Radionuclide Labeling Of Nanobody EG2and Their Multimers And Preliminary Exploration Of Targeting Ability To EGFR

Objective:Epidermal growth factor receptor (EGFR) is one of tumor molecular targets widely applied in clinical. A variety of molecular probes including Nanobody have been developed for the detection of tumor EGFR. Nanobody is the smallest antigen-binding fragment with complete functions, but its binding affinity is low. To improve the affinity of Nanobody, we fused anti-EGFR Nanobody EG2to three self-associating peptides, right-handed coiled coil (RHCC), cartilage oligomeric matrix protein (COMP) and C4b binding protein a-chain (C4bpa), to make three multimeric antibodies, EG2-RHCC, EG2-CoMP and EG2-C4bpα. This study aimed to radiolabel Nanobody EG2, EG2-RHCC, EG2-COMP and EG2-C4bpa with99mTc, respectively, and evaluate their targeting efficiency and pharmacokinetics in tumor xenografts.Methods:The precursor [99mTc(CO)3(OH2)3]+was prepared by adding1ml fresh [TcO4]-saline solution into a Tricarbonyl vial. Nanobody EG2(100μg,2mg/ml), EG2-RHCC (100μg,1mg/ml), EG2-COMP (100μg,2mg/ml) and EG2-C4bpa (100μg,1mg/ml) were radiolabeled with prepared precursor [99mTc(CO)3(OH2)3]+(400-500μl), respectively, followed by purification on a PD-10column. In vitro cell saturation binding, blocking, uptake and efflux studies with99mTc-Nanobody EG2,99mTc-EG2-RHCC,99mTc-EG2-COMP and99mTc-EG2-C4bpa were performed in EGFR overexpression A431and EGFR lower-expression OCM-1cells. Single photon emission computed tomography (SPECT) imaging and biodistribution studies were carried out in99mTc-Nanobody EG2-,99mTc-EG2-RHCC-,99mTc-EG2-COMP-and99mTc-EG2-C4bpa-injected mice bearing A431-and OCM-1-derived tumors.Results:We successfully labeled Nanobody EG2, EG2-RHCC, EG2-COMP and EG2-C4bpa with radionuclide99mTc using Tricarbonyl kits with radiochemical yields of60-71%,69-84%,78-80%and30-50%, respectively. The stability and binding specificity of the four99mTc-labeled compounds were tested in vitro assay. The four99mTc-labeled compounds has good stability either in serum or in phosphate buffered saline (PBS) and their radiochemical purities were greater than90%at24h. In vitro cell binding studies showed that the four kinds of99mTc-labeled compounds could specifically bind to EGFR. Single positron emission computed tomography (SPECT) imaging in A431tumor xenografts showed that radioactivity accumulation at the tumor location could be visible clearly at3h after99mTc-EG2injection (2.71±0.45%ID/g); the ratios of tumor-to-blood (T/B) and tumor-to-muscle (T/M) at3h were4.03±0.44and15.35±3.85, respectively; then tumor imaging gradually fades with time. The A431tumor could be visible as early as3h after99mTc-EG2-COMP injection; the highest tumor uptake (6.96±1.23%ID/g; T/M=5.15±1.33) was achieved at6h and the radioactivity retention could still be visible at24h post-injection. The A431tumor could be visible as early as2h and the radioactivity retention could be observed at18h after99mTc-EG2-C4bpa injection; the highest tumor uptake (3.78±0.25%ID/g; T/M=3.69±0.48) was achieved at10h post-injection. The A431tumor image could be blocked in the presence of100μg excess corresponding unlabeled antibodies. However, the A431tumor image could not be visible after99mTc-EG2-RHCC injection and results from biodistribution showed that the radioactivity uptake in the tumor was low at the time points examined (6h:2.05±0.2%ID/g).Conclusions:Our results suggest that Nanobody, as a kind of small molecular antibody fragment, has good ability to target EGFR with advantages of fast imaging and rapid clearance through the kidneys. In vitro and in vivo studies have demonstrated that EG2-COMP also has a good ability to target EGFR. As a multivalent antibody with moderate molecular weight, it has great application potential in future tumor diagnosis and therapy. Due to its large molecular weight, the EG2-C4bpa may not be suitable for tumor diagnosis. However, our study demonstrated the targeting ability and specificity of EG2-C4bpa, which offers basis for potential therapeutic applications, especially radioimmunotherapy. Although EG2-RHCC has higher binding affinity and specificity in vitro, however, their properties in vivo remain to be further studied. In short, Nanobody and multimers have good application prospect and will play an increasingly important role in future radioimmunoimaging and radioimmunotherapy.