@article{open2992,
          volume = {43},
          number = {4},
           month = {April},
          author = {Yogesh  Rathore  and Jaya  Shukla  and Ishita  Laroiya and Amar  Deep  and Tamanna  Lakhanpal  and Rajender Kumar  and Harmandeep  Singh  and Amanjit Bal and Gurpreet  Singh  and Krishan Gopal  Thakur  and B R  Mittal },
            note = {The copyright of this article belongs to Wolters Kluwer},
           title = {Development 68Ga trastuzumab Fab and bioevaluation by PET imaging in HER2/neu expressing breast cancer patients},
       publisher = {Wolters Kluwer},
            year = {2022},
         journal = {Nuclear Medicine Communications },
           pages = {458--467},
        keywords = {68Ga trastuzumab Fab; breast cancer; HER2/neu; immunoPET; trastuzumab Fab},
             url = {http://crdd.osdd.net/open/2992/},
        abstract = {Introduction: Receptors on breast cancer cells play a crucial role in the management of patients. Trastuzumab is a widely used drug for the treatment of HER2/neu expressing tumors. ImmunoPET with trastuzumab is not feasible due to slow pharmacokinetics. Fragment of antigen-binding (Fab) radiolabeled with positron emitters can be used for immunoPET.

Methods: Fab has been generated by papain digestion and conjugated with the bifunctional chelating agent NOTA. The SDS-PAGE and MALDI-TOF were used to see the integrity of Fab and conjugated Fab. In-vitro stability and target specificity for HER2/neu receptors were performed in plasma and receptor binding with bio-layer interferometry (BLI) techniques. Radiolabeling was standardized with 68GaCl3 and PET imaging was performed in seven patients showing 18F fluorodeoxyglucose (18F-FDG) uptake and correlated with HER2/neu expression by immunohistochemistry.

Results: Fab production was optimized at molar ratio 23:1 of trastuzumab and papain at 37 ?C with a constant stirrer at 850 rpm for 22-24 h, at pH 8. Conjugation with NOTA was standardized at molar ratio 1:25 of trastuzumab Fab and NOTA. Molecular mass of trastuzumab Fab-NOTA was found approximately 46.3 kDa ({\texttt{\char126}}1/3 of intact antibody). Trastuzumab Fab-NOTA showed radiolabelling efficiency of 48-70\% with incubation time 15 min at 37-40 ?C and pH 4.5-5.0. BLI demonstrated the affinity of trastuzumab, trastuzumab Fab and trastuzumab Fab-NOTA towards HER2/neu receptor with KD of {\ensuremath{<}}1pM, {\texttt{\char126}}0.5nM and {\texttt{\char126}}20nM, respectively. All immunohistochemistry proven patients showed uptake in primary breast lesion and lymph nodes.

Conclusion: Trastuzumab Fab-NOTA is suitable for radiolabelling with 68Ga and ImmunoPET imaging of HER2/neu receptor.}
}