RIKEN Accel. Prog. Rep. 51 (2018)

Ⅰ. HIGHLIGHTS OF THE YEAR

One-pot three-component double-click method for synthesis of [67Cu]-labeled
One-pot three-component
double-click
method† for synthesis of
biomolecularradiotherapeutics[67 Cu]-labeled biomolecular radiotherapeutics†

K. Fujiki,＊1 S. Yano,＊2 T. Ito,＊3 Y. Kumagai,＊3 Y. Murakami,＊3 O. Kamigaito,＊2 H. Haba,＊2 and K. Tanaka＊1,＊4,＊5
K. Fujiki,∗1 S. Yano,∗2 T. Ito,∗3 Y. Kumagai,∗3 Y. Murakami,∗3 O. Kamigaito,∗2 H. Haba,∗2 and K. Tanaka∗1,∗4,∗5
A one-pot three-component double-click process tomitting theranostic radionuclide having a half-life of 62 h,
preparetumor-targeting
agentsforcancerradiotherapy
iswhich
compatible
with radioimmunotherapy,
could beA
one-pot
three-component
double-clickprocessto62 method
h, is
which
is synthesis
compatible
withradioimmunotherapy,
three-component
double-clickforsynthesis
ofof[67
Cu]-labeled
One-potOne-pot
three-component
double-click
methodfor[67Cu]-labeleddescribed
By utilizing DOTA
achieved
mixingby
DOTAor
NOTA-attachedalbuminand
prepare here.
tumor-targeting
agents (orforNOTA)cancer containing
radiothercould be byachieved
mixing
DOTAor
NOTA-attachedbiomolecularradiotherapeutics††
biomolecularradiotherapeutics
apy is described
By utilizing
DOTA (oralbumin andanti-IGSF4
antibody
with RIspurificationand subtetrazines(DOTA: here.
1,4,7,10-tetraazadodecane-1,4,7,10anti-IGSF4
antibody
with RIs
and subsequentNOTA) containing
tetrazines(DOTA:
1, 4, 7, ＊310sequentpuriﬁcation
Amiconﬁltration;1,＊4,a＊565
separate
tetraacetic
acid, K.NOTA:
1,4,7-triazacyclononane-1,4,7by
Amiconfiltration;
experimentZn was＊＊3＊2 aby2
Fujiki,＊＊1 S.
Yano,＊
T. Ito,＊3 Y. Kumagai,
Y. Murakami,
O. Kamigaito,
H.separate
Haba,＊2 andK. Tanaka＊with
＊1
＊3
＊
＊1,＊
65
674,＊5
2 Zn was ＊2
conducted
for 1).
comparison
tetraazadodecane-1,
4,
7,2 T.10-tetraacetic
acid,＊3(TCO:
NOTA:
experimenttriacetic
TCO-substituted
aldehyde
conducted
forwithcomparison
(Fig.
2 and
Cu andK. acid)
Fujiki,and
S.the
Yano,
Ito, 3 Y. Kumagai,
Y. Murakami,
O. Kamigaito,
H. Haba,and
K. Table
Tanaka67 be pro(Fig.
2 and
Table
1). 67 Cu
1, 4, 7-triazacyclononane-1,
4, 7-triacetic
acid)
and
the to65
andcouldtrans-cyclooctene),
the twothree-component
click
reactions,the tetrazine
Zn mitting
couldbeproducedin havingthe 65a70Zn
Zn(d,αn)Cuh, andA one-pot
double-clickprocess
theranostic
radionuclide
half-life
of 62
70
67
nat
65
nat
65the
TCO-substituted
aldehyde(TCO:
trans-cyclooctene),
Zn(d,
αn)
Cu
and
Cu(d,
x)
Zn
duced
in
ligation(aninverseelectron-demand
Diels-AlderCu(d,x)Zn
reactions
at
the
AVF
cyclotron.
Our
preparetumor-targeting agentsfor cancerprocess
radiotherapy
whichtheranostic
is compatible
with radioimmunotherapy,
couldofbeworkA one-pot
three-component
double-click
to is mitting
radionuclide
having a half-life
62reh,
1) reactions,
the
two
click
the
tetrazine
ligation(aninactions
at
the
AVF
cyclotron.
