In Vitro And In Vivo Study On The Treatment Of Non-small Cell Lung Cancer With Radionuclide Labeled PD-L1 Nanobody

ObjectiveRadiopharmaceutical therapy is an emerging,effective and less toxic cancer therapy,especially for metastatic cancer,which can overcome the radiation exposure damage to normal tissues caused by traditional radiotherapy.In addition,it has the ability to activate anti-tumor immunity,therefore it is applied in combination with immunotherapy to treat immune-resistant tumors.Most previous studies mainly used proteins with large molecular weight such as monoclonal antibodies as radionuclides’carriers.However,large molecular weight makes it difficult for antibodies to penetrate solid tumors,and slow metabolism of radionuclide-labeled antibodies led to inevitable blood and bone marrow toxicity.In recent years,nanobody has become a suitable carrier for molecular imaging and radionuclide therapy due to its excellent in vivo properties,such as high targeting affinity,considerable tissue penetration,and rapid blood clearance.Since programmed cell death ligand 1（PD-L1）is overexpressed in some non-small cell lung cancers（NSCLCs）,PD-L1 nanobody can be as a carrier to be labeled by radionuclide to realize tumor targeted therapy.In this study,radioactive ¹³¹I-labeled PD-L1 nanobody（Nb109）was used to explore its targeting effect,biological effect and anti-tumor efficacy evaluation on NSCLCs in vitro and vivo.Methods1.The expression of PD-L1 in NSCLCs（H460 and A549）were analyzed by western blot,immunofluorescence and immunohistochemical staining.2.PD-L1 nanobody（Nb109）was labeled with radioactive ¹³¹I by chloramine-T method.The labeling rate,radiochemical purity and stability of ¹³¹I-Nb109 in phosphate buffer saline（PBS）and fetal bovine serum（FBS）solution（37℃）were detected by paper chromatography.The molecular weight and purity of ¹³¹I-NB109 were analyzed by SDS polyacrylamide gel electrophoresis（SDS-PAGE）.The octanol-water partition coefficient was determined to evaluate its lipid-and water-solubility.3.In vitro uptake of ¹³¹I-Nb109 in H460 and A549 cells were conducted to verify its ability to target PD-L1.Different time points（1 h,2 h,4 h）and nanobody concentration（100 n M,50 n M,25 n M,12.5 n M,6.25 n M,3.125 n M,1.5625 n M,0 n M）were set and free ¹³¹I was used as the negative control.4.The toxic effects of ¹³¹I-Nb109 and ¹³¹I on H460 cells were evaluated by CCK8 test and clonal formation assay.The expression levels of high mobility group box 1（HMGB1）,heat shock protein 70（HSP70）and adenosine triphosphate（ATP）were detected by western blot and ATP assay kit after H460 cells were treated with ¹³¹I-Nb109.5.H460 tumor-bearing mice were constructed.When the tumor volume reached about500 mm³,SPECT/CT scan and in vivo biodistribution experiments were performed to verify the targeting ability of ¹³¹I-Nb109 to PD-L1 in vivo.H460 tumor-bearing mice were randomly divided into three groups when the tumor volume reached approximately100 mm³,and then they were intratumorally injected with 20μL PBS,¹³¹I and ¹³¹I-Nb109（300μCi）,respectively.Tumor volume and body weight were monitored the next day.At day 14,mice were euthanized,and blood was taken to detect liver and kidney function as well as blood routine,and tumors and important organs were taken for pathological analysis.Results1.Western blot,immunofluorescence and immunohistochemical staining showed that PD-L1 expression was positive in H460 cells and negative in A549 cells.2.¹³¹I-Nb109 was synthesized successfully.The labeling rate was 69.51～98.06%and the radiochemical purity was over 99%.¹³¹I-Nb109 was stable during 72 h in vitro（PBS:81.17%±0.02%,FBS:96.55%±0.01%）.The molecular weight of ¹³¹I-Nb109 with high hydrophilicity was about 14 k Da and its purity was very high.3.Compared with A549 cells,the uptake rate of ¹³¹I-Nb109 in H460 cells was higher and it increased with time（1 h:0.18%±0.02%,2 h:0.20%±0.02%,4 h:0.37%±0.08%）,while H460 cells had almost no uptake on free ¹³¹I.4.CCK8 and clonal formation experiments showed that ¹³¹I-Nb109 had toxic effects on H460 cells,and relative cell viability decreased with radioactivity increasing.The release of HMGB1,HSP70 and ATP increased after H460 cells were treated with ¹³¹I-Nb109.5.SPECT/CT imaging and in vivo distribution experiments showed that ¹³¹I-Nb109 had higher targeting ability on H460 tumors compared with free ¹³¹I group.The radioactivity was still concentrated in the tumor site at 48 h.¹³¹I-Nb109 significantly inhibited tumor growth without toxic side effects via intratumoral injection in contrast with control group.ConclusionIn this study,hydrophilic radiopharmaceutical ¹³¹I-Nb109 was synthesized successfully.The labeling method was simple and easy to perform.¹³¹I-Nb109 had high labeling rate,radiochemical purity and stability.It showed good ability to target PD-L1-positive NSCLCs and inhibited tumor growth in vitro and vivo.It also induced tumor cells to release damage-related molecular patterns regarded as biomarkers of immunogenic cell death.The results of this study are expected to inspire the development of more novel radiopharmaceuticals to treat NSCLCs.