Note: Descriptions are shown in the official language in which they were submitted.
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SYNTHESIS OF BIOLOGICAL COMPOUNDS LABELED WITH THE ALPHA
EMITTER Ac-225
Technical field
[0001] The present invention generally relates to an improved protocol for the
synthesis of biological compounds labelled with the alpha emitter Ac-225.
Background Art
[0002] Radiotherapeutic treatment of cellular disorders, including cancer and
infectious diseases is widely documented in literature. A variety of methods
have
been developed in order to utilise radionuclides in radiotherapy, including
targeted
radiotherapy, pre-targeted radiotherapy and the use of radionuclides in the
form of
bone-seeking complexes.
[0003] Targeted alpha therapy is a site directed treatment modality for
cellular
disorders, including cancer and infectious diseases, using alpha radiation to
selectively destroy targeted cells, e.g. tumour cells, fungal cells or
bacteria. The
principle of targeted alpha therapy is based on the coupling of alpha-emitting
radionuclides to targeting moieties, e.g. monoclonal antibodies or peptides,
that
recognise a structure in, on or near a target. Due to the short radiation path
length
of alpha particles in human tissue (<100 pm), targeted alpha therapy has the
potential of delivering a highly cytotoxic radiation dose to targeted cells,
while
limiting the damage to surrounding healthy tissue.
[0004] It is well known within the art that radioconjugates are of high
interest for
clinical applications. In fact, those compounds are advantageous for
therapeutic
and diagnostic applications because they are complexed with radioactive metal
ions. These types of complexes may e.g. be used to carry radioactive metals to
tumour cells which may be targeted for example by the specificity of an
attached
antibody.
[0005] Although a number of methods to synthesise radioconjugates are known,
they generally are subject to some drawbacks, either because they require
multiple preparation steps in the presence of the radionuclide and/or because
the
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preparation times are long and/or because the yields obtained in terms of
radioconjugate are modest.
Technical problem
-
[0006] It is an object of the present invention to provide an improved
method for
preparing radioconjugates, in particular radioconjugates labeled or chelated
with
actinium-225 (Ac-225). This improved method should allow for a more efficient
and
fast manufacture of radioconjugates useful in diagnostic and clinical
applications.
General Description of the Invention
[0007] Hence, in order to overcome the above-mentioned drawbacks of the
existing methods, the present invention proposes a method for producing a
radioconjugate labeled with radionuclide actinium-225 (Ac-225) comprising the
following step of:
(C) chelating radionuclide Ac-225 with a conjugated chelate compound in a
chelation reaction mixture to obtain a radioconjugate labeled with Ac-225,
wherein the pH of the chelation reaction mixture is comprised between 7.1 and
10,
preferably between 8.0 and 9.5, more preferably about 9Ø
[0008] It has indeed been surprisingly found that when working at relatively
basic to fairly basic pH values, the chelating reaction kinetics is
significantly
improved. As a consequence, the major advantage of the present method with
respect to known methods is that the operation within the particular pH range
indicated above not only drastically reduces the reaction times needed, but at
the
same time allows for high yields of radioconjugate. Generally, the chelating
reaction yields obtained are well above 80 %, often even above 90 % of the
initial
reactants, although the reaction time is less than a tenth or less of that of
comparable methods. A further advantage of the method is that the operation
within the temperature range indicated allows for synthesis of radioimmuno-
conjugates containing heat sensitive biological compounds such as antibodies
or
fragments thereof. The present method therefore represents an easy, efficient
and
useful one-step express chelation process for the preparation of radio(immuno)-
conjugates.
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[0009] Hence, the chelation reaction (also sometimes referred to as
"labeling") in
step (C) is preferably effected or allowed to run for only 3 to 30 minutes at
a
- temperature between 30 and 60 C, preferably for about 15 minutes
(such as 12 to
18 minutes) at a temperature between 35 and 45 C, such as at about 40 C.
[0010] A further advantage of the present method is that it only comprises one
radiochemical step (in which the radionuclide is involved). This is generally
a
benefit as it reduces unwanted (and unnecessary) losses of part of the
prepared
radioconjugates' activity due to the relatively short half-life of such
radionuclides.
