galectin-9 as a new biomarker of acute-on-chronic liver failure

Galectin-9 (Gal-9) expression in patients with acute-on-chronic liver failure and its correlation with prognosis remain unclear. This study investigated the relationship between liver failure prognosis and Gal-9 expression analysis in patients with acute-on-chronic liver failure. Patients with acute-on-chronic liver failure attributable to hepatitis B and those with chronic hepatitis B were included in this single-center prospective cohort study. The Gal-9 levels in the acute-on-chronic liver failure group were significantly higher than those in the chronic hepatitis B group, and there was an upregulation of Gal-9 and T-cell immunoglobulin domain and mucin domain-3 expressions in peripheral blood T cells. Gal-9 was localized in the regenerative areas of liver tissues in patients with acute-on-chronic liver failure, co-localizing with Kupffer cells. Kaplan–Meier survival curves showed that patients with Gal-9 levels < 9.6 ng/ml had a worse prognosis, with the area under the receiver operating characteristic curve (AUC-ROC) being similar to that of the Model for End-Stage Liver Disease score. The combined ROC curve of the two had better predictive performance, with an AUC of 0.945. High Gal-9 levels in liver regenerative areas can serve as a prognostic marker, indicating a better prognosis for patients with hepatitis B virus-acute-on-chronic liver failure.

Novel Immune Checkpoint Inhibitor Targets in Advanced or Metastatic Renal Cell Carcinoma: State of the Art and Future Perspectives.

Immune checkpoint inhibitors (ICI) have become the cornerstone of treatment in renal cell carcinoma (RCC), for both metastatic disease and in an adjuvant setting. However, an adaptive resistance from cancer cells may arise during ICI treatment, therefore many studies are focusing on additional immune checkpoint inhibitor pathways. Promising targets of immunotherapeutic agents under investigation include T cell immunoglobulin and ITIM domain (TIGIT), immunoglobulin-like transcript 4 (ILT4), lymphocyte activation gene-3 (LAG-3), vaccines, T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and chimeric antigen receptor (CAR) T cells. In this review of the literature, we recollect the current knowledge of the novel treatment strategies in the field of immunotherapy that are being investigated in RCC and analyze their mechanism of action, their activity and the clinical studies that are currently underway.

Higher frequency of peripheral blood CD103+CD8+ T cells with lower levels of PD-1 and TIGIT expression related to favorable outcomes in leukemia patients.

Leukemia is a prevalent pediatric life-threatening hematologic malignancy with a poor prognosis. Targeting immune checkpoints (ICs) to reverse T cell exhaustion is a potentially effective treatment for leukemia. Tissue resident memory T (TRM) cells have been found to predict the efficacy of programmed death receptor-1 inhibitor (anti-PD-1) therapy in solid tumors. However, the IC characteristics of TRM cells in leukemia and their relationship with prognosis remain unclear. We employed multi-color flow cytometry to evaluate the frequencies of CD103+CD4+ and CD103+CD8+ T cells in the peripheral blood (PB) of patients with acute myeloid leukemia and B-cell acute lymphoblastic leukemia compared to healthy individuals. We examined the expression patterns of PD-1 and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) within the circulating CD103+ T cell subsets affected by leukemia. To further elucidate the immunological landscape, we assessed the differentiation status of CD103+ T cells across various disease states in patients with leukemia. Our findings showed a significant increase in the frequency of CD103+CD8+ T cells in the PB of patients with leukemia who had achieved complete remission (CR) compared to those in the de novo (DN) and relapsed/refractory (RR) stages. This increase was accompanied by a notable decrease in the expression levels of PD-1 and TIGIT in CD103+CD8+ T cells in the CR stage. Additionally, our analysis revealed a higher proportion of CD103+CD8+ T cells in the central memory (TCM) and effector memory (TEM) subsets of the immune profile. Notably, the proportions of CD103+ naïve T cells, CD103+ TEM, and CD103+ terminally differentiated T cells within the CD8+ T cell population were significantly elevated in patients with CR compared to those in the DN/RR stages. The data indicate that circulating higher frequency of CD103+CD8+ T cells with lower expression of PD-1 and TIGIT are associated with favorable outcomes in patients with leukemia. This suggests a potential role of TRM cells in leukemia prognosis and provides a foundation for developing targeted immunotherapies.

Biomechanics Model to Characterize Atomic Force Microscopy-Based Virus-Host Cell Adhesion Measurements.

We present a model for virus-cell adhesion that can be used for quantitative extraction of adhesive properties from atomic force microscopy (AFM)-based force spectroscopy measurements. We extend a previously reported continuum model of viral cell interactions based on a single parameter representing adhesive energy density by using a cohesive zone model in which adhesion is represented by two parameters, a pull-off stress and associated characteristic displacement. This approach accounts for the deformability of the adhesive receptors, such as the Spike protein and transmembrane immunoglobulin and mucin domain (TIM) family that mediate adhesion of SARS-CoV-2 and Ebola viruses, and the omnipresent glycocalyx. Our model represents receptors as a Winkler foundation and aims to predict the pull-off force needed to break the adhesion between the virus and the cell. By comparing the force-separation curves simulated by the model and experimental data, we found that the model can effectively explain the AFM pull-off force trace, thus allowing quantification of the adhesion parameters. Our model provides a more refined understanding of viral cell adhesion and also establishes a framework for interpreting and predicting AFM force spectroscopy measurements.

