BACKGROUND: Lymphocyte activation gene 3 (LAG-3) has been considered as the next generation of immune checkpoint and a promising prognostic biomarker of immunotherapy. As with programmed cell death protein-1/programmed death-ligand 1 and cytotoxic T-lymphocyte antigen-4 inhibitors, positron emission tomography (PET) imaging strategies could benefit the development of clinical decision-making of LAG-3-related therapy. In this study, we developed and validated 68Ga-labeled cyclic peptides tracers for PET imaging of LAG-3 expression in bench-to-bedside studies.
METHODS: A series of LAG-3-targeted cyclic peptides were modified and radiolabeled with 68GaCl3 and evaluated their affinity and specificity, biodistribution, pharmacokinetics, and radiation dosimetry in vitro and in vivo. Furthermore, hu-PBL-SCID (PBL) mice models were constructed to validate the capacity of [68Ga]Ga-CC09-1 for mapping of LAG-3+ lymphocytes infiltrates using longitudinal PET imaging. Lastly, [68Ga]Ga-CC09-1 was translated into the first-in-human studies to assess its safety, biodistribution and potential for imaging of LAG-3 expression.
RESULTS: A series of cyclic peptides targeting LAG-3 were employed as lead compounds to design and develop 68Ga-labeled PET tracers. In vitro binding assays showed higher affinity and specificity of [68Ga]Ga-CC09-1 in Chinese hamster ovary-human LAG-3 cells and peripheral blood mononuclear cells. In vivo PET imaging demonstrated better imaging capacity of [68Ga]Ga-CC09-1 with a higher tumor uptake of 1.35±0.33 per cent injected dose per gram and tumor-to-muscle ratio of 17.18±3.20 at 60 min post-injection. Furthermore, [68Ga]Ga-CC09-1 could detect the LAG-3+ lymphocyte infiltrates in spleen, lung and salivary gland of PBL mice. In patients with melanoma and non-small cell lung cancer, primary lesions with modest tumor uptake were observed in [68Ga]Ga-CC09-1 PET, as compared with that of [18F]FDG PET. More importantly, [68Ga]Ga-CC09-1 delineated the heterogeneity of LAG-3 expression within large tumors.
CONCLUSIONS: These findings consolidated that [68Ga]Ga-CC09-1 is a promising PET tracer for quantifying the LAG-3 expression in tumor microenvironment, indicating its potential as a companion diagnostic for patients stratification and therapeutic response monitoring in anti-LAG-3 therapy.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

BACKGROUND: Lymphocyte activation gene 3 (LAG-3) is expressed on activated immune cells and has emerged as a promising target for immune checkpoints blockade. However, conflicting findings have been reported regarding the association between LAG-3 expression in tumors and patient prognosis, indicating the need for further investigation into the significance of LAG-3 expression levels in tumor therapies. In this study, 68Ga-NOTA-XH05, a novel peptide-based positron emission tomography (PET) tracer targeting LAG-3, was constructed to non-invasively detect LAG-3 expression in melanoma after CpG oligonucleotide (CpG) treatment and explore the relationship between LAG-3 expression and therapeutic effect.
METHODS: The tracer 68Ga-NOTA-XH05 was identified by high-performance liquid chromatography after being prepared and purified. Cell uptake and blocking essays were performed to verify the specificity of the tracer in vitro. The expression of LAG-3 in B16-F10 subcutaneous tumors was monitored by flow cytometry, and its correlation with the tracer uptake was analyzed to evaluate the tracer specificity. PET imaging and biodistribution studies were conducted after CpG treatment of unilateral or bilateral B16-F10 subcutaneous tumor models to assess the ability of 68Ga-NOTA-XH05 in monitoring immunotherapy efficacy and the abscopal effect of CpG.
RESULTS: Following purification, 68Ga-NOTA-XH05 exhibited high radiochemical purity and specificity. Flow cytometry analysis revealed a positive correlation between LAG-3 expression in tumors and the uptake of 68Ga-NOTA-XH05. In B16-F10 bearing mice treated with CpG, PET imaging using 68Ga-NOTA-XH05 demonstrated a higher tumor to blood ratio (TBR) compared with the control group. Furthermore, TBR values obtained from CpG-treated mice allowed for differentiation between responders and non-responders. In a bilateral subcutaneous tumor model where only right-sided tumors were treated with intratumoral injection of CpG, TBR values of left-sided tumors were significantly higher than those in the control group, indicating that 68Ga-NOTA-XH05 could effectively monitor the systemic effect of local CpG injection.
CONCLUSIONS: Our findings highlight the detection capability of 68Ga-NOTA-XH05 in assessing LAG-3 expression levels within tumors and evaluating response to immunotherapy, thereby suggesting promising clinical translational prospects.

