Preclinical Evaluation Of Tumor HER2-targeting Molecular Imaging And Chemotherapy

Objective:Malignant neoplasms incidence and mortality are rapidly growing world wide,which is a serious threat to human health.Early specific diagnosis,individual accurate treatment and timely efficacy evaluation are important aspects to improve the survival of tumor patients.Tumor molecular imaging and targeted individual therapy based on molecular imaging have opened up a new field of accurate diagnosis and treatment of tumors and become a hot spot in tumor research.Human epidermal growth factor receptor2(HER2)belongs to the epidermal growth factor receptor(EGFR)family.HER2 gene amplification and protein overexpression are closely related to the high invasiveness,recurrence and mortality of many types of cancer,which makes HER2 a specific target for tumor diagnosis and treatment.HER2affibody with high affinity to HER2 plays an important role in precision medicine of tumor.In this research,we prepared ¹⁸F labeled HER2 affibody,evaluated the character of the radiolabeled HER2-targeting probe and explored its value as novel agent for PET imaging of HER2-positive tumors.At the same time,we synthesized HER2 affibody-cytotoxic drugs conjugate and performed the therapeutic efficacy and imaging assessment of the HER2-targeting chemotherapy drug Z_HER2:V2-pemetrexed in HER2-positive xenograft.Methods:1.HER2 affibody was synthesized by Fmoc/t Bu peptide solid-phase synthesis,and the NOTA sequence was introduced at its N-terminus for[¹⁸F]Al F labeling.In vitro stability of[¹⁸F]Al F-NOTA-HER2 affibody was determined in saline and in 5%human serum albumin(HSA)at 37?at different time points.Cellular uptake study,retention kinetics and internalization of[¹⁸F]Al F-NOTA-HER2 affibody were performed on NCI-N87(HER2+)and MKN74(HER2-)cells blocked with NOTA-HER2affibody.Saturation binding experiment was performed in NCI-N87 cell line by adding[¹⁸F]Al F-NOTA-HER2 affibody molecular probe with different concentration gradients and the Kd value was determined.2.The BALB/c female mice bearing NCI-N87 or MNK74 tumor xenografts were used in this research.In vivo stability was studied by analyzing the radiochemical purity of[¹⁸F]Al F-NOTA-HER2 affibody molecular probe in blood collected at 30min after the injection of radioactive tracers.Biodistribution studies were performed on NCI-N87 and MKN74tumor models.The mice were sacrificed and dissected in groups(3randomized mice per group)at 30min,60min and 120min post injection.Tumor and important organs were collected immediately after cleaned,weighted and measured for the radioactivity using a?-well counter after decay correction.The results were expressed as the percentage injected dose per gram of tissue(%ID/g).The biological distribution and metabolism of[¹⁸F]Al F-NOTA-HER2 affibody in mice were observed.3.Six randomized mice bearing NCI-N87 or MKN74 tumors were intravenously injected with[¹⁸F]Al F-NOTA-HER2 affibody molecular probe.For blocking study,500?g of cold NOTA-HER2 affibody was co-injected for each of the three randomly selected NCI-N87 tumor models.The PET images in mice bearing NCI-N87 and MKN74 tumors were obtained at 30min,60min and 120min post injection.After the scanning,the target to nontarget ratios(T/NT ratios)were measured and compared.4.Z_HER2:V2-pemetrexed conjugate was synthesized using manual solid phase peptide synthesis.Pemetrexed was linked to the N-of the HER2:V2affibody by conjugation.Compound Z_HER2:V2-pemetrexed was labeled with^99mTc using the ligand exchange method based on the-GGGC chelator.In this context,HER2+A549 lung cancer xenografts were treated using Z_HER2:V2-pemetrexed,pemetrexed or physiological saline.Therapeutic efficacy was monitored by SPECT imaging using the ^99mTc-labeled Z_HER2:V2-pemetrexed conjugate and further confirmed by performing apoptosis assays using flow cytometry analysis,hematoxylin-eosin(H&E)staining.To evaluate the expression of HER2 in tumor tissues,immunohistochemistry(IHC)was performed,accompanied by quantitative analysis using flow cytometry.A toxicological evaluation was also conducted.Results:1.