Introduction: In this study, a new agent consisting of HER2-specific affibody Z(HER2:v2) and chemotherapy drug pemetrexed was synthesized to develop a new targeted drug. Its biological characteristics and anticancer efficacy were assessed in cells level and xenografts models by radiolabeling with technetium-99m. Methods: After the Z(HER2:v2)-pemetrexed conjugate was synthesized, radiolabeling of the conjugate was performed using its C-terminal 4 amino acids (Gly-Gly-Gly-Cys) as the chelating moiety. The radiochemical yield of the [Tc-99m]Tc-Z(HER2)(:v)(2) -pemetrexed was identified by instant thin-layer chromatography (ITLC). Stability of the radiolabeled conjugate was investigated both in vitro and in vivo. In vitro binding affinity and cell internalization study of the probe were performed in A549 cells (HER2-positive). Tumor uptake was evaluated by in vitro uptake assay in A549 cells and H23 cells (HER2-negative), and by in vivo biodistribution and SPELT imaging in A549 and H23 tumor-bearing mice. The antitumor efficacy of the Z(HER2:v2) -pemetrexed conjugate was evaluated in cells and xenograft models. Results: The Z(HER2:v2) -pemetrexed was successfully synthesized and conjugated with technetium-99 m, and acquired the radiochemical yield of 97.0 +/- 0.3%. The stability of [ Tc-99m]Tc-Z(HER2:v2)-pemetrexed was good in both physiological saline and human serum. The radiolabeled agent displayed excellent HER2-binding specificity and affinity in vitro, and was gradually internalized into the cells. Biodistribution study revealed obvious tumor uptake in A549 xenografts (percentage injected dose per gram, 2.6 +/- 1.0%ID/g at 4 h postinjection), while the uptake in HER2-negative H23 tumors was much lower (0.2 +/- 0.1%ID/g at 4 h postinjection, P < 0.01). SPEC' imaging exhibited an intensity in the A549 xenograft which could be blocked by excess Z(HER2:v2) pemetrexed. Treatment with Z(HER2:v2) -pemetrexed significantly impaired the tumor growth (P < 0.05), with less weight loss than pemetrexed. Conclusion: [Tc-99m]Tc-Z(HER2:v2) -pemetrexed showed desirable property and HER2-specificity. The Z(HER2:v2)pemetrexed conjugate could inhibit tumor growth of HER2-positive lung adenocarcinoma and may have the potential to become a targeted drug for lung cancer. Advances in knowledge and implications for patient care: The compound described herein performs HER2-targeting with favorable anticancer efficacy and offers the potential of novel targeting strategies for further tumor therapy. (C) 2018 Elsevier Inc. All rights reserved.