U.S. patent application number 10/766057 was filed with the patent office on 2004-09-23 for receptor binding conjugates.
Invention is credited to Henriksen, Gjermund, Larsen, Roy H..
Application Number | 20040184990 10/766057 |
Document ID | / |
Family ID | 19904060 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040184990 |
Kind Code |
A1 |
Larsen, Roy H. ; et
al. |
September 23, 2004 |
Receptor binding conjugates
Abstract
The present invention relates to a receptor binding conjugate
which consists of an antibody, a radionuclide and folate or a
folate derivative, wherein or not the conjugate possesses dual
binding ability. The present invention also relates to a method and
a kit to prepare, as well as a method to use, such conjugates.
Furthermore, the use of a conjugate according to the present
invention to prepare a pharmaceutical solution is disclosed.
Inventors: |
Larsen, Roy H.; (Bekkestua,
NO) ; Henriksen, Gjermund; (Mjondalen, NO) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
19904060 |
Appl. No.: |
10/766057 |
Filed: |
January 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10766057 |
Jan 28, 2004 |
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09731301 |
Dec 5, 2000 |
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6740304 |
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Current U.S.
Class: |
424/1.49 ;
530/391.1 |
Current CPC
Class: |
A61K 51/0459 20130101;
A61K 51/1093 20130101; A61K 51/0493 20130101; A61P 35/00 20180101;
A61K 51/1045 20130101; C07K 16/30 20130101; C07K 2317/76 20130101;
C07K 2317/21 20130101; C07K 2317/40 20130101; C07B 59/008
20130101 |
Class at
Publication: |
424/001.49 ;
530/391.1 |
International
Class: |
A61K 051/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 1999 |
NO |
19995978 |
Claims
What is claimed is:
1. Receptor binding conjugate, characterized in that it consists of
three components; (1) an antibody or antibodies preferably of IgG
or IgM class, or fragments or constructs (e.g. minibody) thereof,
(2) a radionuclide or a mixture of radionuclides and (3) folate or
a folate derivative, wherein or not the conjugate possesses a dual
target binding ability.
2. Receptor binding conjugate according to claim 1, characterized
in that the antibody or antibodies are polyclonal IgG without dual
binding ability.
3. Receptor binding conjugate according to claim 1, characterized
in that the radionuclide can be selected from the group consisting
of alpha emitters, beta emitters, gamma emitters, position emitters
and/or x-ray,
4. Receptor binding conjugate according to claim 1, characterized
in that the radionuclide is an alpha emitter, such as .sup.211AT,
.sup.212Bi, .sup.213Bi, .sup.212Pb, .sup.225Ac, .sup.223Ra,
.sup.224Ra or .sup.227Th.
5. Receptor binding conjugate according to claim 1, characterized
in that the radionuclide is an beta emitter, such as .sup.131I,
.sup.90Y or .sup.153Sm.
6. Receptor binding conjugate according to claim 1, characterized
in that the radionuclide is .sup.211At or .sup.125I.
7. Method to prepare a receptor binding conjugate, characterized in
that it consists of three components; (1) an antibody or antibodies
preferably of IgG or IgM class, or fragments or constructs (e.g.
minibody) thereof, (2) a radionuclide or a mixture of radionuclides
and (3) folate, wherein the method uses standard procedures for
radionuclide labelling and folate labelling of the antibody.
8. Method to prepare a receptor binding conjugate, characterized in
that it compromises preparing a conjugate consisting of 1-50 folate
units attached to an antibody, fragment or construct, labelled with
any radionuclide useful for radiotherapy, with or without using a
linker between the radionuclide and the antibody, fragment or
construct, and performed so that the radiolabelling may be carried
out before, simultaneous, or after the folate labelling of the
antibody, fragment or construct.
9. Method according to claim 8, characterized in that the
radionuclide can be selected from the group consisting of alpha
emitters, beta emitters, gamma emitters, positron emitters, and/or
x-ray.
10. Method according to claim 8, characterized in that the
radionuclide is an alpha emitter such as .sup.211At, .sup.211`Bi,
.sup.213Bi, .sup.212Pb, .sup.225Ac, .sup.223Ra, .sup.224Ra or
.sup.227Th.
11. Method according to claim 8, characterized in that the
radionuclide is an beta emitter such as .sup.131I, .sup.90Y, or
.sup.153Sm.
12. Method according to claim 8, characterized in that the
radionuclide is an emitter useful for radioguided surgery, e.g.
.sup.125I.
13. Method according to claim 8, characterized in that the antibody
or antibody fragment is a polyclonal human antibody, or polyclonal
antibody from other species.
14. Method according to claim 8, characterized in that the antibody
or antibody fragment is a murine or chimeric or fully humanized
monoclonal antibody with or without binding affinity towards an
antigen other than the folate binding protein.
15. Method according to claim 8, characterized in that the antibody
or antibody fragment itself has binding affinity towards an antigen
other than the folate binding protein, i.e., the conjugate may
thereby have dual-receptor affinity when folate is conjugated to
it.
16. Use of a folate-antibody-radionuclide conjugate according to
claim 1, to prepare a pharmaceutical solution suitable for
injection or infusion into mammals including humans.
17. Use of a folate-antibody-radionuclide conjugate according to
claim 16, to prepare a pharmaceutical solution suitable for
injection or infusion into mammals including humans by intravenous,
and/or regional, and/or intratumoural route of administration.