[0011] The chelation reaction mixture in step (C) preferably comprises a
buffer
or buffer system to control the pH. The buffer(s) in step (C) may be chosen
among
those known to be appropriate for the pH range of 7.1 to 10, such as 3-
{[tris(hydroxylmethyl)methyl]amino}propanesulfonic acid (TAPS); N,N-bis(2-
hydroxyethyl)glycine (Bicine); tris(hydroxymethyl)aminomethane (Tris, also
referred to as tris(hydroxymethyl)methylamine); N-tris(hydroxymethyl)-
methylglycine (Tricine), etc. In a particularly preferred embodiment, the
chelation
reaction mixture comprises tris(hydroxymethyl)aminomethane (Tris) as a buffer.
[0012] The "conjugated chelate compound" (also called "conjugate" herein) is a
chelate compound conjugated (i.e. generally covalently linked) to a biological
compound.
[0013] A "chelate compound" or "chelate" or "chelator" useful in the present
invention are so-called bifunctional chelators which are compounds having the
double functionality of sequestering metal ions combined to the ability to
covalently
bind a biological compound. Useful chelate compounds may thus be any
appropriate chelating agent capable of reacting with a biological molecule,
such as
one or more selected from diethylene triamine pentaacetic acid (DTPA);
ethylene
diamine tetraacetic acid (EDTA); 1 14,7,10-tetraazacyclododecane-N,N',N",N"-
tetraacetic acid (DOTA); p-isothiocyanatobenzy1-1,4,7,10-tetraazacyclododecane-
1,4, 7, 10-tetraacetic acid (p-SCN-Bz-DOTA); 1,4,7, 10-tetraazacyclododecane-
N ,N',N"-triacetic acid (DO3A); 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetrakis(2-
propionic acid) (DOTMA); 3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-
carboxy-13-phenyl-tridecanoic acid (B-19036); 1,4,7-triazacyclononane-N, N',
N"-
triacetic acid (NOTA); 1,4,8, 11-tetraazacyclotetradecane-N, N', N",N"-
tetraacetic
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acid (TETA); triethylene tetraamine hexaacetic acid (TTHA); trans-1,2-
diaminohexane tetraacetic acid (CYDTA); 1,4,7,10-tetraazacyclododecane-1-(2-
. hydroxypropy1)-4,7,10-triacetic acid (HP-DO3A); trans-cyclohexane-
diamine
tetraacetic acid (CDTA); trans(1,2)-cyclohexane dietylene triamine pentaacetic
-
acid (CDTPA); 1-oxa-4,7,10-triazacyclododecane-N,N',N"-triacetic acid
(OTTA);
1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetrakis{3-(4-carboxyl)-butanoic
acid};
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetic acid-methyl amide);
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonic acid);
2 ,2', 2"-(10-(2-(2, 5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-
tetraazacyclo-
dodecane-1,4 ,7-triyOtriacetic acid (DOTA-NHS ester) and derivatives thereof.
[0014] A "biological compound" in the context of the present invention may be
any appropriate naturally, synthetically, or recombinantly obtained or
prepared
compound selected from a protein, a peptide, an antibody or an antigen-binding
fragment thereof, a protein comprising antigen-binding polypeptide sequences
of
an antibody, a monoclonal antibody, a fraction of a monoclonal antibody, such
as
a variable region thereof, a protein comprising an antigen binding sequence of
an
antibody, a polynucleotide, or any derivative of these compounds.
[0015] Thus, the term "radioconjugate" as used herein refers to chelate
compound conjugated to a biological compound, wherein the chelate compound
has been complexed with a radionuclide, such as Ac-225. The term
"radioimmunoconjugate" more particularly refers to such a radioconjugate if
the
biological compound is a compound capable of using antibody-antigen bonding
for
the targeting described in the introductive part.
[0016] The conjugated chelate compound can be directly used in step (C).
However, if necessary, the conjugated chelate compound may also be first
prepared by reacting a chelate compound with a biological molecule in a
preliminary conjugation step. Hence, in a further aspect, the method described
herein preferably additionally comprises before said step (C) the following
preparation step:
(A) conjugating a biological compound with a chelate compound in a conjugation
reaction mixture to obtain a conjugated chelate compound.