Identification of a Mnlrig-1 involved in testis reproductive immunity in the oriental river prawn Macrobrachium nipponense

The testis evolves a highly organized testicular microenvironment to support spermatogenesis. However, the knowledge about it is limited in crustacean. In this study, we identified a member of immunoglobulin superfamily (IgSF) from Macrobrachium nipponense testis and explored its roles as a potential pattern recognition receptor (PRR) involved in reproductive immunity. Based on the domains it contains and homology analysis result, we designate it as leucine-rich repeats and immunoglobulin-like domains protein-1 (MnLrig-1). The Mnlrig-1 comprises a 3288 bp open reading frame (ORF) encoding a 1095 amino acid protein. MnLrig-1 is consisted of one signaling peptide; one LRR_NT domain; eight LRR domains; five LRR_TYP domains; one LRR_CT domain; three IGc2 regions; one transmembrane region, and C-terminal cytoplasmic tail, sharing similar domains with orthologs in other crustacean species. MnLrig-1 is widely expressed in various tissues of M. nipponense. Mnlrig-1 is significantly induced by LPS, PGN, Aeromonas hydrophila, and Vibrio alginolyticus challenge in the testis at 3 h and maintained a high level from 3 h to 24 h. Additionally, two recombinant immunoglobulin domains of MnLrig-1 are obtained, while only one domain shows direct binding affinity towards LPS, PGN, Escherichia coli, A. hydrophila, Staphylococcus aureus, and Bacillus subtilis in vitro. Moreover, silencing Mnlrig-1 results in a significant upregulation of three anti-lipopolysaccharide factors (ALFs) in the testis. These results reveal the potential role of MnLrig-1 as a PRR involved in the testis reproductive immunity in M. nipponense. The insights gained from this study will expand our understanding of immune system in crustacean and may have implications for aquaculture and disease management in crustaceans.

Directed protein engineering identifies a human TIM-4 blocking antibody that enhances anti-tumor response to checkpoint inhibition in murine colon carcinoma

Abstract Background T-cell immunoglobulin and mucin domain containing molecule-4 (TIM-4) is a scavenger receptor best known for its role in recognizing dying cells. TIM-4 orchestrates phagocytosis allowing for cellular clearance of apoptotic cells, termed efferocytosis. It was previously shown that TIM-4 directly interacts with AMPKα1, activating the autophagy pathway, leading to degradation of ingested tumors, and effectively reducing antigen presentation. Methods This study sought to identify a novel human TIM-4 antibody that can prevent phagocytosis of tumor cells thereby allowing for more antigen presentation resulting in anti-tumor immunological response. Using phage display panning directed against human TIM-4, we engineered a novel human TIM-4 antibody (SKWX301). Combination of in vitro phagocytosis assays and cell viability assays were used to test functionality of SKWX301. To examine the effect of SKWX301 in mouse models, we employed a syngeneic mouse model. CT26 cells were subcutaneously injected into BALB/c mice and tumor growth and mouse survival were analyzed. Results SKWX301can prevent human macrophage phagocytosis of cancer cells in vitro. Combination of low dose SKWX301 and anti-PD1 antibody significantly inhibited tumor growth and increased overall survival in mice. This demonstrates that SKWX301 is effective in both human in vitro models and mouse in vivo models. Conclusion Our study has demonstrated a rapid antibody discovery approach and identified a novel human TIM-4 antibody that can serve as a therapeutic for antitumor immunity to improve cancer therapy.

TIM-3 expression on monocyte-derived dendritic cells

The T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), an inhibitory checkpoint receptor, has been identified as a crucial regulator of cellular immune responses. TIM-3 has been discovered as a receptor involved in the negative regulation of T cells. Recent studies have demonstrated that TIM-3 is expressed on innate immune cells, including dendritic cells (DCs), even at a higher level than T cells. In the tumor microenvironment, the majority of DCs have a monocytic origin. Models for studying such DCs in vitro are DC cultures generated from monocytes in the presence of growth factors. The present study aimed to investigate the expression of TIM-3 in IFNα-induced monocyte-derived DCs (IFN-DCs) and the impact of DC activation on TIM-3 expression. DCs were obtained by culturing the adherent fraction of mononuclear cells from healthy donors for 4 days in the presence of GM-CSF and IFNα, followed by LPS addition for 24 hours. Human double-stranded DNA (dsDNA, 5 μg/mL) was added as an activation stimulus to intact IFN-DCs at the stage of maturation, along with LPS. Expression of the membrane TIM-3 molecule was determined by flow cytometry, and the level of expression of TIM-3 mRNA – by real-time RT-PCR with reverse transcription. Intact donor IFN-DCs expressed the membrane TIM-3 molecule at a high level (more than 70% of cells). The addition of LPS as a maturation stimulus almost halved the expression of TIM-3 (pW &lt; 0.05) without affecting the expression of HAVCR2/TIM-3 mRNA. Exogenous dsDNA (along with LPS) increased the expression of HAVCR2/TIM-3 mRNA by more than three times (pW = 0.05) with a decrease in the number of TIM-3+DCs (pW = 0.003). Our findings indicate the presence of mechanisms that support expression of this inhibitory checkpoint receptor under conditions of DC activation. Further studies of the regulation of TIM-3 expression by monocyte-derived dendritic cells will expand the understanding of the biological significance of inhibitory receptors on DCs from the point of view of the immune response, as well as, in the future, increase the effectiveness of current approaches in cancer immunotherapy using IFN-DCs and inhibitors of checkpoint molecules.