Experimental Study Of A Nuclear Medicine Diagnosis And Treatment Integrated Probe Targeting Tumor PD-L1 In A Mouse Colon Cancer Model

Objectives Immune checkpoint inhibitors(ICIs)PD-1/PD-L1 block therapy is one of the first approved immunotherapies for cancer.Clinical studies have found that some patients do not respond well to single ICI treatment.The level of PD-L1 expressed by tumor cells before treatment is related to the therapeutic effect.Currently,immunohistochemistry(IHC)is used to detect tumor PD-L1 expression for patient screening,but IHC requires tumor biopsy which is invasive and unable to reflect tumor heterogeneous PD-L1 expression.In this study,we radiolabeling aPD-L1 antibodies with long half-life radionuclide 89Zr for PET imaging for the noninvasive detection of tumor PD-L1 expression.In addition,previous studies have confirmed that the combination of radiotherapy and immunotherapy can improve the synergistic anti-tumor effect.In this study,the therapeutic radionuclide 177Lu labeled ?PD-L1 antibody was used to conduct radioimmunotherapy(RIT)and radio-assisted immunotherapy studies to further explore the therapeutic effect and anti-tumor mechanism.Methods:In order to adapt to the long circulation time of ?PD-L1 antibody,89Zr(t1/2=78.41 hours)and a chelating agent of p-SCN-Bn-DFO were selected for radiolabeling,and the PD-10 chromatography column was used to purify the product.After filtering through 0.22 ?m sterile filter,the radiochemical purity and stability in vitro,and the acute toxicity test of mice were carried out.C57BL/6 mice were implanted with MC38 tumor cells subcutaneously on the right shoulder and perform parallel multi-point static microPET imaging to compare the distribution and time-activity curve(TAC)of three different aPD-Ll antibodies(Y001,Y002,Y003)to screen for antibody with high tumor-specific uptake.Then radiolabel the antibody with 177Lu and divide the tumor-bearing mice into 6 groups:(1)PBS control group;(2)5 mg/kg Y003 antibody group;(3)10 mg/kg Y003 antibody Group;(4)177Lu-DOTA-Y003 3.7 MBq radioimmunotherapy group;(5)177Lu-DOTA-Y003 11.1MBq radioimmunotherapy group;(6)177Lu-DOTA-Y003 3.7 MBq+5 mg/kg Y003 RIT combined with immunotherapy group to evaluate the treatment effect.The body weight of the mice was measured every 2 days and the main organs of the mice were taken for HE staining to evaluate the treatment toxicity after treatment.Flow cytometry and immunofluorescence staining were performed at specific time points to explore the anti-tumor mechanism.Results:The labeling of 89Zr-DFO-?PD-L1 is simple with high yield,the radiochemical purity>95%,the stability in vitro at room temperature is good,and there is no obvious toxicity to mice.The results of microPET imaging of tumor-bearing mice with three different ?PD-L1 antibodies(Y001,Y002,Y003)showed that:tumor uptake of 89Zr-DFO-Y001 is low,most of the radioactivity is retained in the liver;tumor uptake of 89Zr-DFO-Y002 is significantly higher than 89Zr-DFO-Y001,and radioactive uptake of liver is about half of 89Zr-DFO-Y001:89Zr-DFO-Y003 has the highest tumor uptake,tumor uptake can be seen 12 hours after injection,then increased to a maximum of 40%ID/g after 120 hours.It was found that the uptake of 89Zr-DFO-Y003 was mainly concentrated in the tumor and the uptake of major organs such as liver and kidney was low.It was found that 177Lu-DOTA-Y003 3.7 MBq+5 mg/kg Y003 RIT combined with immunotherapy group achieved the best therapeutic effect,significantly inhibiting tumor growth and prolonging the survival time of mice,and the mice in this group had no obvious weight loss and major organ morphology changes,indicating this combine treatment is safe.The investigation of the anti-tumor mechanism found that the tumor infiltration CD4+and CD8+T cells increased after combined treatment,and there was no significant change in Treg cells,indicating that combined treatment can change the immune microenvironment of tumor and improve the synergistic anti-tumor effect.Conclusion:The PET imaging probe 89Zr-DFO-aPD-L1 targeting tumor PD-L1 expression was successfully synthesized.The synthesis and labeling steps were simple,the radiochemical purity was high,and the drug stability was good in vitro.MicroPet imaging of tumor-bearing mice suggests that Y003 antibody has high tumor uptake and PET/CT imaging based on 89Zr-DFO-Y003 helps non-invasively detect tumor PD-L1 expression.Subsequently,we labelled Y003 with radioactive nuclide 177Lu for the RIT study,which confirmed that RIT can upregulate the expression of tumor PD-L1,and that RIT combined with immunotherapy can change the tumor microenvironment and produce synergistic antitumor effect in MC38 mouse colorectal cancer.