18. Method to use a conjugate described in claim 1, to target cells
expressing the folate binding protein by means of injection or
infusion into mammals including human subjects for the purpose of
imaging the receptor containing cells (tissues).
19. Method to use a conjugate described in claim 1, to target cells
expressing the folate binding protein by means of injection into
human subjects for the purpose of delivering potentially
therapeutic radiation to malignant cells (tissues) expressing
folate binding receptor(s).
20. Method to use a conjugate described in claim 1, to target cells
expressing the folate binding protein by means of injection into
human subjects for the purpose of delivering potentially
therapeutic radiation to malignant cells (tissues) expressing
receptor(s) for folate when the malignant tissue is brain-, lung-,
cervix-, ovary- or breast cancer.
21. Use according to claim 16 in combination with folate- and
radiolabelled IgG, IgM or fragments thereof and several
radioimmunoconjugates and/or other forms of radio pharmaceutical
therapy, chemotherapy, external beam therapy, or surgery to treat
malignancies expressing folate binding protein.
22. Kit for the preparation of the folate labelled antibody or
antibody fragment according to claim 1, comprising a vial
containing a folic acid solution and a vial containing a coupling
activating agent (e.g. 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide) in solution, which can be used by combing the two
vials content prior to adding it to a solution containing the
antibody or antibody fragment.
23. Kit according to claim 22, for the preparation of folate and
radioisotope labelled antibody or antibody fragments comprising two
vials and with a third vial containing a solution with the
radioisotope or a ligand, that is either prelabelled or can be
labelled subsequently with the radioisotope that can be attached by
means of covalent binding or chelation to the antibody or antibody
fragment.
24. Preparation of pharmaceutical solutions suitable for
radiotherapy or radiodetection based on the dual-binding-ability
principle where the active component is a conjugate consisting of
(1) IgM, IgG or fragments and constructs (e.g., estrogen or
derivative or testosterone or an derivative) with receptor affinity
other than that of the antibody itself.
Description
[0001] The present invention relates to a receptor binding
conjugate which consists of an antibody, a radionuclide and folate
or folate derivative, wherein or not the conjugate possesses dual
binding ability. The present invention also relates to a method and
a kit to prepare, as well as a method to use such conjugates.
Furthermore, the use of a conjugate according to the present
invention to prepare a pharmaceutical solution is disclosed.
[0002] The use of folate and folate derivatives to target tumours
expressing folate binding protein (FBP), a
glycosyl-phosphatidyl-inositol- -linked cell membrane protein
involved in cellular uptake of oxidised folates via endocytosis,
has attracted attention among researchers [Kranz et al, 1996; Reddy
et al, 1998; Shinoda et al, 1998; Trippet et al, 1999]. As several
types of human cancer cells have been shown to overexpress FBP,
this receptor may be a possible target for delivery of therapeutic
radioisotopes conjugated with folate. It has been shown that
various types of low-molecular weight folate-chelate-radionuclide
conjugates could be used to target cells expressing FBP both in
vitro and in vivo [Mathias et al, 1998]. However, the
pharmacokinetics may not favour small molecules as they generally
are rapidly eliminated from the body, and there by exposing folate
receptors in the kidneys to higher concentration
radiophamaceuticals. Furthermore, the tumour uptake would be
limited, as the blood concentration in the target tissues decreases
rapidly.
[0003] In a previous study, shinoda et al. (1998) evaluated folate
conjugated bovine serum albumin (BSA) labelled with the
radionuclide indium-111, and found that there was a significant
difference in pharmacokinetics and biodistribution of non-folate
compared to the folate labelled BSA. A high liver uptake and rapid
blood clearance indicated that the folate labelled version of
.sup.111In-BSA was not particularly suitable for radionuclide
delivery to tumour cells expressing folate binding protein.
[0004] The combination of folate-antibody-radionuclide has to our
knowledge not been presented before, although the combination of
folate and antibody has been evaluated for other uses. Kranz, et
al. (1996) conjugated folate to an anti-effector cell antibody with
the intent of (1) binding the folate-antibody to folate binding
protein on the target cells and (2) affecting lysis of the targeted
cells due to the antigen binding portion of the antibody interacted
with an effector cell. However, the FBP receptor system has not yet
been successfully used to target radionuclides to cancer cells.
[0005] In order to target radionuclides to the FBP containing
tumours in locoregional settings in particular, it may be
advantageous to use larger carriers that do not diffuse too rapidly
from the region in which they are injected. Monoclonal antibodies
have been raised against the FBP (Campbell et al., 1991), but the
problem with monoclonal antibodies is that they usually contains
whole protein or peptide sequences from non-human species, and are
therefore to a various degree immunogenic when used in humans. This
may hamper the use of monoclonal antibodies in protocols requiring
repetitive injections because of the appearance of human
anti-mouse-antibodies or human anti-chimeric-antibodies, which
causes immunocomplexation and unfavorable biodistribution of
radioimmunoconjugates (Bruland et al., 1995; Meredith et al., 1993)
Also, monoclonal antibodies and their fragments are usually only
just monovalent or bivalent (i.e. not multivalent) with respect to
antigen combining sites.
[0006] It is therefore the object of the present invention to
provide an effective receptor selective tumour targeting agent
(conjugate) prepared from a carrier molecule (i.e. an antibody), a
radionuclide and folate which is chemically linked to the carrier.
