PET Imaging Study Of Monoclonal Antibody Molecular Probes Targeting PD-1/PD-L1 Signaling Pathway In Bone And Soft Tissue Sarcoma

Objective:The PD-1/PD-L1?programmed cell death-1/programmed cell death ligand-1?signaling pathway has become a hot spot in tumor immunotherapy research in recent years.Bone and soft tissue sarcomas have a high degree of malignancy,complex and diverse morphological classification,and poor sensitivity to radiotherapy and chemotherapy.Immunotherapy may be a new direction for the development of treatments for bone and soft tissue sarcomas.Studies have shown that tumor cells can escape immune surveillance by suppressing T cells through the PD-1/PD-L1 axis.PD-1/PD-L1 drugs can specifically act on T cells and enhance the immune killing function of T cells in the body,thus achieving"breakthrough"effects in tumor treatment.However,because patients respond differently to these drugs,only a subset of patients receive clinical benefit.Therefore,there is a need to improve the efficacy of immunotherapy,and there is an urgent need for further screening of patients with positive expression.Nuclear medicine and molecular imaging technology provide a good means for early diagnosis and prognosis prediction of tumors.The use of radioactive molecular probes can noninvasively allow detection and evaluation of the expression level of PD-1 protein and PD-L1 receptor protein in tumors.This strategy will further guide the use of immunotherapy in tumor patients.In this study,PD-1/PD-L1-targeted antibodies were modified and radiolabeled,and two noninvasive PET molecular probes for PD-1/PD-L1-specific imaging were developed.Molecular Imaging of PD-1 protein and PD-L1 receptor protein in tumors was performed to prevent the effects of invasive procedures,which can be used to create pathological sections,and treatments on the expression level of PD-1/PD-L1 in the body.Methods:The positron nuclide ¹²⁴I was produced with the HM-20 medical cyclotron.First,experimental research on a PD-1 monoclonal antibody molecular probe was performed.JS001?toripalimab?is a humanized Ig G monoclonal antibody that can strongly inhibit the PD-1 receptor.In this study,we used different iodine isotopes(^{nat/124/125/131}I)to label the JS001 monoclonal antibody to prepare probes and perform quality control to target the h PD-1 receptor protein.The EC50 values of JS001 before and after ^natI labeling were compared.The team used PHA?phytohemagglutinin?to stimulate T cell activation and used Western blotting and flow cytometry to detect h PD-1 overexpression in T cells.Cell experiments?cell uptake,Kd measurement?were then performed in vitro to evaluate the specific targeting affinity of the ¹²⁵I-JS001 probe for T cells in vitro.A human PD-1 C57BL/6 tumor-bearing mouse model was generated,and the expression of the corresponding h PD-1 receptor protein was verified by polymerase chain reaction?PCR?and Western blot analysis.In addition,a small amount of muscle and spleen tissue was taken from humanized PD-1C57BL/6 mice for gene sequencing.After immune PET imaging verification, pathological staining,including immunohistochemistry?IHC?and immunofluorescence?IF?,was used to determine the expression of h PD-1 in tumor tissues.Second,experimental research on a PD-L1 heavy chain antibody molecular probe was performed.We used phage display technology to generate a new type of anti-h PD-L1 heavy chain antibody?HCAb?,named Nb6,that has a high affinity for h PD-L1.The research team used a series of iodine isotopes(^nat/124/125I)to label anti-h PD-L1 Nb6 probes and perform quality control to target the h PD-L1 receptor protein.The ELISA method was used to compare the EC50 values of anti-h PD-L1Nb6 before and after ^natI labeling.The expression of h PD-L1 in OS-732 osteosarcoma cells was determined by flow cytometry and immunofluorescence