Fibrinogen-like protein 1 (FGL1) contributes to the proliferation and metabolism of hepatocytes; however, as a major ligand of the immune checkpoint, its role in the liver regional immune microenvironment is poorly understood. Hepatocytes specifically and highly expressed FGL1 under normal physiological conditions. Increases in hepatic CD8+ T and NK cell numbers and functions were found in Fgl1-deficient (Fgl1-/-) mice, but not in the spleen or lymph node, similar to findings in anti-FGL1 mAb-treated wild-type mice. Furthermore, Fgl1 deficiency or anti-FGL1 mAb blockade restrained liver metastasis and slowed the growth of orthotopic tumors, with significantly prolonged survival of tumor-bearing mice. Tumor-infiltrating hepatic CD8+ T and NK cells upregulated the expression of lymphocyte activation gene-3 (LAG-3) and exhibited stronger antitumor activities after anti-FGL1 treatment. The antitumor efficacy of FGL1 blockade depended on cytotoxic T lymphocytes and NK cells, demonstrated by using a cell-deficient mouse model and cell transfer in vivo. In vitro, FGL1 directly inhibited hepatic T and NK cells related to the receptor LAG-3. In conclusion, hepatocyte-derived FGL1 played critical immunoregulatory roles in the liver and contributed to liver metastasis and tumor growth by inhibiting CD8+ T and NK cell functions via the receptor LAG-3, providing a new strategy for liver cancer immunotherapy.

UNASSIGNED: Tuberculosis (TB) persists as a global health challenge, with its treatment hampered by the side effects of long-term combination drug therapies and the growing issue of drug resistance. Therefore, the development of novel therapeutic strategies is critical. This study focuses on the role of immune checkpoint molecules (ICs) and functions of CD8+ T cells in the search for new potential targets against TB.
UNASSIGNED: We conducted differential expression genes analysis and CD8+ T cell functional gene analysis on 92 TB samples and 61 healthy individual (HI) samples from TB database GSE83456, which contains data on 34,603 genes. The GSE54992 dataset was used to validated the findings. Additionally, a cluster analysis on single-cell data from primates infected with mycobacterium tuberculosis and those vaccinated with BCG was performed.
UNASSIGNED: The overexpression of LAG-3 gene was found as a potentially important characteristic of both pulmonary TB (PTB) and extrapulmonary TB (EPTB). Further correlation analysis showed that LAG-3 gene was correlated with GZMB, perforin, IL-2 and IL-12. A significant temporal and spatial variation in LAG-3 expression was observed in T cells and macrophages during TB infection and after BCG vaccination.
UNASSIGNED: LAG-3 was overexpressed in TB samples. Targeting LAG-3 may represent a potential therapeutic target for tuberculosis.