The conjugate should also simultaneously retain a significant
antigen combining ability for the original antigen; i.e. the
conjugate possesses dual binding ability. Furthermore, it is an
object of the present invention to provide a method to prepare and
use such conjugates. These objects have been obtained by the
present invention, characterised by the enclosed claims.
[0007] The present invention relates to a new class of folate
derivatives (conjugates), as well as a method to prepare and use
such conjugates. The conjugate consists of (1) an antibody or
antibodies preferably of IgG or IgM class, or fragments or
constructs (e.g. minibody) thereof, (2) a radionuclide and (3)
folate. According to the present invention, the conjugate contains
only human proteins/peptides and may have increased avidity, due to
the possibility of conjugating multiple folates per antibody, and
thereby increasing the probability of binding the conjugate to the
target cells. The antibody which is used is either an inert
antibody, or a monoclonal antibody with affinity towards a tumour
associated antigen. In the first case, a conjugate with FBP
affinity alone is constructed, whereas in the second case a
conjugate with potentially dual reseptor affinity is made, as the
antigen retains a significant antigen combining ability for the
original antigen, as well as binding ability for folate to FBP. The
principal advantage of dual receptor affinity is that the abundance
of target cells without at least one receptor for the conjugate
will be reduced, increasing the chances of a succesfull targeting.
To our knowledge we are the first to propose the use of
folate-antibody-radionuclide to deliver radiation to tumour
cells.
[0008] The present invention will now be described in more detail,
with reference to figures and examples.
[0009] FIG. 1 Binding assay for tritium labelled folate
(.sup.3H-FA; upper graph) and for folate and iodine-125 labelled
polyclonal human IgG (.sup.125I-FA-HIgG; lower graph),
[0010] FIG. 2 Folate-TP IgG-.sup.125I binding to HELA-S3 cells;
10-50 ng/ml range,
[0011] FIG. 3 Binding assay of Folate-HIgG-.sup.125I on HELA-S3
cells; 1-10 ng/ml range,
[0012] FIG. 4 Folate-TP3 IgG-.sup.125I binding to OVCAR-3 cells;
0,1-20 ng/ml range,
[0013] FIG. 5 Specific conjugate binding to cells,
[0014] FIG. 6 Binding assays of folate labelled Vs non-folated TP-3
IgG monoclonal antibody on OHS cells.
[0015] In order to develop a new class of folate derivatives, i.e.
to create a new compound (conjugate) that could, if preferred,
contain only human protein/peptides, that is multivalent to FBP and
that possesses or does not possesses dual binding ability, the
possibility of using antibodies labelled with folate and
radioisotope were elucidated. The advantages of using antibodies in
such conjugates are twofold: The larger size of the antibody,
compared to small molecular weight compounds, affects the
pharmacokinetics of the conjugates, i.e., if injected intravenously
it causes a slower blood clearance and sustained concentration of
radiopharmaceutical and therefore potential higher tumour uptake;
if injected intracavitary it slows down the clearance of
radioisotope from the region [Larsen et al. 1995]. Dependent of the
degree of folate conjugation (i,e, the avidity, or number of
receptor combining units), this conjugate can be multivalent with
respect to FBP combining binding sites. Furthermore, by conjugating
folate to antibodies having antigen binding ability towards an
antigen different from FBP, a conjugate which can bind to two
different receptors (i.e. have dual binding ability) is created,
For example, if an antibody which is raised against an antigen such
as e.g. the anti-osteosarcoma antibody TP-3, is labelled with
folate and radioisotope, it can bind to osteosarcoma cells
expressing the antigen by conventional antibody antigen
interaction, but also to cells expressing FBP via the folate
groups. This dual binding ability principle may be important
because the cellular expression of FBP, as well as antigen, may
vary in the target cells and subpopulations. Therefore, if the
antibody itself also binds to the tumour cells via a different
receptor, the probability of achieving a therapeutically sufficient
targeting of all of the tumour cells increases. Thus, this
principle can be used for targeting two different cell populations
each present with only one of the receptors, or it could be used to
increase the targeting probability of cells expressing both of the
receptors. This type of conjugate can be used in many ways,
including for the targeting of therapeutic or diagnostic
radioisotopes to cancer cells in vivo.
[0016] The dual binding ability principle may also be used for
other receptor binding molecules than folate E.g. oestrogen- and
testosterone derivatives could be conjugated to antibodies with
affinity for breast or prostate cancer providing a conjugate with
dual binding ability for breast cancer or prostate cancer cell.
[0017] Additionally we have studied the use of monoclonal
antibodies against an osteosarcoma associated antigen in order to
show that when radiolabelled and folate labelled, these antibodies
could posses dual receptor affinity, i.e., affinity for both the
FBP and the osteosarcoma associated antigen.
[0018] When we evaluated folate labelled human IgG radiolabelled
with astatine-211 and iodine-125 we found, surprisingly, that
folate labelled vs. non-folated radiolabelled IgG did not show
significant difference in biodistribution in mice. We therefore
conclude that folate-antibody-radionuclide conjugate can be useful
for in vivo tumour targeting.
[0019] The use of the folate-antibody-radioisotope conjugate may be
exploited both using intravenous injections as well as for
locoregional applications. E.g., conjugates based on short lived
radionuclides (i.e. t.sub.1/2 of a few hours) may be promising for
the treatment of intraperitoneally metastasised ovarian cancer,
since this type of cancer has a high probability of overexpressing
FBP, and since the clearance of antibodies from the region is
relatively slow ensuring a high concentration of
radiopharmaceutical in the tumour affected area.
