Anda di halaman 1dari 17

COMPARISON OF

BIFUNCTIONAL
64
CHELATES FOR Cu
ANTIBODY IMAGING

EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR


IMAGING

Va n e s s a Pro z z o
P H M 1 1 15
Fe b r u a r y 7 ,
2 0 15

BACKGROUND
The high target affi nity of monoclonal antibodies
(mAbs) is advantageous for diagnostic imaging and
radiotherapy
mABs are of particular interest in the development of
molecular imaging agents because they can be used
to
measure antigen expression noninvasively in the body
stratify patient populations for specifi c treatments
monitor therapeutic response

By attaching a radioisotope onto a mAb, an agent for


therapy and/or diagnostics can be obtained
However, the large size and sensitive functional
groups on mAbs can be problematic for
radiopharmaceutical development

64

Cu

Is a radioisotope being investigated for


radiopharmaceuticals
It is desirable because its physical properties align
with the pharmacokinetic properties of mAbs
6 4 Cu half-life (t 1 / 2 = 12.7 h) is suitable for imaging 2448 h after administration, allowing the antibody
enough time to localize to the tumor
6 4 Cu decay generates + emissions used in PET
imaging as well as potentially therapeutic
emissions
A bifunctional chelate (BFC) is used to chelate the
64
Cu and link it to the antibody, however current
BFCs have poor in vivo stability or harsh reaction
conditions are required for conjugation

STUDY AIM
To compare trastuzumab conjugates to two novel BFCs,
p-SCN-PCTA and p-SCN-Bn-Oxo-DO3A , directly with a
DOTA analog (p-SCN-Bn-DOTA) to examine their
potential use for antibody imaging with 6 4 Cu

EXPERIMENTAL DESIGN
Bioconjugate Preparation:
The bioconjugates were
prepared by incubating
trastuzumab with one of
the BFCs in HEPES buff er at
pH 8.5, at room
temperature for 20 hours
This facilitated the linking
of amine functional groups
in trastuzumab with thiol
groups in BFCs via the
formation of a thiourea

HPLC: remove impurities


Protein Concentration Determination: Coomassie
Protein Assay
Chelate Concentration Determination:
Radiolabelling with an excess of high specifi c activity 111 In
spiked with a nonradioactive In 3+ standard
Quenched with DTPA after 3 hours
HPLC used to determine the %In bound to the bioconjugate
compared to In-DTPA
# of chelates/monoclonal antibody = %In bound to the
bioconjugate [ In3+]/[mAb]

EXPERIMENTAL DESIGN
RADIOLABELING & BIOCONJUGATE STABILITY:
6 4 Cu (100-400 MBq) was added to one of the
bioconjugates in NaAc buff er pH 5.5
Incubated at room temperature for 30 min- 2 h
Purifi cation by size exclusion chromatography
HPLC was done before purifi cation to determine
radiochemical yield (RCY) and after purifi cation to
determine radiochemical purity of the fi nal product
Each of the bioconjugates was added to mouse serum
and incubated at 37C
HPLC: to determine loss of 6 4 Cu to serum proteins

EXPERIMENTAL DESIGN
F l o w Cy to m et r y:
Used to determine the affi nity of bioconjugates compared to trastuzumab using
HER2/neu-expressing cell lines and vector as a control

Cell Binding Studies:


Carried out in triplicate with each of the 6 4 Cu-radiolabelled bioconjugates in each
of the cell lines
Experiments for control and nonspecifi c uptake

P E T I m a g i n g a n d B i o d i s t r i b u ti o n S tu d i e s :
HER2-negative and HER2-positive tumors were grown subcutaneously on Rag2
mice
Mice were injected intravenously with one of the 3 radiolabelled bioconjugates
and were imaged at 24 and 48 hours post-injection
Mice were euthanized & the per unit activity present in tissues was determined

RESULTS

Coomassie Protein Assay Results:


Bioconjugate Used

Average # of Chelates per


trastuzumab

PCTA-Trastuzumab

1.5 0.4

Oxo-DO3A-Trastuzumab

1.3 0.2

DOTA-Trastuzumab

1.7 0.5

%
RA D I O C H E M I C A

YIELD vs
REACTION
TIME
L

SPECIFIC ACTIVITY

BIODISTRI
BUTION
STUDIES
Fi g . 4
Biodistribution
of 64Curadiolabelled
OXO - D O 3 A ( r e d ) , P C TA (blue) and
D O TA - ( y e l l o w )
at 24 h (A) and
40 h (B) post
injection in
LCC6HER2
xe n o g r a ft (so l i d )
or LCC6vector
( c h e c ke r e d )
t u m o r- b e a r i n g
mice

CONJUGATE STABILITY

64

Cu-Radiolabelled
Bioconjugates

% Intact after 24
HR

% Intact after 40 HR

PCTA-Trastuzumab

>95

80

Oxo-DO3ATrastuzumab

>95

78

DOTA-Trastuzumab

54

26

TRANSVERSE PET IMAGES

CONCLUSION
Both novel BFCs have advantageous properties:
They were found to label under mild conditions more
rapidly compared to DOTA-trastuzumab
Post-radiolabelling purifi cation is not needed
Showed high stability in mouse serum
They have higher tumor uptake compared to 6 4 CuDOTA-Trastuzumab
These BFCs may allow for earlier imaging protocols
with better tumor to background ratios
Confi rmation of these results with other antibodies
would be worthwhile

CRITIQUES
Not enough data shown for stability of the
bioconjugates in the presence of mouse serum
Incorrect referencing of data
Images were unclear Shape of mouse?

YOU DECIDE
Accept
Accept with minor revision
Accept with major revision
Reject