Our
workprovidesa
described
here.
Byutilizing
DOTA
(or
NOTA)
containing
achievedbymixing
DOTAor
NOTA-attachedalbuminandcycloaddition)
providesnew and with
operationallysimple methodforand theRIKEN
clickradiotherapy(a rapidis
prepare tumor-targeting
agentsfor cancer
which is acompatible
radioimmunotherapy,
couldnew
be
1)
67
2)-8)
67 antibody
tetrazines (DOTA:
1,4,7,10-tetraazadodecane-1,4,7,10anti-IGSF4
withmethod
RIs and for
subsequent
verse
electron-demand
Diels-Alder
cycloaddition)
and
Cuand
to
and
operationallyintroducing
introducing
6π-azaelectrocyclization),
could
simultaneously
proceedCu tosimple
biomolecules,
which
is purification
animportantdescribed
here.
By utilizing
DOTA(orNOTA) containing
achievedby mixing
DOTAor NOTA-attachedalbumin65
2–8)
tetraacetic
acid,
NOTA:
1,4,7-triazacyclononane-1,4,7by
Amiconfiltration;aseparate
experiment
with
Zn
was
the
RIKEN
click
(a
rapid
6π-azaelectrocyclization),biomolecules,
which
is
an
importantprocessforpreparunder
mild conditions
to 1,4,7,10-tetraazadodecane-1,4,7,10afford the covalent attachment ofprocess for antibody
preparingwith
clinically
relevant tumor-targeting
tetrazines(DOTA:acid)
RIs
triacetic
and the TCO-substituted aldehyde (TCO: anti-IGSF4
conducted for comparison(Fig. and
2 andsubsequent
Table 1). 67purification
Cu and65
could
simultaneously
proceedunder
mild conditions
toing
clinically
relevant atumor-targeting
agents.
theDOTAor trans-cyclooctene),
NOTA,
which
forms
a
bioavailable
stableagents.
65
70
67
tetraacetic
acid,
NOTA:
1,4,7-triazacyclononane-1,4,7by
Amiconfiltration;
separate
experimentZn wasthe two click reactions, the tetrazine
Zn could be produced in theZn(d,αn)with
Cu and67
aﬀord
thecovalent
attachment
of
the
DOTA
or
NOTA,
nat
65
complexcopper(II),biomolecules
such
as albumin
triaceticwith
acid)and theTCO-substituted
aldehyde(TCO:(Fig.AVF
2 andTable Our1). work
Cu andligation(an toinverseelectron-demand
Diels-Alder conducted
Cu(d,x)forZncomparison
reactions at thecyclotron.
70
67 forwhich
formscycloaddition)a antibody
bioavailable
stablecomplexwithcopperandanti-IGSF4
without
altering
theirtheactivities
new produced
and operationallymethod
and
the
RIKEN
click
(a rapid 65Znprovides
trans-cyclooctene),
the 1)twoclick
reactions,tetrazine
could a bein the simple
Zn(d,αn)Cu and
2)-8)
67
nat
65
(II),
to
biomolecules
suchasalbuminandanti-IGSF4
introducing
6π-azaelectrocyclization),
could
simultaneously
proceedCu
tobiomolecules,
which
is
an
important(Fig.
1).
ligation(an inverse electron-demand Diels-AlderCu(d,x) Zn reactions at the AVF cyclotron. Our work
under
conditions
to activities
afford the covalentattachment ofprocess for preparing clinically relevant tumor-targeting
1) mild
antibody
without
altering(Fig.(a1).
cycloaddition)
providesa new and operationally simple method forandthe their
RIKEN
click
rapid
the DOTAor 2)-8)
NOTA, whichforms
a bioavailable
stableagents.
67
Subsequently,
theradiolabelingof
DOTAorNOTAintroducing
6π-azaelectrocyclization),
could
simultaneously
proceedCu to biomolecules, which is an importantcomplex with copper(II), to biomolecules such as albumin
attached
albuminandanti-IGSF4
antibody(atumorunder mild conditions
toaffordthecovalent
attachmentofprocessforpreparingclinically relevant tumor-targeting
and anti-IGSF4 antibody
without altering their−activities
67
targetingwithas aa bioavailable
promising βstable/γthe DOTAantibody)or
NOTA,
which Cu
forms
agents.