[0020] The folate-antibody-carrier molecule can be used to
transport different isotopes, e.g., alpha- and/or beta-emitters for
therapy, and x-ray and/or gamma emitters and/or positron emitters
for tumour scintigrapby and positron-emission-tomography (PET),
respectively Halogen, as described in the experimental section, as
well as metal radionuclides, can be conjugated to the
folate-antibody, the metal radionuclides by means of bifunctional
chelators with coupling reactivity towards, e.g. amino groups (e.g.
lysine) in the proteins. Examples of alpha emitters are .sup.211At.
.sup.212Pb (as a chemical linked generator nuclide), .sup.212Bi,
.sup.213Bi, .sup.223Ra, .sup.224Ra, .sup.225 Ac and .sup.227Th.
Examples of beta-emitters are .sup.67Cu, .sup.90Y, .sup.131I,
.sup.153Sm, .sup.166Ho and .sup.186Re. Examples of x-ray emitters
and PET nuclide are .sup.99mTc and .sup.18F. A special application
would be in radioguided surgery e.g. with .sup.125I-labelled
antibody-folate conjugate.
[0021] In general, folate can be coupled to amino groups on ligands
by generating an activated ester in the .alpha.- or
.gamma.-carbonyl position of folate. Several ways of generating
activated coupling reagents from folate exist (Readdy et al.,
1998). Folate conjugated in the .alpha.-position may not show the
same receptor activity as the one conjugated in .gamma.-position.
It is well known that folate can be coupled to antibodies using the
EDC-method (Readdy et al., 1998). This method is not selective for
the .alpha.- or .gamma.-carbonyl position of folate, but the
.gamma.-position is sterically favoured when EDC-method is used.
The conjugation of several folates per carrier molecule would
ensure that at least one .gamma.-conjugated folate is incorporated
(Reddy et al., 1998).
[0022] The methods of generating folate-antibody-radionuclide
conjugates used herein showed that both amine coupling of
radioactive coupling reagent and iodogen labelling of tyrosine
could be used to generate a product with folate targeting
properties. Probably most general methods for antibody
radiolabelling are compatible with folate-labelling. When using
amino coupling reagens the radiolabelling yield may be reduced if
the antibody is heavily prelabelled with folates due to a reduction
in available lysine amines. Radiolabelling can principally be
performed before or after folate labelling of the antibody.
[0023] The folate-antibody-radionuclide conjugate according to the
invention is used to prepare a pharmaceutical solution suitable for
injection or infusion into mammals, including humans. This solution
is administered to the patient in need thereof via all systemic
administration routes known in the art. Examples are intravenous,
regional and/or intratumoural routes. Consequently the preparation
according to the invention is composed of the active conjugate,
optionally adjuvants, pharmaceutically acceptable modifiers,
solvents and vehicles, and comprises injection fluids and/or
infusion fluids suitable for the selected administration route.
[0024] The radioimmunoconjugate according to the invention is
further used in combination with other radioimmunoconjugates,
folate- and radiolabelled IgG and IgM or fragments thereof and/or
other forms of radiopharmaceutical therapy, chemotherapy, external
beam therapy or surgery to treat malignancies expressing folate
binding protein.
[0025] The folate-antibody-radionuclide conjugate is also used in a
method to imageing the receptor containing tissues or cells
expressing the folate binding protein, or delivering potentially
therapeutic radiation to malignant cells expressing the folate
binding protein. Wherein the malignant tissue is selected from the
group comprising e.g. brain, lung, cervix, ovary and breast
cancer.
[0026] In accordance with the invention it is provided a kit for
preparation of the folate labelled antibody or antibody fragment
comprising a vial containing a folic acid solution and a vial
containing a coupling activating agent (e.g.
1-ethyl-3-(3-diethylaminopropyl)cabodii- mide) in solution and
optionally a third vial containing a solution with the radioisotope
or a ligand, either prelabelled or which can be subsequently
labelled with the radioisotope by means of covalent binding or
chelation to the antibody or antibody fragment.
[0027] The present invention also provides a preparation of a
pharmaceutical solution suitable for radiotherapy or radiodetection
based on the dual-binding-ability principle where the active
component is a conjugate consisting of (1) IgM, IgG or fragments
and constructs thereof that is labelled with (2) a radionuclide and
(3) in general a molecule (e.g., estrogen or an derivative or
testosteron or an derivative) with receptor affinity other than
that of the antibody itself.
Best Mode
[0028] The receptor binding conjugate according to the invention
consists of three components; (1) an antibody or antibodies
preferably of IgG or IgM class, or fragments or constructs (e.g.
minibody) thereof, (2) a radionuclide or a mixture of radionuclides
and (3) folate, wherein or not the conjugate possesses a dual
target binding ability, wherein the radionuclide may be selected
from .sup.211At or .sup.125I.
[0029] The method to prepare a receptor binding conjugate
consisting of three components; (1) an antibody or antibodies
preferably of IgG or IgM class, or fragments or constructs (e.g.
minibody) thereof, (2) a radionuclide or a mixture of radionuclides
and (3) folate, comprises using standard procedures for
radionuclide labelling and folate labelling of the antibody.
[0030] Generally the preparation conditions must be optimized for
each antibody labelled because of differences in the number of, and
localization of (e.g. antibody combining sites or the non binding
region) lysine units.
