Note: Descriptions are shown in the official language in which they were submitted.
215595~
WO 94/19021 PCT/FR94/00183
-- 1 --
PROTEIN CONJUGATES, COMPOSITIONS CONTAINING THEM AND
THEIR APPLICATIONS AS DRUGS
The present invention relates to protein
conjugates with specific affinity towards appropriate
target cells, and more particularly towards cancer
cells, as well as to their applications as anti-cancer
drugs (cytolytic properties).
The transport of active molecules to specific
biological sites was suggested by Erlich more than 100
years ago. The molecules used are more effective and
have less pronounced side effects when their activity
is manifested only on contact with the target organs or
cells. This is particularly important in carcinology,
where toxic or highly antigenic substances are
administered.
The greatest progress in this field was
obtained with molecules possessing both a non-specific
portion and a portion possessing a particular affinity
for a specific type of cell.
Since the development of monoclonal antibodies
(mca), one of the aims of biotechnology has been the
synthesis of immunotoxins, that is to say mca's
specific for cells of one type, which are coupled to an
agent capable of destroying the said cells. In general,
an mca is prepared so as to have a specificity for an
antigen present at the surface of the target cell, such
that the mca will bind to the target cell, and, when it
is incorporated into the said cell, it will transport
its toxic conjugate into the cell.
The toxic conjugate is usually ricin or the
subunit A of ricin. The subunit B binds to the
galactose residues which are widely present at the cell
surface, while the subunit A enzymatically inactivates
ribosomes. It has been estimated that a single ricin
molecule can inactivate all the ribosomes in a cell.
The invaded cell then dies, since it is incapable of
producing new proteins.
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Besides ricin, other agents may be incorporated
into the immunotoxins; there may be mentioned, in
particular, bacterial or plant toxins (or the active
fragments thereof), radioactive atoms, and agents used
in anti-cancer chemotherapy.
In all cases, the immunotoxins thus formed
direct the active substance (the toxin or the anti-
cancer agent) to the pathological area and thereby lead
to the removal of the target cells.
The specific toxin/antibody conjugates form
very active immunotoxins, but they also possess a non-
specific toxicity which restricts their application in
vivo.
Moreover, toxin fragments, which lack sites
capable of recognizing the intended targets and
conjugated to antibodies, are commonly employed- as
immunotoxins. Such hybrid molecules such as, for
example, those which contain a deglycosylated ricin-A
chain, are thus rendered highly specific. However,
their activity is lower than that of immunotoxins
comprising complete ricin and this activity depends on
the intensity at which endocytosis takes place.
Indeed, translocation of the immunotoxin into
the target cell depends essentially on the surface
antigen recognized by the antibody component of the
immunotoxin.
This is an important step in the action of
immunotoxins which, in a great many cases, does not
lead to endocytosis: consequently, intracellular
translocation of the immunotoxin does not therefore
take place. In such cases, the treatment thus results
in failure. Furthermore, many antigenic labels, present
at the cell surface, are also secreted into the
extracellular medium. The effectiveness of the
immunotoxin is consequently very much reduced on
account of the effect of competition between the
surface antigens and the secreted antigens.
In the particular case of antitumour therapy,
the results obtained using immunotoxins consisting of
21~595~
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cytotoxic substances linked to tumour-specific
antibodies did not prove to be as conclusive as might
be hoped from theory. Indeed, this theory assumed that
these antibodies must have conducted the cytotoxic
molecule into the cell. However, it is apparent that
either the effectiveness of the cytotoxic molecule is
lessened, or the affinity of the antibody is lessened,
or both.
Moreover, the penetration of the cytotoxic
substances into the target cells often proved to be
very problematic and the cytotoxic effect thus very
much reduced.
Furthermore, it has been discovered that many
products obtained by conjugation between antibodies and
such cytotoxic molecules cannot be used as drugs for
reasons of solubility or diverse incompatibilities.
Among other difficulties, the choice of
cytotoxic agent is often problematic. Indeed, this
agent should be relatively non-toxic once conjugated
and should become much more toxic after it has been
transported to its site of action, where intracellular
enzymes release the toxic substance into the tumour
cells.
The objective consequently set forth by the
Applicant was to have available a composition, of
therapeutic aim, capable of acting only on specific
targets (cancer cells) and capable of promoting
endocytosis (stimulation of the transport of substances
across cell membranes).
