Synthesis of Site-Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion.
Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope64Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Yiming
Y
Wu
Department of Nuclear Medicine, Peking University Cancer Hospital & Institute , Beijing 100142, China.
Hua
H
Zhu
Department of Scientific Research, Peking Union Medical College Hospital, Chinese Academy of Medical Science , Beijing 100073, China.
Bo
B
Zhang
Department of Nuclear Medicine, Peking University Cancer Hospital & Institute , Beijing 100142, China.
Fei
F
Liu
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Jingxian
J
Chen
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Yufei
Y
Wang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Yan
Y
Wang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Ziwei
Z
Zhang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Ling
L
Wu
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Longlong
L
Si
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Huan
H
Xu
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Tianzhuo
T
Yao
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Sulong
S
Xiao
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Qing
Q
Xia
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Lihe
L
Zhang
Department of Nuclear Medicine, Peking University Cancer Hospital & Institute , Beijing 100142, China.
Zhi
Z
Yang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Demin
D
Zhou
D000818:Animals; D048588:Benzoxazines / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D060326:Chemistry Techniques, Synthetic; D003301:Copper Radioisotopes / Q000737:chemistry*; D004355:Drug Stability; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D008187:Lutetium / Q000737:chemistry; D016513:Mice, SCID; D049268:Positron-Emission Tomography / Q000379:methods*; D016499:Radioimmunotherapy / Q000379:methods*; D011868:Radioisotopes / Q000737:chemistry; D000069283:Rituximab / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics
C497620:2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one; D048588:Benzoxazines; D002614:Chelating Agents; D003301:Copper Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; D018796:Immunoconjugates; D011868:Radioisotopes; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D000069283:Rituximab; D008187:Lutetium