I. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Influence of antibody stabilization with sodium ascorbate onradioimmunotherapy with an 211 At-conjugated anti-tissue factor
antibody†
H. Takashima,∗1,∗2 Y. Koga,∗1,∗2 S. Manabe,∗3,∗4,∗5 K. Ohnuki,∗6 R. Tsumura,∗1 T. Anzai,∗1 N. Iwata,∗1Y. Wang,∗2 T. Yokokita,∗2 Y. Komori,∗2 D. Mori,∗2 H. Haba,∗2 H. Fujii,∗6 Y. Matsumura,∗7 and M. Yasunaga∗1Alpha radiation is characterized by higher linear transfer compared to other types of ionizing radiation and arange of 50–100 µm in tissue. Therefore, the selectivetumor accumulation of alpha emitters exerts potent antitumor eﬀects without serious toxicity against normal
cells adjacent to the tumor. Astatine-211 (211 At) is analpha emitter, its high production yield is suﬃcient toprepare 211 At-labeled radiopharmaceuticals for administration at clinical doses.
Tissue factor (TF), a transmembrane glycoprotein initiating the extrinsic blood coagulation cascade, is overexpressed in tumors such as gastric cancer, pancreaticcancer, and malignant gliomas.1,2)
Immunoglobulin G (IgG) selectively accumulate in tumor via the enhanced permeability and retention (EPR)eﬀect.3) In addition, the antigen-antibody reaction enhances the tumor accumulation of antibodies.2) Therefore, we focused on applying anti-TF monoclonal antibodies (mAbs) established by us to armed antibodies
such as antibody-drug conjugate (ADC)4–6) and antibody labeled with therapeutic radionuclides.
We synthesized 211 At in the 209 Bi(α, 2n)211 At reaction using the RIKEN azimuthally varying ﬁeld (AVF)
cyclotron, and we labeled an anti-TF mAb with the radionuclide as previously reported.7)
To evaluate 211 At-conjugaed anti-TF mAb, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ﬂow cytometry analyses.
In the SDS-PAGE analysis, 211 At-conjugated anti-TFmAb was smeared, which was in contrast to band patterns in 211 At-unlabeled anti-TF mAbs. Consequently,
ﬂow cytometry analysis revealed that the binding activity of the astatinated mAb was disturbed comparedwith 211 At-unlabeled mAbs. These ﬁndings suggest thatanti-TF mAb was denatured by the 211 At-induced radiochemical reaction. Then, to protect anti-TF mAb fromdenaturation, we puriﬁed astatinated mAbs using an elution buﬀer containing sodium ascorbate (SA), which is afree radical scavenger. In the SDS-PAGE analysis, bandpatterns were demonstrated in 211 At-conjugated anti-TFmAbs eluted in phosphate-buﬀered saline (PBS) contain†∗1
∗2
∗3

∗4
∗5

∗6

∗7Condensed from the article in Cancer Sci. 112, 1975 (2021)
Developmental Therapeutics, EPOC, National Cancer Center
RIKEN Nishina Center
Laboratory of Functional Molecule Chemistry, Pharmaceutical Department and Institute of Medicinal Chemistry, Hoshi
University
Research Center for Pharmaceutical Development, Tohoku
University
Glycometabolic Biochemistry Laboratory, RIKENDivision of Functional Imaging, EPOC, National Cancer Center
Department of Immune Medicine, National Cancer Center Research Institute, National Cancer Center

Purification
without SA

211At-induced

Disturbed binding activity
Disturbed antitumor effect

211At-induced

Maintained binding activity
Maintained antitumor effectantibody denaturation (+)

211At labelingPurification
with SA

antibody denaturation (‒)

Fig. 1. Summary of 211 At-induced antibody denaturationand protective eﬀect of sodium ascorbate.

ing 0.6 or 1.2% SA and 211 At-unlabeled mAbs. Consequently, the binding activities of the astatinated anti-TFmAbs in the SA solution were comparable to those of211
At-unlabeled mAbs.
211
At-conjugated anti-TF mAbs stabilized with SA exerted greater cytocidal eﬀects on gastric cancer cells than
the astatinated mAb eluted in PBS. Similar to ADC,
the cytotoxicities of the stabilized immunoconjugates depended on the level of TF in the cancer cell.
To evaluate in vivo toxicities, we observed the bodyweight loss in mice administered SA, 211 At-conjugatedanti-TF mAb, or free 211 At. Although body weightloss was observed in mice administered PBS containing
1.2% SA, the loss was transient and the radioprotectant
seemed tolerable. Body weight loss after the administration of the astatinated anti-TF mAb in PBS containing
1.2% SA was milder than free 211 At dissolved in 1.2%SA solution.
211At-conjugated anti-TF mAb eluted in PBS containing 1.2% SA showed signiﬁcantly greater antitumor effects in a high TF-expressing gastric cancer xenograftmodel than the non-stabilized immunoconjugate. Similar to ADC, the antitumor eﬀect of the astatinated antiTF mAb in 1.2% SA soution depended on the TF expression on the cell membrane of cancer cells.
In summary, SA protected the astatinated anti-TF
mAb from 211 At-induced antibody denaturation, which
resulted in the maintained binding and antitumor activities of the immunoconjugate (Fig. 1). Without intolerable side eﬀects, 211 At-conjugated anti-TF mAb eluted
in PBS containing 1.2% SA showed potent antitumor effects in gastric cancer xenograft models dependent on
the level of TF on the cancer cell membrane.
References

1) Y. W. van den Berg et al., Blood 119, 924 (2012).
2) H. Takashima et al., Sci. Rep. 7, 12341 (2017).
3) Y. Matsumura et al., Cancer Res. 46, 6387 (1986).
4) Y. Koga et al., Int. J. Cancer 137, 1457 (2015).
5) R. Tsumura et al., J. Control. Release 284, 49 (2018).
6) R. Tsumura et al., Oncol. Rep. 45, 329 (2021).
7) H. Takashima et al., RIKEN Accel. Prog. Rep. 53, 172 (2020).

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