(Fig.
1).
e-mittingtheranostic
radionuclide
havinghalf-lifeofcomplex withcopper(II),to biomoleculessucha asalbuminFig. 2. Radiolabelings of DOTA or NOTA-attached
and anti-IGSF4 antibody without altering their activities
Fig. 2. Radiolabelingsof antibody.
DOTA or NOTA-attached albualbumins
and anti-IGSF4(Fig. 1).
mins and anti-IGSF4 antibody.

67
Table 1.
RadiochemicalyieldsCu and 65Zn.
Fig.
2. Radiolabelingsof (RCY)DOTA ofor NOTA-attached
65
Entryalbumins
Chelator-attached
Added [ Zn] orRCYb
and anti-IGSF4 antibody.
67 of 67
a Cu and 65 Zn.
Table 1. Radiochemicalyields(RCY)biomolecules
[ Cu]
(%)
Table 1. Radiochemical yields (RCY)of 67Cu and 65Zn.
(Radioactivities)
65
EntryDOTA-albumin
[(300
Zn]
orNOTA-attached
RCYb 8065
2. Chelator-attachedRadiolabelings
DOTA
or
1 Fig.
7b of [Added
Zn]
kBq)
67
a
[ Cu]
(%)
albumins
and biomolecules
anti-IGSF47b
antibody.
2
DOTA-albumin[67Cu] (11 MBq)
72
(Radioactivities)
67
3 1 DOTA-anti-IGSF4[
65 Cu] (11 MBq)
DOTA-albumin 7b[ Zn] (30067
kBq)
80 51
Table21. Radiochemicalof MBq)Cu and 6572Zn.
mAb 9a yields
DOTA-albumin
7b (RCY)[67Cu] (1165
Entry
Chelator-attached
Added
[MBq)
Zn]
or 51 RCY
DOTA-anti-IGSF4[[6767Cu]
4 3 NOTA-albumin
8Cu](11
(11
MBq)
19 b
67
a
67
mAb 9abiomoleculesCu]MBq)(%)
5
NOTA-anti-IGSF4[ Cu][ (1174NOTA-albumin
8[67
Cu] (11 MBq)
19
(Radioactivities)
mAb 9b67
NOTA-anti-IGSF4[65(11 MBq)
7a) 1 567 7b[65Cu]
Zn]were(300110
kBq)
SpecificDOTA-albumin
activities
Zn
MBq/μg80
andmAbof9b Cu and 67
2
DOTA-albumin
7b
[
Cu]
(11
MBq)
72
a)
67
65
125
MBq/μg,respectively.
Fig. 1. (a) One-pot three-component click labeling ofSpecific activities of Cu and Zn
were 110 MBq/μg and67b) 3
DOTA-anti-IGSF4
Cu] (11MBq) from 51the(Radiochemical
obtained
125 MBq/μg,
respectively. yield) [ wasalbumin and anti-IGSF4
antibodythree-component
as a cancer-targeting
Fig. 1. (a) One-pot
click labeling of RCY
b)
mAb
9a
RCY
(Radiochemicalyield)
wasobtainedfromthe theradioactivityofthepurifiedradiolabeledproduct
againstalbuminandanti-IGSF4
antibody
asacancer-targeting
agent. (b) Affinities of intact and labeled anti-IGSF4
67
65 NOTA-albumin
67the purified
radioactivityofradiolabeledproduct
against the19
4
8
[
Cu]
(11
MBq)
agent.
(b)
Affinitiesofintactandlabeledanti-IGSF4
added
[
Zn]
or
[
Cu].
antibodies to IGSF4 analyzed by ELISA. DMF =[65Zn] or [67Cu].
to IGSF4 analyzed by ELISA. DMF =5added NOTA-anti-IGSF4[67Cu] (11 MBq)
7
N,N-dimethyl antibodies
formamide,
ELISA = enzyme-linked
N,N-dimethyl formamide, ELISA = enzyme-linked
mAb
9b
References
immunosorbent
assay.
immunosorbent
assay.
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67
65
Specific
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Cu andZn J.were110
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IGSF4
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＊2∗1＊3