Lymphocyte activation gene 3 (LAG-3) has attracted much attention as a potentially valuable immune checkpoint. Individual identification of LAG-3 expression at screening and during treatment could improve the successful implementation of anti-LAG-3 therapies. HuL13 is a human IgG1 monoclonal antibody that binds to the LAG-3 receptor in T cells. Here, we used [89Zr]Zr-labeled HuL13 to delineate LAG-3+ T-cell infiltration into tumors via positron emission tomography (PET) imaging. A549/LAG-3 cells, which stably express LAG-3, were generated by infection with lentivirus. The uptake of [89Zr]Zr-DFO-HuL13 in A549/LAG-3 cells was greater than that in the negative control (A549/NC) cells at each time point. The equilibrium dissociation constant (Kd) of [89Zr]Zr-DFO-HuL13 for the LAG-3 receptor was 8.22 nM. PET imaging revealed significant uptake in the tumor areas of A549/LAG-3 tumor-bearing mice from 24 h after injection (SUVmax = 2.43 ± 0.06 at 24 h). As a proof of concept, PET imaging of the [89Zr]Zr-DFO-HuL13 tracer was further investigated in an MC38 tumor-bearing humanized LAG-3 mouse model. PET imaging revealed that the [89Zr]Zr-DFO-HuL13 tracer specifically targets human LAG-3 expressed on tumor-infiltrating lymphocytes (TILs). In addition to the tumors, the spleen was also noticeably visible. Tumor uptake of the [89Zr]Zr-DFO-HuL13 tracer was lower than its uptake in the spleen, but high uptake in the spleen could be reduced by coinjection of unlabeled antibodies. Coinjection of unlabeled antibodies increases tracer activity in the blood pool, thereby improving tumor uptake. Dosimetry evaluation of the healthy mouse models revealed that the highest absorbed radiation dose was in the spleen, followed by the liver and heart wall. In summary, these studies demonstrate the feasibility of using the [89Zr]Zr-DFO-HuL13 tracer for the detection of LAG-3 expression on TILs. Further clinical evaluation of the [89Zr]Zr-DFO-HuL13 tracer may be of significant help in the stratification and management of patients suitable for anti-LAG-3 therapy.

OBJECTIVE: To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells.
METHODS: Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjμgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 μg/mL.
CONCLUSIONS: The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid β precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson\'s disease and related α-synucleinopathies.

BACKGROUND: Progesterone receptor (PR) serves as a crucial prognostic and predictive marker in breast cancer. Nonetheless, the interplay between PR and the tumor immune microenvironment remains inadequately understood. This investigation employs bioinformatics analyses, mouse models, and clinical specimens to elucidate the impact of PR on immune microenvironment and identify potential targets for immunotherapy, furnishing valuable guidance for clinical practice.
METHODS: Analysis of immune infiltration score by Xcell between PR-positive and PR-negative breast cancer tumors. Construction of overexpression mouse progesterone receptor (mPgr) EMT-6 cell was to explore the tumor immune microenvironment. Furthermore, anti- Lymphocyte-activation gene 3 (LAG3) therapy aimed to investigate whether PR could influence the effectiveness of immune treatments.
RESULTS: Overexpression mPgr inhibited tumor growth in vitro, but promoted tumor growth in Balb/c mouse. Flow cytometry showed that the proportion and cytotoxicity of CD8+T cells in tumor of overexpressing mPgr group were significantly reduced. The significant reduction in overexpressing mPgr group was found in the proportions of LAG3+CD8+ T cells and LAG3+ Treg T cells. Anti-LAG3 treatment resulted in reduced tumor growth in EV group mouse rather than in overexpressing mPgr group. Patents derived tumor fragment (PDTF) also showed higher anti-tumor ability of CD3+T cell in patents\' tumor with PR <20% after anti-human LAG3 treatment in vitro.
CONCLUSIONS: The mPgr promotes tumor growth by downregulating the infiltration and function of cytotoxic cell. LAG3 may be a target of ER-positive breast cancer immunotherapy. The high expression of PR hinders the sensitivity to anti-LAG3 treatment.

LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6 nM) and MHC-II binding (IC50 of 6.2 nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.