[0031] Pretreatment of the IgG
[0032] The batch of antibody is subject to buffer exchange using a
Sephadex PD-10 column (Pharmacia) pre-equilibrated with the buffer
used for giving the optimal folate labelling conditions for that
particular antibody, (e.g., 0.05 M borate at pH 8.5). The antibody
is eluted through the column and, if required, diluted with buffer
to obtain a concentration suitable for performing the labelling
steps (e.g., 1-50 mg/ml).
[0033] The process which is described in detail in the enclosed
examples, meant to be clairifying but not limiting, consists of the
following steps:
[0034] Folate labelling.
[0035] The general procedure is described in the experimental
section. Firstly, folate is conjugated to the antibody (typically
1-20 folate per antibody). Coupling activated folate in solution is
added to antibody and the and the reaction is left to proceed for
less than 10 s to more than 2 days. The reaction may be quenched
e.g., by adding excess of glysine in borate buffer. The resulting
conjugate is purified using gel filtration separation (size
exclusion e.g., Sephadex G-25 PD-10 column). Secondly, the folate
labelled antibody is radiolabelled with a radionuclide. The final
product is purified by gel filtration using, e.g., a Sephadex G-25
PD-10 column.
[0036] Radiolabelling may be performed subsequent to folate
conjugation. Standard radiolabelling procedures, as described in
detail in the experimental section, can be used, Radiolabelled
conjugates can be prepared with specific activity ranging from less
than 1 Bq/mg to several. GBq/mg of antibody.
EXAMPLES
Example 1
Conjugation of Folate to Human IgG
[0037] Pre-treatment of the IgG
[0038] Commercially available human IgG (HIgG) (Gammanorm:
Pharmacia & Upjohn) at an initial concentration of 165 mg/ml in
buffer of glycine, NaCl and NaAc was used.
[0039] H-IgG was separated from small molecular weight components
by eluting the mixture on a Sephadex G-25 column, (PD-10,
Pharmacia) preequillibrated with PBS.
[0040] The IgG concentration was determined by absorbance readings
at=280 nm using an extinction coefficient for the protein of 224
000.
[0041] The elution profile of purified HIgG on size exclusion HPLC
using TSK-G3000 SW.sub.x1 column (Toso Haas) showed essentially one
peak corresponding to the IgG.
[0042] (HPLC system: Shimadzu, LC-10AT pump, SPD-M10A. Diode array
detector).
[0043] Conjugation of Folic Acid
[0044] To ensure reactivity, a stock solution of folic acid was
prepared by dissolving commercially available folic acid (Sigma) in
dimethyl sulfoxide with a H.sub.2O content less than 0.05%. The
solution was then cannulated into a bottle containing activated 4A
sieves (Fluka) and stored under an argon atmosphere in the dark for
6-10 hours.
[0045] .sup.3H-Folic acid (Amersham Pharmacia Biotech,
Buckinghamshire, England) was included as a tracer in the folic
acid preparation and was added as an aqueous solution of the
potassium, salt of .sup.3H-Folic acid (1% with respect to citric
acid) and thereafter desiccated at 8 mTorr for 3 days. The folic
acid containing .sup.3H-folic acid was thereafter treated as
described above, The folic acid was activated for coupling to the
HIgG by adding 4-10 mol equivalents of
1-ethyl-3-(3-dimethylaminoprop- yl) carbondiimide to the folic acid
solution and incubating for 30 min at ambient temperature.
Thereafter, a 20-60-fold molar excess of the activated folio acid
was added to H-IgG (15 mg/ml) in PBS and allowed to react for 30-60
min. The reaction was quenched by adding 0.2 ml of 0.3 M glycine in
PBS/borate, pH 8.5. The folic-acid HIgG conjugate (FA-HIgG) was
separated from unreacted material using a PD-10 column
preequillibrated in PBS. Individual fractions was assayed for the
presence of HIgG aggregates/dimers/fragments or low molecule or
weight components by size exclusion HPLC.
[0046] The extent of folic acid conjugation was determined by the
.sup.3H-content of the purified FA-HIgG as measured by liquid
scintillation counting (Beckmann LS 6500) combined with the
determination of the protein concentration spectrophotometrically;
the protein concentration was determined by the absorption at 280
nm correcting for the contribution to the absorption that is due to
folic acid at this wavelength as calculated from the extinction
coefficient and the concentration of folic acid obtained from the
.sup.3H measurements.
Example 2
Preparation of .sup.125I-FA-HIgG and .sup.211At-FA-HIgG
[0047] Radioiodination by the Iodogen Method
[0048] FA-HIgG in PBS (typically 2-5 mg/ml) was iodinated by the
Iodogen method using standard methodology (Fraker et al., 1978) The
radiolabelled product was purified by PD-10 and assayed on HPLC
size exclusion column. This study indicated that the yield of
radiolabelled product was similiar if the antibody was labelled
with folate before or after radiolabelling.
[0049] Radiolabelling using the
N-succinimidyl-3[.sup.125I]iodobenzoate (ATE-Method)
[0050] Folate-labelled antibody or antibody alone, at typically
concentrations 1-10 mg/ml in borate buffer (pH 8-9), were added to
a dry vial containing either
N-succinimidyl-4-[.sup.211At]astatobenzoate or
N-succinimidyl-3-[.sup.125I]iodobenzoate. The two labelling
reagents had been prepared as described (Larsen et al., 1994b).