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[0017] This step may be done using any known method useful to link a chelator
to a biological compound [e.g. Mirzadeh et al. 1990]. Furthermore, in this
conjugation reaction, the chelate and the biological compounds may be any
appropriate compounds, such as those already cited above. Preferably, the
chelate compound is selected from DOTA and its functional derivatives. The
biological compound is preferably a protein, a peptide, an antibody or a
derivative
thereof, particularly preferably a monoclonal antibody such as Lintuzumab
(HuM195), Rituxinnab (trade names Rituxane and MabTherae), Cetuximab
(Erbituxe), Trastuzumab (Herceptine), mAb2556 (anit-gp41) mAb c595 (anti-
MUC1), anti-CD38-MAb M0R03087, MX35, F8 (specific to EDA fibronectin), L19
(specific to EDB fibronectin) and F16 (specific to domain A1 of tenascin C).
[0018] The conjugation reaction in step (A) is conducted for a time sufficient
to
obtain an adequate conjugation yield. The time necessary depends among others
on the pair of reactants and the temperature at which the reaction takes
place. In
general, the reaction conditions useful for this step comprise reaction times
from
30 minutes to 48 hours at temperatures between 15 and 40 C, preferably from 6
hours to 18 hours at temperatures between 25 and 35 C.
[0019] It is generally necessary or at least preferable to also control the pH
of
the conjugation mixture. A pH range which is useful for a particular
conjugation
reaction will depend among others on the pair of reactants; however, a
particularly
useful range of pH will be pH values from 7 to 10, more preferably 8 to 9.5 or
even
between 8.5 and 8.9. Appropriate buffers may be used to keep the pH in the
selected range, such as those mentioned above or preferably bicarbonate or
phosphate buffers.
[0020] If the method comprises a conjugation step (A), a purification of the
conjugated chelate compound may be useful to eliminate unreacted chelate and
biological compounds before proceeding further to the actual chelating step
(C).
[0021] In a preferred embodiment of the method, the method thus further
comprises between steps (A) and (C) the following step of:
(B) purifying the conjugated chelate compound obtained in step
(A).
[0022] This step may be effected using any one or more of the known
techniques, such as filtration, size exclusion chromatography, affinity
purification,
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centrifugation, extraction, adsorption, dialysis, etc. A particularly
preferred
technique comprises ultrafiltration with a molecular weight cut-off of at
least 10000
= Da, more preferably of at least 20000 Da, even more preferably of at
least 30000
Da. Depending on the compounds used in the conjugation step (A), the cut-off
= may even be at least 40000 Da or more.
[0023] Generally it will be desirable or even necessary to also adjust and
control
the pH during the purifying step (B). In such cases it may be advantageous to
add
a buffer or buffer system. A preferred buffer or buffer system is 4-(2-
hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES), bicarbonate or sodium acetate or
combinations thereof. Preferably, the conjugation is performed at pH 8.5 ¨ 8.9
in
bicarbonate buffer (0.15 M NaCI / 0.05 M NaHCO3). This buffer may also be used
in the first washing steps (e.g. 3x), then the product may be washed several
times
with the buffer system in which the final product is stored, e.g. 0.15 M NaCI
/ 0.05
M Na-acetate, pH 7.2.
[0024] In a further aspect, further auxiliaries and additives may be added to
the
chelation reaction mixture if necessary or deemed useful. In one aspect,
radioprotectants or stabilizers are further added to this chelation reaction
mixture.
Such radioprotectants or stabilizers may be chosen for example for povidone
(polyvinylpyrrolidone, PVP), ascorbic acid, benzyl alcohol, cysteamine,
cystamine,
propylene glycol, dextran, and gentisic acid, preferably ascorbic acid or
gentisic
acid.
[0025] The radioprotectant(s) or stabilizer(s), preferably gentisic acid
and/or
ascorbic acid, is/are added in the chelation reaction mixture in step (C)
either from
the start before the actual chelation reaction begins, at any time during said
chelation reaction or at the end of said chelation reaction. In a preferred
aspect,
the radioprotectant(s) or stabilizer(s) is/are added at the end of the
chelation
reaction (or in other words immediately after the reaction), i.e. generally
after
about 10 to 15 minutes.
[0026] In a still further aspect, the method further comprises after step (C)
the
following step:
(D) quenching the chelation reaction of step (C) by adding a quenching
compound
to the reaction mixture.