staining.Cell uptake and Kd measurements were used to assess the specific targeting affinity of the¹²⁵I-anti-h PD-L1 Nb6 probe for OS-732 in vitro.An OS-732 tumor-bearing mouse animal model was generated and verified by polymerase chain reaction?PCR?and Western blot analysis.Finally,the ¹²⁴I-anti-h PD-L1 Nb6 probe targeting the h PD-L1receptor protein was verified by immuno PET imaging and corresponded to pathological staining.Results:In the first part,PD-1 receptor-targeted PET probes were developed:in vitro,when comparing ^natI-JS001 and JS001,the EC50 values before and after JS001 labeling were 0.89±0.15 ng/m L and 1.03±0.14 ng/m L,respectively.There was no statistically significant difference in the EC50 values before and after labeling.After 2hours of incubation,the uptake of the ¹²⁵I-JS001 probe by activated T cells was 5.63times that of the unactivated T cells.Coincubation with excess JS001 reduced the uptake of the ¹²⁵I-JS001 probe by T cells.After 2 hours of incubation,probe uptake in the unblocked group was 1.91 times that of the blocked group.The results showed that the affinity of ¹²⁵I-JS001 and T cells reached 4.26 n M after PHA?phytohemagglutinin?stimulation.Through gene sequencing,it was confirmed that the humanized PD-1 C57BL/6 mouse PD-1 gene sequence was highly similar to the human PD-1 gene sequence.The sequence similarity between the sequenced sample and human c DNA was>99.9%.The sequence similarity between the sequenced sample and ordinary mouse c DNA was 68.23%.Humanized PD-1 C57BL/6 tumor-bearing mice carrying S180 sarcoma were verified by h PD-1 immuno PET imaging.In the humanized PD-1 C57BL/6 S180 sarcoma mouse model,the uptake of probes in the tumor area of the ¹²⁴I-JS001 group at different time points was significantly higher than that in the blocked group or the ¹²⁴I-h Ig G group.This validates the specific targeting of the ¹²⁴I-JS001 probes.IHC and IF staining confirmed that PD-1 receptor protein was expressed on the surface of T cells in tumor tissues.In the second part,PD-L1 receptor-targeted PET probes were developed:when comparing the EC50 values of the two groups of anti-h PD-L1 Nb6 and ^natI-anti-h PD-L1 Nb6,no significant difference was found.The EC50 values before and after anti-h PD-L1 Nb6 labeling were 0.65±0.08 ng/m L and 0.70±0.09 ng/m L,respectively.Coincubation with excess precursors?anti-h PD-L1 Nb6?reduced the uptake of ¹²⁵I-anti-h PD-L1 Nb6 by OS-732 cells.After 2 hours of incubation,probe uptake in the unblocked group was 8.73 times that of the blocked group.The affinity of ¹²⁵I-anti-h PD-L1 Nb6 to OS-732 cells was 2.19 n M,which showed good targeting ability.To conduct in vivo studies,we successfully generated an OS-732 osteosarcoma tumor-bearing mouse model and verified it by immuno PET imaging.The ¹²⁴I-labeled probe specifically targeted the h PD-L1 receptor on the surface of human osteosarcoma cell membranes and could be detected by PET/CT imaging for up to120 hours in vivo.Finally,pathological staining confirmed the expression of h PD-L1receptor protein on the surface of OS-732 tumor cell membranes,which is also consistent with the PET imaging results.Conclusion:Both ¹²⁴I-JS001 and ¹²⁴I-anti-h PD-L1 Nb6 can be used for noninvasive PET imaging in vivo.The ¹²⁴I-JS001 radiotracer has the potential in the noninvasive monitoring of PD-1-positive tumors and the guidance of tumor-specific personalized immunotherapy,which may provide new strategies for guiding patients to choose PD-1 tumor immunotherapy.In addition,¹²⁴I-anti-h PD-L1 Nb6 may provide a novel strategy for the clinical screening of h PD-L1-positive patients,helping to identify patients who can benefit from immunotherapy for malignancies such as osteosarcoma.¹²⁴I-anti-h PD-L1 Nb6 may become an important tool for implementing immunotherapy in malignant tumors,including osteosarcoma.