After 15 min. of gentle agitation of the vial 0.3 ml 0.2 M glycine
in borate (pH 8-9) was added and the vial agitated for additional 5
min. Thereafter the solution was transferred to a Sephadex G-25
PD10 column (Pharmacia) and the radiolabelled IgG was eluted using
a PBS buffer (pH 7.4).
[0051] Results: The overall labelling yield were in the range of
35-60% using this method. These procedures were used for TP-3,
Rituximab and human polyclonal IgG as well as TP-1 F(ab').sub.2 and
could be performed in similar fashion with N-succinimidyl-[.sup.211
At]astatobenzoate (Larsen et al., 1994).
[0052] In an experiment to determine protein bound fraction of
.sup.211At and .sup.125I the purified product were evaluated using
methanol precipitation of the protein. More than 97% of the
radioactivity precipitated for the .sup.125I-FA-HIgG,
.sup.211At-FA-HIgG, .sup.125I-HIgG and .sup.211At-HIgG
preparations.
Example 3
Biodistribution of .sup.125I-FA-HIgG and .sup.211At-FA-HIgG Versus
.sup.125I-HIgG and .sup.211At-HIgG
[0053] Methods: A single FA-HIgG preparation with an average
FA/HIgG ratio of 2,3 was used for the preparation of both
.sup.125I-FA-HIgG and .sup.211At-FA-HIgG and for comparison
.sup.125I-HIgG and .sup.211At-HIgG were also prepared as described
in example 2.
[0054] White female Balb/C mice with a body weight in the range of
16-20 g were used in the biodistribution experiment. The compounds
were administered by intraperitoncal injection of 500 .mu.l of the
preparation to each animal. The compounds were administered in
paired label arrangement (.sup.211At-FA-HIgG vs. .sup.125I-HIgG and
.sup.125I-FA-HIgG vs. .sup.211At-HIgG) using approximately 100 kBq
of .sup.211At and 50 kBq of .sup.125I for each animal. Animals were
sacrificed by cervical dislocation and the tissue distribution
determined at 1, 6 and 24 hours using groups of 3 mice at each time
point and performing the experiments in two series, reversing the
label vs. antibody type (with respect to folate). The radioactivity
contents of the tissue samples were measured on a NaI (TI)
well-type detector. Firstly, the window and threshold of the
detector were adjusted in order to measure .sup.211At
disintegrations with no detectable crossover from .sub.125I.
Secondly, after a time period corresponding to 20 half-lives of
.sup.211At, the tissue samples were measured again for
quantification of the .sup.125I content. Samples of radiolabelled
HIgG preparations with a single nuclide and mixtures of the
nuclides were used as references.
[0055] Results: Table 1 shows the distribution ratio between folate
labelled and non-folated radiolabelled HIgG. The ratios were kept
between 0.7-1.9 at all points indicating only minor distribution
changes in a few tissues as the effects of the folate label on the
antibody. No significant effect of the folate label was determined
for the kidneys which is considered a sensitive tissue because of
folate binding receptors. In general, the folate labelling had no
major impact of the biodistribution of radiolabelled antibody
indicating folate labelled radioimmunoconjugates may be useful for
in vivo applications.
[0056] Conclusion: R nal clearance of the folate labelled IgG
appeared to be slow preventing accumulation at folate binding
receptors in the kidneys. Folate labelling also had no major impact
on the biodistribution of radiolabelled HigG in other tissues,
indicating that folate- and radiolabelled IgG could be useful tools
for cancer treatment.
1TABLE 1 Ratio of distribution.sup.1 of folate labeled vs non
folated radiolabeled human IgG performed in "paired label" fashion
using astatine-211 and iodine-125. Tissue 1 h 6 h 24 h Blood 1.2
.+-. 0.3 1.2 .+-. 0.5 0.7 .+-. 0.3 Kidney 1.2 .+-. 0.7 0.9 .+-. 0.3
0.9 .+-. 0.2 Liver 1.5 .+-. 0.2 1.5 .+-. 0.6 0.8 .+-. 0.3 I. P. Fat
1.2 .+-. 0.1 1.1 .+-. 0.4 1.1 .+-. 0.3 Neck 1.9 .+-. 1.2 1.0 .+-.
0.6 1.5 .+-. 0.8 Lung 1.2 .+-. 0.3 1.0 .+-. 0.1 0.8 .+-. 0.1 Muscle
1.3 .+-. 0.1 0.9 .+-. 0.1 0.8 .+-. 0.1 Heart 1.3 .+-. 0.4 1.0 .+-.
0.1 0.8 .+-. 0.3 Sm. Intestine 1.3 .+-. 0.4 1.2 .+-. 0.2 0.9 .+-.
0.1 L. Intestine 1.9 .+-. 0.2 1.6 .+-. 0.2 1.0 .+-. 0.1 Spleen 1.5
.+-. 0.1 1.3 .+-. 0.3 1.0 .+-. 0.2 Stomach 1.3 .+-. 0.1 1.3 .+-.
0.9 1.4 .+-. 1.3 Femur 1.7 .+-. 0.6 1.0 .+-. 0.1 0.7 .+-. 0.1
.sup.1The experiment were performed in two series, 3 animals per
time points in one serie. Combination of radiolabel and antibody
type (folate labeled vs non-folated) was reversed between the two
series.