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[0027] The quenching reaction, also referred to as termination reaction, may
be
useful to scavenge possibly unreacted (unchelated) radionuclide. This
quenching
. or termination may be done by adding a quenching compound, such as a
chelator.
These chelators may be one or more of those cited above, however they need not
' to be bifunctional chelators, because they only need the
functionality of
sequestering metal ions, in particular Ac-225. One particularly appropriate
quenching compound is for example diethylenetriaminepentaacetic acid (DTPA).
[0028] For any one or more of the steps described herein, it might be
necessary
to heat the corresponding reaction mixture. Such a heating may be performed
using any conventional method or apparatus, such as a heating block or
equivalent alternatives. Preferably, the heating is performed using microwave
heating, especially for step (C).
Brief Description of the Drawings
[0029] Preferred embodiments of the invention will now be described, by way of
example, with reference to the accompanying drawings in which:
- Fig. 1 is a diagram showing the radiolabeling yield for synthesis of Ac-
225-
DOTA-rituximab according to the method according to the present invention
(full
squares) in comparison with the prior art described by Simon et al. (US
20120220754) (shown as empty circles);
- Fig. 2 is a diagram showing the stability of 225Ac-DOTA-rituximab
radioconjugate
in human serum at 37 C under 5% CO2 atmosphere;
- Fig.3 is a diagram showing the scatchard analysis of the binding affinity
of Ac-
225-DOTA-rituximab to K422 lymphoma cells synthesized according to the
method of the present invention.
[0030] Further details and advantages of the present invention will be
apparent
from the following detailed description of several not limiting examples and
embodiments with reference to the attached drawings.
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Examples
[0031] Conjugation reaction:
[0032] A solution of 0.34 mg of p-SCN-Bn-DOTA in 1 ml 0.15 M NaCI / 0.05 M
NaHCO3 (pH 8,5 ¨ 8,9) is added to a reaction vial containing 5 mg of
monoclonal
antibody rituximab (anti-CD20) dissolved in 1 ml of 0.15 M NaCI / 0.05 M
NaHCO3
(pH 8,5 ¨ 8,9). The mixture is stirred for 18 hours at 25 C. For removal of
unconjugated p-SCN-Bn-DOTA chelate, the reaction mixture is subsequently
filtered through an ultrafiltration unit with 30 kD cutoff (Amicon) until
approximately
0.3 ml are left on top of the filter. Subsequently 1 ml of 0.15 M NaCI / 0.05
M
NaHCO3 (pH 8,5 ¨ 8,9) is added to the filtration unit and passed through the
filter
until 0.3 ml of solution are remaining on top of the filter. This step is
repeated three
times. Subsequently 1 ml of 0.15 M NaCI / 0.05 M sodium acetate (pH 7,2) is
added to the filtration unit and passed through the filter until 0.3 ml of
solution are
remaining on top of the filter. This step is also repeated three times.
Finally the
purified conjugated monoclonal antibody (DOTA-rituximab) is taken up in 1.5 ml
0.15 M NaCI / 0.05 M sodium acetate (pH 7,2).
[0033] Characterisation of the conjugated antibody:
[0034] The final concentration of the DOTA-rituximab conjugate in 0.15 M NaCI
/
0.05 M sodium acetate is analyzed by spectrophotometry or using a
colourimetric
method for protein assay. The ratio of DOTA-chelate molecules per molecule of
monoclonal antibody is determined by spectrophotometry as described in
[Dadachova E, Chappell LL and Brechbiel M.: "Spectrophotometric method for
determination of bifunctional macrocyclic ligands in macrocyclic ligand-
protein
conjugates." (Nucl Med Biol. 1999;26(8):977-82)], by radiometric titration or
mass
spectrometry (e.g. MALDI-MS).
[0035] Radiolabeling (chelation) procedure:
[0036] 0.1 mg of DOTA-rituximab in 0.02 ml 0.15 M NaCI / 0.05 M sodium
acetate (pH 7,2) is added to a reaction vial containing 0.5 ml of 0.1 M TRIS
buffer
(pH 9,0). Subsequently 10 pl of Ac-225 stock solution in 0.1 M HCI containing
0.1
mCi to 0.5 mCi (3.7 MBq to 18.5 MBq) Ac-225 are added to the reaction vial.
