Peptide PET Tracer Targeting LAG3 For Evaluation Of Immunotherapy In Malignant Melanoma

Objective:The rapid development of immunotherapy has made it an important method for treating tumors.But it has a low response rate and may have serious adverse reactions,so it is urgently needed to enrich the treatment methods,select patients suitable for treatment and evaluate the efficacy timely.Lymphocyte activation gene 3（LAG3）is a new target for immunotherapy and its expression is increased in the activated T cells.In this study,a new molecular tracer ⁶⁸Ga-NOTA-XH05 was constructed by labeling XH05 with positron radionuclide ⁶⁸Ga to explore its application in positron emission computed tomography（PET）to noninvasively detect LAG3 expression in tumors,evaluate the efficacy of immunotherapy and monitor the response of melanoma to XH05 treatment.The aim of this study is to provide a new tracer and a new approach for the decision making and efficacy evaluation of immunotherapy regimens for malignant melanoma.Methods:（1）The tracer ⁶⁸Ga-NOTA-XH05 was prepared,and was identified by high performance liquid chromatography（HPLC）after purified.The ⁶⁸Ga-NOTA-XH05 PET imaging and biodistribution of B16-F10 subcutaneous tumor models were studied.The expression of LAG3 in the tumors was monitored by flow cytometry,and its correlation with the uptake of the tracer was analyzed to evaluate the specificity of the tracer.（2）PET imaging and biodistribution were conducted after Cp G oligonucleotide（Cp G-ODN,Cp G）used for the treatment of unilateral or bilateral B16-F10 subcutaneous tumor models,to evaluate the ability of ⁶⁸Ga-NOTA-XH05 monitoring the efficacy of immunotherapy and the abscopal effect of Cp G.Hematoxylin-eosin staining（HE staining）,immunofluorescence staining and flow cytometry were used to verify the biological processes reflected by the tracer.（3）After treatment with XH05 alone or combined with Cp G,the tumor growth was monitored to evaluate its efficacy.⁶⁸Ga-NOTA-XH05 PET imaging was used to continuously monitor the changes of LAG3 in the tumors.HE staining and immunofluorescence staining were used to detect the histological changes after treatment,and the results were compared with those of PET imaging to analyze the differences and similarities in monitoring the therapeutic effect of XH05.Results:（1）After purification,the radiochemical purity of ⁶⁸Ga-NOTA-XH05 was over99%and it had high stability in vitro.In PET imaging of B16-F10 subcutaneous tumor model,the tumors were clearly visualized.The mean fluorescence intensity of LAG3 in tumor cells was detected by flow cytometry and it was positively correlated with the uptake of ⁶⁸Ga-NOTA-XH05（R²=0.5213,P=0.0184）,indicating that the uptake of⁶⁸Ga-NOTA-XH05 could reflect the expression level of LAG3 in tumor.（2）⁶⁸Ga-NOTA-XH05 PET imaging of the mice with B16-F10 subcutaneous tumors was performed after intratumoral injection of Cp G.The tumor blood ratio（TBR）was2.234±0.386,which was significantly higher than that of the control（TBR=1.348±0.301,P=0.0001）.The result of biodistribution was similar to the imaging result.According to the tumor growth rate,mice in Cp G treatment group were divided into responders and nonresponders,and the TBR of the nonresponders was significantly higher than that of the responders（2.537±0.404 vs.1.991±0.101,P=0.0341）.Immunofluorescence staining confirmed that the expression of LAG3 in the tumors of the nonresponders was higher than that of the responders,and both were higher than that of the control,indicating that⁶⁸Ga-NOTA-XH05 could effectively distinguish the responders from the nonresponders.In bilateral B16-F10 subcutaneous tumors model,only the right tumors were treated with intratumoral injection of Cp G,and the growth rate of bilateral tumors decreased,confirming the abscopal effect of intratumoral injection of Cp G.On day 2 after first treatment,⁶⁸Ga-NOTA-XH05 PET imaging showed increased TBR of the right tumors,but no significant difference displayed between the left tumors and the control.On day 8,uptake of the left tumors was also significantly higher than that of the control,and even higher than that of the right tumors.The LAG3 immunofluorescence staining of tumor tissue was consistent with the imaging results,indicating that ⁶⁸Ga-NOTA-XH05 can effectively monitor the abscopal effect of Cp G.（3）After XH05 treatment,the tumor growth rate of some mice was slower than that of the control,and the tumor growth rate of some mice in the XH05+Cp G combined treatment group was slower than that of the Cp G treatment group,indicating that XH05 was effective in the treatment of some mice.HE staining of the tumor showed substantial necrosis,and immunofluorescence staining showed decreased proliferation of the tumor cells.Continuous ⁶⁸Ga-NOTA-XH05 PET imaging of the mice during treatment showed that the TBR of mice responding to XH05treatment was lower during and after the treatment than that of the control or Cp G treatment group,and that the TBR of the XH05 monotherapy responders increased to be similar to that of the XH05 nonresponders and the control after stopping the treatment for a period of time.The LAG3 immunofluorescence staining results of tumor tissues were consistent with the last PET imaging results,but could not distinguish between the responders and nonresponders in XH05 treatment group,indicating that⁶⁸Ga-NOTA-XH05 has certain advantages in dynamically monitoring LAG3 expression to evaluate the efficacy.Conclusion:In this study,PET imaging tracer ⁶⁸Ga-NOTA-XH05 constructed by labeling LAG3 targeting peptide XH05 with ⁶⁸Ga was successfully developed.This tracer had high radiochemical purity and good stability,and its uptake of the tumors was positively correlated with LAG3 expression level,which could be used to detect LAG3 expression level in patients before anti-LAG3 treatment.Further PET imaging was performed on the melanoma bearing mouse model after immunotherapy.The results showed that⁶⁸Ga-NOTA-XH05 could evaluate the immunotherapy response and monitor the abscopal effect of the therapy.This study also attempted to apply high-dose XH05 to the treatment of melanoma in mice,and found that it can slow down the growth rate of tumor.Meanwhile,⁶⁸Ga-NOTA-XH05 could monitor the change process of LAG3 expression in the tumors during the treatment of XH05.These results confirmed that ⁶⁸Ga-NOTA-XH05could play a role in the selection of patients before anti-LAG3 treatment,the evaluation of efficacy after immunotherapy,and the monitoring of LAG3 changes caused by novel drugs,and had good clinical translational value.