[¹⁸F]Al F-NOTA-HER2 affibody molecular probe was manually prepared within 30min.The maximum non-decayed corrected radiochemical yield of[¹⁸F]Al F-NOTA-HER2 affibody molecular probe was up to 32.69%with radiochemical purity of more than 98%.[¹⁸F]Al F-NOTA-HER2 affibody was stable in saline and in 5%human serum albumin(HSA)at 37?with radiochemical purity>90%within 4h.[¹⁸F]Al F-NOTA-HER2 affibody molecular probe showed significantly higher uptake,retention kinetics and internalization in NCI-N87 cells than in MKN74 cells at 5min,30min,60min and 120min(P=0.000,0.000,0.000,0.000;0.000,0.000,0.000,0.000;0.000,0.000,0.000,0.000).The uptake in NCI-N87 cells was blocked by 6 times and 35 times excess NOTA-HER2 affibody at 60 min and 120min(P=0.000,0.000 and P=0.000,0.000),indicating that the uptake of[¹⁸F]Al F-NOTA-HER2 affibody molecular probe in NCI-N87 cells was HER2-specific.The Kd value of[¹⁸F]Al F-NOTA-HER2 affibody molecular probe to HER2 in NCI-N87 cells was 52.25±3.68n M,indicating that the tracer binds to HER2with high affinity.2.[¹⁸F]Al F-NOTA-HER2 affibody molecular probe was injected to mice and a single peak was observed of blood at 30 min,with the radiochemical purity preserved more than 90%.In distribution study,NCI-N87 tumors showed significantly higher uptake than that of MKN74 tumors at each time points(P=0.003,0.016,0.010).The highest renal uptake of[¹⁸F]Al F-NOTA-HER2 affibody molecular probe proved that the kidney was its excretory organ.The low uptake in non-target organs and the rapid blood clearance make[¹⁸F]Al F-NOTA-HER2 affibody molecular probe suitable for imaging.3.The PET imaging results were consistent with the biodistribution data.The specific accumulation of radioactivity in the HER2-positive NCI-N87tumors could be clearly visible at 30min after injection and maintained within120 minutes.No significant radioactive aggregation was observed in MKN74tumors within the time tested.The high uptake in NCI-N87 tumors could be blocked by co-injection of excess NOTA-HER2 affibody at 30min,60min and120min(P=0.003,0.016,0.010),indicating that the uptake of[¹⁸F]Al F-NOTA-HER2 affibody molecular probe in NCI-N87 tumors was HER2-specific.4.HER2+A549 lung xenografts were treated using Z_HER2:V2-pemetrexed,pemetrexed or physiological saline.Imaging with ^99mTc-Z_HER2:V2-pemetrexed demonstrated that in HER2+A549 models,Z_HER2:V2-pemetrexed showed better antineoplastic effects than pemetrexed on day 14 after treatment.Compared with pemetrexed,the results from the pathological and flow cytometry analyses also revealed that Z_HER2:V2-pemetrexed exhibits high antitumor activity against A549 tumors,inducing necrosis and apoptosis(P=0.015).Tumor flow cytometry and immunohistochemical HER2 assay showed no significant difference in HER2 expression between Z_HER2:V2-pemetrexed and pemetrexed(P=0.602,0.072).In addition,the clinical signs of toxicity in the Z_HER2:V2-pemetrexed treated group were reduced compared with those in the pemetrexed treated group.Conclusions:1.[¹⁸F]Al F-NOTA-HER2 affibody molecular probe was obtained with good radiochemical purity within 30 min.[¹⁸F]Al F-NOTA-HER2 affibody molecular probe can selectively accumulate in HER2-positive cells with high binding affinity and targeting specificity.[¹⁸F]Al F-NOTA-HER2 affibody molecular probe is expected to become a new tracer for HER2-targeted molecular imaging.2.[¹⁸F]Al F-NOTA-HER2 affibody could rapidly localize in HER2+xenografts and remove from blood circulation predominantly by urinary excretion.3.The[¹⁸F]Al F-NOTA-HER2 affibody molecular imaging can provide optimize contrast in models with HER2+xenografts and maybe a feasible approach for the non-invasive real-time detection of the HER2 status.[¹⁸F]Al F-NOTA-HER2 affibody is expected to become a new tracer for HER2-targeted molecular imaging.4.Imaging using ^99mTc-Z_HER2:V2-pemetrexed compound could be used as an effective noninvasive visualization technique to monitor the therapeutic response of Z_HER2:V2-pemetrexed.The Z_HER2:V2-pemetrexed conjugate encompasses promising targeted antitumor activity against HER2-positive lung adenocarcinoma,with reduced side effects compared with pemetrexed.Hence,the Z_HER2:V2-pemetrexed conjugate may serve as a novel molecular agent with tremendous clinical breakthrough potential in the diagnosis and treatment of HER2-positive tumors.