The subject of the present invention is protein
conjugates, of the type comprising a non-specific
portion and a portion possessing a particular affinity
for target cells of a specific type, characterized in
that the portion possessing a particular affinity for
target cells of a specific type (also referred to as
substance for specific transport to the target cell) is
~-foetoprotein, and in that the non-specific portion of
the said conjugate is chosen from the group which
comprises cytotoxic substances chosen from animal
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toxins, plant toxins, cytolytic enzymes and low
molecular weight anti-cancer active principles and are
selected from carboxyphosphamide, amboclorine,
doxorubicin, bleomycin, cisplatin, vinblastine,
calichemicine and methotrexate or substances which
modify the metabolism of cancer cells, such as
nucleotide sequences, which express a protein and the
anti-sense nucleotide sequences.
Surprisingly, such conjugates retain some of
the properties of ~-foetoprotein and are, in
particular, readily incorporated into cells which carry
the Q'- foetoprotein receptor on their surface, by a
mechanism of endocytosis which stimulates penetration
of the non-specific portion (cytotoxic substance,
nucleotide sequence) and are only active towards
cancerous cell lines such as lymphomas, hepatomas,
neuroblastomas, melanomas, astrocytomas, terato-
blastomas and carcinomas of embryonic, ovarian,
testicular or presacral type.
Equally surprisingly, besides the fact that the
non-specific portion (cytotoxic substance, enzyme,
anti-cancer active principle) and Q~-foetoprotein retain
their effectiveness, the conjugate has an activity with
respect to target cells which is significantly higher
than that obtained with the non-conjugated non-specific
portion.
Another subject of the present invention is
pharmaceutical compositions, characterized in that they
comprise, as active principle, at least one protein
conjugate in accordance with the invention and at least
one pharmaceutically acceptable vehicle.
The ~-foetoprotein - cytotoxic substance
protein conjugates chosen from anti-cancer active
principles are particularly able to be used for the
preparation of a drug intended for use in the treatment
of cancers and especially in the treatment of
leukaemias, lymphomas, hepatomas, neuroblastomas,
melanomas and astrocytomas.
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The ~-foetoprotein-vinblastine, ~-foetoprotein-
doxorubicin and ~-foetoprotein-calichemicine conjugates
are preferably particularly able to be used for the
preparation of a drug intended for use in chronic
myeloid leukaemia.
Apart from the preceding arrangements, the
invention also comprises other arrangements, which will
emerge from the description which follows, referring to
examples of implementation of the process which forms
the subject of the present invention.
It should, however, be understood that these
examples are given solely by way of illustration of the
subject of the invention, of which they in no way
constitute a limitation.
EXAMPLE 1: Production of ~-foetoprotein
a) Preparation:
Amounts of ~-foetoprotein of the order of a
milligram may be prepared by immunoaffinity techniques.
(1) The first step consists therefore in
preparing this protein in a pure enough state and in
sufficient quantity (about 100 ~g) to immunize mice and
prepare monoclonal antibodies which will then be used
to purify the ~-foetoprotein in large amounts in a
single step.
~-Foetoprotein may be prepared from blood of
the human umbilical cord or from cancer cells in
culture (hepatomas, teratocarcinoma). The method of
production consists, in effect, in separating this
protein from the other proteins which accompany it,
according to methods which are standard and known per
se, such as chromatography by ion exchange, by
affinity, ~y exclusion on gel or by hydrophobic
interaction, preparative electrophoresis, dialysis and
ultrafiltration.
In a first step, the starting material, whole
blood or cell suspension, is centrifuged and then
subjected to a dialysis or an ultrafiltration in order
to remove mineral salts and low molecular weight
molecules. The total proteins are then purified by
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chromatography followed by preparative electrophoresis,
so as to avoid any contamination by foreign proteins
and in particular by albumin.
More precisely, 10 ml of human foetal umbilical
serum (12 weeks of gestation) are dialysed against a
pH 7.0 buffer solution (6.5 mM bis-tris propane), for
24 hours, through a regenerated cellulose membrane with
a cutoff threshold of 6000-8000 daltons. The dialysed
serum is then placed on a Cibacron Blue 2-Sepharose~
CL-6B column (Pharmacia) 26 mm in diameter and 70 cm in
length, in the same buffer. The aim of this operation
is to remove the majority of the albumin which remains
bound to the gel.