Example 4
Binding Assay of Folic Acid and Folic Acid Conjugates
[0057] Immobilised folate binding protein (FBP) was used to
investigate the binding characteristics of the folic acid
conjugates. FBP (Sigma) with a binding capacity of 2.5 g folic acid
per milligram protein was immobilised on high protein binding 96
well EIA/RIA assay plates (Costar Corning Inc. NY, USA)
[0058] Investigation of the Immobilisation of Folate Binding
Protein
[0059] FBP was labelled with .sup.125I by the Iodogen method and
the labelled protein was isolated by eluting the reaction mixture
on a PD-10 column. Purified .sup.125I-FBP in concentrations ranging
form 5-20 g/ml in PBS were added in to the 96 well assay plates and
incubated on a rotating rack at 4.degree. C. overnight. The liquid
in the wells were removed (L1) and the wells were thereafter washed
four times with PBS (L2) (room temp.). After this, the wells were
added 2M NaOH and were allowed to stand approximately 1 h at room t
temperature before the liquid was removed (L3). The immobilised
quantities of the various concentrations of quadruplicates of
.sup.125I FBP was determined as the ratio of L3/L1+L2 after
measuring the radioactivity of the respective samples by liquid
scintillation counting. Conditions that resulted in an average of 1
g FBP immobilised on the wells were used in the binding assays of
.sup.3H-FA and .sup.125I-FA-HIgG.
[0060] Binding of .sup.3H-FA to Immobilised Folate Binding
Protein
[0061] Wells were incubated with a solution of FBP in PBS and
washed three times with PBS after removal of the incubation
mixture. One half of the number of coated wells were added a 5 M
solution of folic acid in PBS, the other half were added PBS only.
After 1 h at room temperature, the liquid was removed and
thereafter folic acid in concentrations ranging from 0,1 to 2.8 nM
(6 parallels) containing .sup.3H-FA was added to the wells and
incubated at 4 C overnight. The specifically bound fraction of
folic acid was determined by a similar method as described for
.sup.125I-FBP, correcting for the fraction of .sup.3H-FA retained
in the wells that were preincubated with folic acid.
[0062] Binding of .sup.125I-FA-HIgG to Immobilised Folate Binding
Protein
[0063] Wells were incubated with a solution of FBP in PBS and
washed two times with PBS after removal of the incubation mixture
and subsequently with a solution of HIgG in PBS (10 g/ml). Half of
the number of coated wells were added a 5 M solution of folic acid
in PBS, the other half were added PBS only. After. 1 h at room
temperature, the liquid was removed and the wells were added
.sup.125I-FA-HIgG in concentrations ranging from 0,2(4) to 13,(3)
nM with respect to the conjugated folic acid (5 parallels). The
specifically bound .sup.125I-FA-HIgG was determined as described
above for .sup.3H-FA.
[0064] Determination of Binding Properties
[0065] The specifically bound .sup.3H-FA was plotted against the
concentration of .sup.3H-FA in a Scatchard type plot (FIG. 1). For
.sup.125I-FA-HIgG, a similar plot was made assuming the presence of
at least one binding-reactive folic acid derivative on each HIgG
molecule. The following values were determined from the binding
assay:
[0066] K.sub.a was determined from the plot with .sup.3H-FA:
2.times.10.sup.8
[0067] K.sup.a was determined from the plot with .sup.125I-FA HIgG:
0.5.times.10.sup.8 (It should be noted that a two component plot
could also be a possible solution to the points since if only the
three lowest concentration points are considered a significant
higher K.sub.a-value would be obtained).
[0068] Conclusion: The radiolabelled folate-IgG conjugate showed a
significant binding affinity towards the receptor (folate binding
protein).
Example 5
Antigen Binding Ability of Folate- and Radioisotope Labelled TP-3
IgG and TP-1 F(ab').sub.2 on OHS-cells
[0069] The TP-3 and TP-1 monoclonal antibodies (Bruland et al.,
1986) binds to two different epitopes of an cell-surface antigen
expressed on osteosarcoma cells (Bruland et al., 1988) These
antibodies were used to study the effect of folate conjugation on
the antibodies ability to bind to the antigen in vitro. OHS cells
were grown according to standard methods (Larsen et al., 1994a) but
using 10% fetal calf serum in stead of 13%. Cells were harvested
added DMSO and frozen at -80.degree. C.
[0070] Methods: TP-1 F(ab')2 and TP-3 IgG were folate labelled (as
described above) to obtain folate:antibody ratios of 2:1 and 4:1
respectively. Folate-labelled as well as non-folated versions of
the TP-3 and TP-1 F(ab').sub.2 were subsequently radiolabelled
using N-succinimidyl-3-[.sup.125I]Iodobenzoate as described in
example 1. Frozen batches of OHS osteosarcoma cells which on
average expressed 7.4.times.10.sup.5 antigens per cell (Larsen et
al., 1994a) were melted washed and centrifuged twice with PBS to
remove DMSO and suspended in PBS (containing 1% BSA) to a
concentration of 4 million cells per ml. To 4 ml test tubes were
added 0.3 ml of this suspension. Thereafter 0.5 ng of one of the
four conjugates .sup.125I-TP-1 (Fab').sub.2-folate; .sup.125I-TP-1
F(ab').sub.2.sup.; 125I-TP-3 IgG-folate; was added to the tubes.
Sample tubes were subsequently incubated for three hours,
whereafter the tubes were counted for radioactivity on a NaI
counter, washed and centrifuged three times before cell pellet
associated radioactivity was counted. The Retained cell binding
ability (RCBA) was determined as follows:
[0071] RCBA=cell binding fraction of folate labelled RIC/cell
binding fraction of non-folated RIC
[0072] RIC; radioimmunoconjugate
[0073] Results: The RCBA of .sup.125I-TP-3 IgG-folate vs.
