Molecular Probes Based On Nanobodies Targeting CD38 And PD-L2 For Cancer Diagnosis And Tumor Treatment

Cancer is one of the main causes of human death,currently there is still a lack of accurate diagnostic methods for tumors.At the same time,how to achieve accurate evaluation of in individual tumors from patients and accurate screening of immunotherapy beneficiaries is still one of the medical problems to be solved urgently.Because of its real-time,non-invasive characteristic,nuclear medicine imaging can reflect molecular-level information,and has a wider application prospect than clinical pathological examination.To solve such problems,in this study two nuclear medicine probes targeting CD38 and programmed cell death ligand 2（PD-L2）were developed,hoping to be applied for patients selecting and treatment guidance.Daratumumab is an anti-CD38 monoclonal antibody for treatment of multiple myeloma（MM）patients.The tumor CD38 expression level is one of important factors in influencing daratumumab efficacy.Therefore,there is an urgent clinical need for a noninvasive method to evaluate the CD38 levels in cancer patients before,during and after daratumumab treatment.We developed a new CD38-specific molecular imaging probe ^99mTc-CD3813,for noninvasive imaging of CD38 expression by single photon emission computed tomography（SPECT）.We demonstrated the CD38 affinity and specificity of ^99mTc-CD3813,and the Kd of it was 3.14nmol/L.^99mTc-CD3813 SPECT/CT has the capability to visualize subcutaneous and orthotopic myeloma lesions in mice models,getting more exhaustive results than ¹⁸F-fluorodeoxyglucose(¹⁸F-FDG)positron emission tomography（PET）.Experimental verification showed that CD3813 and daratumumab binds to different domain of CD38,which meant there is no competition between them.^99mTc-CD3813 SPECT/CT monitored significant reduction of CD38 expression in the tumor-bearing mice under daratumumab treatment,as evidenced by its reduced tumor uptake（3.04±0.70%ID/cc vs.1.07±0.28%ID/cc,P<0.001）.^99mTc-CD3813 SPECT/CT was also able to detect the increased tumor uptake（0.79±0.29%ID/cc vs.2.12±0.12%ID/cc,P<0.001）due to the upregulation of CD38 levels caused by all-trans retinoic acid（ARTA）.^99mTc-CD3813 is a promising SPECT radiotracer for imaging of CD38-positive tumors,and has clinical potential as a molecular imaging method for evaluation of tumor burden,evaluation of CD38 expression and treatment guidance of patients.In the second project,we developed a human PD-L2 specific nanobody probe for evaluating PD-L2 levels of patients.Although the expression level of tumor programmed cell death ligand 1（PD-L1）was used as a biomarker to predict the response of programmed cell death protein 1（PD-1）and PD-L1 inhibitor treatment,there still were patients with negative PD-L1 expression can get benefit from PD-1 blockade therapy.The latest research showed that some tumors have high PD-L2 expression levels.As another ligand of PD-1,PD-L2 can also mediate immune escape of tumor cells by binding to PD-1.Its existence weakens the effect of PD-L1 blockade therapy.Therefore,PD-L2 is expected to become a new biomarker for predicting the response of PD-1 therapy.We developed a new molecular imaging probe^99mTc-MIRC415,for noninvasive imaging of PD-L2 expression by SPECT.It’s proved that^99mTc-MIRC415 has high radiochemical purity（>99%）and good stability.The Kd of it was16.47 nmol/L.SPECT/CT imaging results showed that the ^99mTc-MIRC415 probe could clearly image the A549^PD-L2 xenograft mouse positive tumor model,and the tumor had the highest uptake of the probe at 2 h after injection,reaching 27.00±3.01%ID/cc.As a PD-L2negative control,A549^WT tumor showed no obvious uptake,only 0.62±0.23%ID/cc 2 h after injection.This result suggested the fast metabolic properties in vivo and targeting specificity of the probe.According to immunofluorescence staining experiments,the PD-L2 expression levels of A549^WT,H1975,H226 and A549^PD-L2 tumor models varied from low to high.SPECT/CT imaging experiments were performed on them respectively,and we found that the tumor uptake of probe was positively correlated with tumor PD-L2 expression levels,which demonstrated the ^99mTc-MIRC415 SPECT/CT imaging could monitor PD-L2 expression in different models.The PD-L2 up-regulation in H226 tumor model intervened by IFNγwas also observed by ^99mTc-MIRC415 SPECT/CT imaging（from 1.48±0.21%ID/cc to 3.14±0.56%ID/cc,P<0.05）,showing that the probe has the ability to monitor the dynamic changes of PD-L2 in vivo.In summary,we expect that nuclear medicine molecular probes that specifically target PD-L2 can be applied to monitor the dynamic PD-L2 expression levels in vivo,enriching the companion diagnostic detection of PD-1/PD-L1 immunotherapy.This method could provide more accurate predictive indicators for individualized tumor immunotherapy.In summary,we developed specific nuclear molecular probes targeting tumor-related markers,which can achieve accurate detection of cancer at the molecular level,hoping to promote the development of personalized diagnosis and treatment for patients.The platform of probe preparation and evaluation established in this study is beneficial for the subsequent development of nanobody probes in cancer diagnosis and treatment.