The
reaction solution is mixed using a vortex mixer. An aliquot of 2 pl is
withdrawn from
reaction mixture and pipetted onto a pH paper to verify the pH is 8.5 ¨ 9Ø
If
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required, the pH is adjusted by addition of 0.1 M sodium hydroxide solution.
Subsequently the solution is heated to 40 C for 15 minutes.
*
[0037] Analysis of the radiolabeling yield using instant thin layer
chromatography
(ITLC):
[0038] At the end of the chelation reaction, an aliquot of 1 pl of the
reaction
mixture is withdrawn for analysis of the radiochemical purity by instant thin
layer
chromatography (ITLC-SG, Agilent) using 0.05 M sodium citrate solution, pH 5.5
as solvent as described in [Essler M, Gartner FC, Neff F, Blechert B,
Senekowitsch-Schmidtke R, Bruchertseifer F, Morgenstern A, Seidl C.
"Therapeutic efficacy and toxicity of (225)Ac-labelled vs. (213)Bi-labelled
tumour-
homing peptides in a preclinical mouse model of peritoneal carcinomatosis."
(Eur J
Nucl Med Mol Imaging. 2012;39(4):602-12)]. The radiolabeling yields obtained
are
typically well above 80 %, often even above 90 % as illustrated in Fig. 1 for
specific activities of 0.1 to 5 mCi Ac-225 per mg monoclonal antibody.
[0039] As illustrated in Figure 1, the radiolabeling yields obtained using the
method described here are significantly higher than the radiolabeling yields
obtained following the prior art described in Simon et al. (US2012/0220754
A1).
[0040] Optional: Purification of the radioimmunoconjugate:
[0041] If deemed necessary, the radioimmunoconjugate can be purified by size
exclusion chromatography using a PD10 column (Biorad). To this end, 0.01 ml of
a
solution containing 1.5 mg/ml DTPA and 0.05 ml of 20% ascorbic acid solution
are
added to the radiolabeling mixture (obtained after the radiolabeling
procedure) and
the entire mixture is loaded onto a PD10 column preconditioned with 10 ml 0.9%
NaCI solution. Subsequently the column is washed with 2.36 ml of 0.9% NaCI
solution. Discard the washings. Add 2 ml 0.9% NaCI solution on the column and
collect the eluate containing the purified radioimmunoconjugate. The
radiochemical purity of the purified radioimmunoconjugate typically exceeds
98%.
[0042] Stabilization:
[0043] In the absence of a suitable radioprotectant the radiochemical purity
of
the Ac-225 labeled radioimmunoconjugate gradually decreases with time due to
radiolytic effects. In order to increase the stability of the
radioimmunoconjugate, a
radioprotectant is added. To this end, following the radiolabeling procedure
step or
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following the optional purification step 1 ml of a 20% solution of ascorbic
acid
adjusted to pH 6 are added to the radioimmunoconjugate. However, the volume
and the pH as well as the concentration of the ascorbic acid may vary.
Furthermore, other radioprotectants may be used instead of the ascorbic acid.
[0044] Serum stability of the radioimmunoconiugate:
[0045] The stability of two samples of Ac-225 labeled DOTA-rituximab
synthesized according to the method described above was studied in human
serum. To this end an aliquot of 0.1 ml of purified radioimmunoconjugate was
added to 1 ml of human serum and incubated at 37 C under 5% CO2 atmosphere.
At various time points, an aliquot of the sample was analyzed by ITLC. The
results
are shown in Figure 2. Both radioimminoconjugates show excellent stability
over
22 days (exceeding two half-lives of Ac-225).
[0046] Binding affinity of the radioimmunoconjuqate:
[0047] The binding affinity of an Ac-225-DOTA-rituximab radioimmunoconjugate
synthesized according to the method disclosed here was investigated towards
K422 lymphoma cells using a saturation binding assay as described in [Mario De
Decker, Klaus Bacher, Hubert Thierens, Guido Slegers, Rudi A. Dierckx, Filip
De
Vos: "In vitro and in vivo evaluation of direct rhenium-188-labeled anti-CD52
monoclonal antibody alemtuzumab for radioimmunotherapy of B-cell chronic
lymphocytic leukemia." (Nuclear Medicine and Biology 35 (2008) 599-604)]. As
shown in Figure 3, the radioimmunoconjugate has preserved an excellent binding
affinity with a value of 30 nM.