The eluted proteins are collected and
chromatography is then performed on a Mono Q column
(Pharmacia FPLC System). The elution is carried out by
a gradient of sodium chloride (0-0.5 M) in the same
buffer as above. The ~-foetoprotein is eluted by 0.35 M
sodium chloride. After concentration by ultrafiltration
on PM-10 membrane with a cutoff threshold of 10,000
daltons (Amicon), the proteins are again
chromatographed by filtration on crosslinked agarose
gel (Superose~ 12, Pharmacia), in a column 25 mm in
diameter and 80 cm in length in a pH 7.0 phosphate
buffer. After having standardized the column, the peaks
corresponding to molecular weights of 70 kDa and
140 kDa are collected. These two fractions are combined
and then subjected to preparative electrophoresis on
polyacrylamide gel: the electrophoresis is carried out
without SDS, starting from a zone of concentration gel
(4% polyacrylamide in the concentration gel and 7.5% in
the separation gel). A Tris-HCl buffer pH 6.7 is used
in the concentration gel and a Tris-glycine buffer
pH 8.3 is used in the separation gel.
The sample is diluted by half with Tris-HCl
buffer pH 6.7. One part of glycerol is added per one
part of sample diluted in the buffer, and one-tenth of
the volume to be deposited of bromophenol blue solution
in TrisHCl buffer pH 6.7 is added, in order to
21559~3
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visualize the migration front. The electrophoresis is
carried out at 15C under 30 mA for about four hours.
In order to determine the location of the
~-foetoprotein on the gel after migration, an
electrophoresis of foetal serum is first carried out
against adult serum under the same conditions. The
concentration of ~-foetoprotein in adult blood is
extremely low. Thus, comparison of the two
electrophoreses makes it possible to locate the band
corresponding to ~-foetoprotein between the albumin and
transferrin in foetal blood.
The gel is stained in part with Coomassie blue
and the band, in its non-stained part corresponding to
the a-foetoprotein, is carefully cut out in order to
avoid contamination with the albumin still present at
this stage in the purification. The gel containing the
~-foetoprotein is homogenized in pH 7.0 phosphate
buffer and subjected to agitation in a tube for 1 hour.
After centrifugation, the supernatant is
collected and chromatographed on Cibacron Blue
2-Sepharose~ as above, but in pH 7.0 phosphate buffer.
The proteins not retained by the gel are collected and
then concentrated by ultrafiltration, as above. These
proteins are then used to immunize mice in order to
obtain antibodies which will allow significant amounts
of ~-foetoprotein to be prepared by immunoaffinity.
(2) At this stage, the ~-foetoprotein is used
to produce antibodies of low anti-~-foetoprotein
affinity. This production takes place by injecting
~-foetoprotein into mice, in the presence of Freund
adjuvant. The popliteal lymphatic ganglions are then
removed from these mice and the cells of these
ganglions are fused with myeloma cells in order to form
hybridomas. The period of immunization is deliberately
chosen to be very brief (one week), in contrast with
the usual methods which last for several months.
Moreover, ganglion cells are used here in order to
carry out the fusion instead of splenocytes.
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This method makes it possible to produce clones
of anti-~-foetoprotein antibodies of low affinity.
More precisely, two approximately four-month-
old BALBtC mice are immunized by injection of 20 ~g of
proteins combined with Freund adjuvant, into a hind
foot, twice with a two-day interval, and then a third
time with 50 ~g of adjuvant-free proteins two days
later.
Three days after the last injection, i.e. nine
days in total, the mice are sacrificed and the
popliteal lymphatic ganglions are removed. They are
homogenized and washed with DMEM medium containing no
foetal calf serum, and the homogenate is used to carry
out a fusion with Sp2/o myeloma cells. These mouse
myeloma cells are conventionally used to produce
hybridomas; they have the property of not growing in
HAT medium.
The Sp2/o cells are cultured on DMEM-Hybrimax
medium with 10~ foetal calf serum. The fusion is
carried out in the presence of PEG 3000, at a
concentration of 50~ in DMEM medium from Fl thymocytes
(CBA x BALB/c). The hybridomas are cultured in HAT
medium. The clones appear in 10-15 days in 75~ of the
cultures and correspond to Sp2/o complemented with
immunocompetent cells.
About 40 different clones produce anti-~-
foetoprotein monoclonal antibodies. 9 of them do not
react with normal human serum by the ELISA technique
and 5 do not react by the method of immunological
analysis after transfer (immunoblotting). Two clones
are chosen in order to separate the ~-foetoprotein in
larger amounts; they possess no cross-affinity with
other serum proteins.
to 50 million hybridoma cells are then
injected into a mouse in order to cause it to develop a
tumour producing monoclonal antibodies. Between the
10th and 17th day after the implantation, the ascitic
fluid is removed in order to recover the antibodies.