.sup.125I-TP-3 IgG was 0.36 while the RCBA for .sup.125I-TP-1
F(ab').sub.2-folate vs. .sup.125I-TP-1 F(ab').sub.2 was 0.62. This
indicates that although some immunoreactivity was lost, antibodies
were still reactive with the osteosarcoma associated antigen.
[0074] Conclusion: Antibodies labelled with folate can in principle
react with both the antigen and folate binding protein, i.e., such
conjugate possesses dual binding ability
Example 6
Binding of Folate- and Radio-labelled Antibodies to OVCAR-3 and
HELA-S3 Cells Expressing Folate Binding Protein. Demonstrating Dual
Binding Ability Using OHS Cells
[0075] Methods: OVCAR-3 cells (human ovarian carcinoma), HELA-S3
(human cervical carcinoma) were used as folate expressing cells.
The cells were cultured in 75 cm.sup.2 plastic flasks supplemented
with RPMI 1640 Medium supplemented with 10% fetal calf serum,
penicillin, streptomycin and glutamine. 10 days prior to harvesting
the culture medium was replaced with folate-free RPMI 1640 medium
supplemented with 10% calf serum to ensure the expression of folate
binding protein before the cells were harvested. Harvested cells
were added DMSO and frozen at -80.degree. C. Prior to use in the
binding assays the frozen cells were melted and immediately added
PBS containing 1% BSA and washed and centrifuged twice to remove
the DMSO. Suspension of cells in PBS with 1% BSA were kept on ice
during incubation with conjugates. Binding assays were performed as
follows; Stock cell suspensions of OVCAR-3 and HELA-S3 of 1 million
cells per ml were made and 0.5 ml were added to 2 ml test tubes.
The tubes were added various amounts of folate-antibody-.sup.125I.
To determine non-specific binding parallel tubes were added
non-folated antibody-.sup.125I at the same concentrations. The
tubes were incubated on a shaker for 3 hours counted for
radioactivity and washed. Finally the cell pellet associated
radioactivity was counted. The following conjugates were evaluated:
folate-TP-1 F(ab').sub.2-.sup.125I (-2 folate per antibody
molecule, labelled with SIB (SIB=N-succinimidyl-3-[.sup.125I-
]iodobenzoate), 50 MBq/mg); TP-1 F(ab').sub.2-.sup.125I
(SIB-labelled, 90 MBq/mg); folate-TP-3-IgG-.sup.125I (.about.4
folate per antibody molecule, SIB-labelled, 70 MBq/mg);
TP-3-IgG-.sup.125I (SIB-labelled, 120 MBq/mg) folate-HIgG-.sup.125I
(iodogen-labelled, 400 MBq/mg).
[0076] An additional experiment was performed by incubating
folate-TP-3-IgG-.sup.125I and TP-3-IgG-.sup.125I
[0077] with OHS cells and using the data for TP-3-IgG-.sup.125I
incubated with HELA-S3 as non-specific control.
[0078] Results: Preliminary data indicates that there are at least
two types of interaction with folate binding protein, One which has
a high affinity and saturates at low concentration (typically below
50 ng/ml) K.sub.as' in the range of 10.sup.10-10.sup.12 was
determined with the limited data points available at these low
concentrations (FIGS. 2-4). In a one point assay, using 0.1 ng/ml
concentration of folate-TP-3-IgG-.sup.125I (and 0.1 ng
TP-3-IgG-.sup.125I as control of non-specific binding) and
5.times.10.sup.5 OVCAR-3 cells in 0.5 ml, it was found that 67% of
the folate-antibody-radionuclide bound specifically to the cells.
At higher concentrations binding of lower affinity appeared to
occur (FIG. 5). Saturation of the binding did not seem to occur
even for conjugate concentrations in .mu.g/ml range. The
cell-binding appeared to be increasing linearly with increases in
the medium concentration of folate-antibody-radionuclide
conjugates. It should be noted that the specific uptake for
folate-TP-3-IgG-.sup.125I on OHS was similar to that on the HELA-S3
and OVCAR-3 when corrected for non-specific binding (FIG. 5). The
affinity of folate-TP-3-IgG-.sup.125I (4 folates per antibody) was
compared to that of non-folated TP-3-IgG-.sup.125I. (FIG. 6). Both
TP-3 versions had a significant affinity towards OHS cells. The
data indicates a reduction in K.sub.a of about five for the folated
version compared to the non-folated version of the TP-3 IgG
antibody, probably due to modification of lysine in the antigen
combining sites.
[0079] Conclusion: Folate-antibody-radionuclide conjugates show a
significant binding to folate binding protein (FBP) on cells
indicating that these conjugates may be used to target
FBP-expressing tumour cells in vivo. Also, as demonstrated by
specific binding of folate-TP-3-IgG-.sup.125I to antigen-positive
OHS cells as well as FBP-positive HELA-S3 and OVCAR-3 cells,
folate-antibody-radionuclide conjugates can possess dual binding
ability.
LITERATURE
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Preparation and quality control of .sup.211At-labelled and
.sup.125I-labelled monoclonal antibodies. Biodistribution in mice
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Aas, M, De Vos, L and Nustad, K. .alpha.-particle radiotherapy With
.sup.211At-labelled monodisperse polymer particles,
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