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.
In this work, hollow nanobox metal-organic framework (HNM) nanocomposites were synthesised and utilised for the first time in a signal decreased electrochemical immunosensor for the ultrasensitive quantitative determination of lymphocyte activation gene-3 (LAG-3) protein, which is a newly discovered biomarker. With the aid of signal materials, namely, SiO 2 -tagged anti-LAG-3 antibody (SiO 2 -Ab 2 ) and the biotin-streptavidin system, the sensor can achieve signal amplification . Encapsulation of tin dioxide-functionalised reduced graphene oxide (rGO-SnO 2 ) and gold and platinum alloys (AuPt alloys) onto the surface of hollow nanobox metal-organic frameworks (MOFs) was performed to prepare rGO-SnO 2 /hollow nanobox-MOFs/AuPt alloys (rGO-SnO 2 /HNMs/AuPt) as the matrix. SiO 2 -Ab 2 , which is used as the signal-decreased label, can be utilised to enhance the distinction of the electrochemical signal after the specific recognition between antibodies and antigens, owing to its large steric hindrance property. In this sensor, this proposed sandwich immunosensor can achieve a high sensitivity, especially in the presence of low concentrations of the LAG-3 protein. Under optimal conditions, this sandwich-designed immunosensor exhibited a sensitive detection of the LAG-3 protein from concentrations of 0.01 ng mL −1 to 1 μg mL −1 , with a lower detection limit of 1.1 pg mL −1 (based on 3σ). We proposed that this ultrasensitive biosensor can be utilised for the detection of the LAG-3 protein in early clinical tumour diagnosis.
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