Ⅰ. HIGHLIGHTS OF THE YEAR
RIKEN
Accel. Prog. Rep. 56 (2023)

RIKEN Accel. Prog. Rep. 56 (2023)

Prevention of radionuclide-induced antibody denaturation maintains
active targeting and maximizes antitumor eﬃcacy in211
At-radioimmunotherapy†
H. Takashima,∗1,∗2 K. Ohnuki,∗3 S. Manabe,∗2,∗4,∗5,∗6 Y. Koga,∗1,∗7 R. Tsumura,∗1 T. Anzai,∗1 Y. Wang,∗2X. Yin,∗2 N. Sato,∗2 Y. Shigekawa,∗2 A. Nambu,∗2 S. Usuda,∗2 H. Haba,∗2 H. Fujii,∗3 and M. Yasunaga∗1
Selective tumor accumulation of alpha emitters withhigh linear energy transfer and a short particle range
in tissue results in potent antitumor eﬃcacy without
serious toxicity. Thus, there is a growing interest in
developing novel target alpha therapies. Astatine-211(211 At) is a promising alpha emitter that is applicableto cancer treatment.
In the preparation process of radioactive antibodies, caution should be exercised in the radionuclideinduced chemical reaction causing antibody denaturation.
We demonstrated that reactive oxygenspecies (ROS) generated from 211 At-induced radiolysis of water denature astatinated antibodies.1) The
radionuclide-induced antibody denaturation disrupts
binding activity and attenuates in vivo antitumor effect. In contrast, sodium ascorbate (SA), a free radical scavenger, successfully quenches ROS and prevents
denaturation, resulting in the maintenance of binding activity and antitumor eﬀect.2) Although we revealed the inﬂuence of radiochemical reaction on 211 Atlabeled antibody as described above, several questions
remain. First, it is unclear whether 211 At-induced denaturation aﬀects the pharmacokinetics of radioactive
antibodies, such as half-life in blood circulation, distribution to normal organs, and tumor accumulation via
active targeting and passive targeting, which are basedon antigen-antibody reaction and enhanced permeability and retention eﬀect, respectively.3) In addition, theprotective eﬀects of SA on the pharmacokinetics havenot been clariﬁed.
In this study, using an 211 At-labeled anti-human epidermal growth factor receptor 2 (HER2) antibody stabilized with SA, a denatured radioactive anti-HER2
antibody, and an 211 At-labeled nontargeted control antibody (anti-CD20 antibody) stabilized with SA, wecompared their residence time in blood circulation, dis†

∗1∗2
∗3
∗4

∗5∗6
∗7

Condensed from the article in Mol. Pharm. 20, 1156 (2023)
Developmental Therapeutics, Exploratory Oncology Research & Clinical Trial Center (EPOC), National Cancer
Center
RIKEN Nishina Center
Division of Functional Imaging, EPOC, National Cancer
CenterLaboratory of Functional Molecule Chemistry, Pharmaceutical Department and Institute of Medicinal Chemistry,
Hoshi University
Research Center for Pharmaceutical Development, Tohoku
University
Glycometabolic Biochemistry Laboratory, RIKEN
Department of Strategic Programs, EPOC, National CancerCenter- S37 -Disrupted active targeting
Low tumor accumulation
Less antitumor effectRadiolysis

Disrupted binding activity

Sodium ascorbate

Maintained binding activity

Maintained active targeting
High tumor accumulation
Greater antitumor effectFig. 1. Graphical abstract of this study.

tribution to normal organs, tumor accumulation, andantitumor eﬀect in a xenograft model with high expression of HER2.
In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, we conﬁrmed that 211 At denatured radioactive anti-HER2 and anti-CD20 antibodies under no protection, and SA successfully stabilized the radioactive antibodies. The binding activity of the denatured radioactive anti-HER2 antibody
was consistently disrupted, whereas the binding activity of the stabilized immunoconjugate was comparable
to the naked antibody. Similarly, the cytocidal eﬀectof the denatured radioactive anti-HER2 antibody on
HER2-positive cancer cells was attenuated more than
the stabilized radioactive antibody.
There is no diﬀerence in blood circulation time aswell as distribution to normal organs between thegroups administered 211 At-labeled anti-HER2 antibody under SA protection and the denatured radioactive anti-HER2 antibody in ex vivo biodistributionstudy. These ﬁndings suggest that 211 At-induced antibody denaturation may not aﬀect tumor accumulation via passive targeting. However, single-photonemission computed tomography and ex vivo biodistribution studies demonstrated that tumor accumulation of 211 At-labeled anti-HER2 antibody stabilizedwith SA was signiﬁcantly higher than that of the denatured radioactive anti-HER2 antibody and 211 Atlabeled nontargeted control antibody under SA protection. In a xenograft model with high expression ofHER2, the stabilized radioactive anti-HER2 antibody
consistently outperformed the denature immunoconjugate and the radioactive nontargeted control antibody. 211 At-induced antibody denaturation hampers

Ⅰ. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 56 (2023)

tumor accumulation via active targeting, attenuating
antitumor eﬃcacy, whereas SA successfully maintains
tumor targeting and antitumor activity. In alpharadioimmunotherapy, active targeting signiﬁcantly increases tumor accumulation of 211 At.
In conclusion, SA-dependent protection that maintains tumor targeting and in vivo antitumor effect will facilitate the clinical application of 211 Atradioimmunotherapy.
References
1) S. Manabe et al., ACS Omega 6, 14887 (2021).
2) H. Takashima et al., Cancer Sci. 112, 1975 (2021).
3) Y. Matsumura et al., Cancer Res. 46, 6387 (1986).

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