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The monoclonal antibodies of IGg type are then
purified by chromatography on Protein-A-Sepharose~
(Pharmacia).
5 ml of ascitic fluid are centrifuged at
3000 rpm for 20 minutes, diluted by half with Tris-HCl
buffer pH 8.1 and placed on a Protein-A-Sepharose~
column. The column is washed with 100 ml of Tris-HCl
buffer pH 8.1 and the immunoglobulins are eluted with
pH 6.0 acetate buffer, then pH 4.5 and finally pH 3.5.
The antibodies with low anti-~-foetoprotein affinity of
one of the clones (lOC3) are eluted at pH 4.5, and
another (7H8) at pH 3.5.
The immunoglobulins are then concentrated by
precipitation with 50~ ammonium sulphate, at 4C, for
18 hours and are centrifuged for 30 minutes at
4000 rpm. The centrifugation pellet is redissolved in a
minimum volume of PBS buffer and dialysed against the
same buffer, for 24 hours. 3 to 5 mg of antibody are
thus obtained per 1 ml of ascitic fluid removed from
the mice.
The immunoglobulins thus purified are then
immobilized on a Sepharose~ gel (Pharmacia) activated
with cyanogen bromide according to the method
recommended by the gel manufacturer.
(3) Preparation of the ~-foetoprotein in larger
amounts may then be undertaken by immunoaffinity
chromatography after the antibodies have been
immobilized on a support such as Sepharose~ activated
with protein A, for example, so as to read the fraction
Fc of Igl, Ig2 and Ig4.
This general method makes it possible to obtain
substantial amounts of ~-foetoprotein in high purity.
When prepared under these conditions, this protein
proves to be stable.
More precisely, a column 25 mm in diameter and
50 cm in length is prepared with Sepharose gel on which
the monoclonal antibody as described above is bound.
The column is loaded with 50 ml of human foetal
serum and the ~-foetoprotein is eluted with a phosphate
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buffer in the form of a gradient of pH ranging from 3.5
to 6Ø The ~-foetoprotein is eluted at pH 6.0, in a
proportion of about 90~.
b) Conjugation of ~-foetoprotein with various
active molecules:
~ -Foetoprotein is a glycoprotein. Accordingly,
the two domains of the molecule, proteinic and osidic,
may be used as binding sites for various substances.
Any substance having a chemically active site may, in
principle, be conjugated with ~-foetoprotein. In a
conventional manner, molecules containing thiol
residues may be linked to ~-foetoprotein by a reaction
between amino groups of the protein and activated
carboxylic esters or with imidates. Molecules
containing carboxylic residues are bound particularly
effectively by carbodiimide derivatives. In general,
the binding to osidic residues is carried out by
modification with sodium metaperiodate, especially when
this involves binding molecules containing free amino
residues.
All these processes are already known per se.
It may thus be envisaged to bind a large number
of substances, such as cytotoxic molecules, genetic
material (nucleotide sequences), inhibitors of genomic
replication (anti-sense DNA for example), enzymes or
alternatively fluorescent agents, with the aim of
locating cancer cells under ultraviolet light.
c) Targets of conjugated ~-foetoprotein:
Any cell having the ~-foetoprotein receptor on
its surface is a possible target for conjugated
~-foetoprotein. This receptor is not present on any
normal or non-foetal cell. Only foetal cells and cancer
cells have a proportion thereof which exceeds 90~, in
more than 70 different cases of cancers (R. Moro et
al., Tumor Biol., 8, 293, 1987).
~1~59~3
-- 1 1 --
EXAMPLE 2: Preparation of conjugates in accordance with
the invention
a) a-Foetoprotein-toxin conjugates
- a-foetoprotein (AFP) and diphtheria toxin
(DT).
To 10 ml of an ~-foetoprotein solution
concentrated to 1 mg/ml in PBS buffer are added 100 mg
of SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate)
dissolved in 160 ml of dimethylformamide. The solution
is then dialysed against PBS buffer or chromatographed
on Sephadex~ G-25 (Pharmacia), after equilibrating with
PBS buffer. The protein thus modified is collected and
concentrated by ultrafiltration on a cellulose membrane
having a cutoff threshold of 10,000 daltons.
10 mg of diphtheria toxin are placed in an
aqueous solution containing 50 mM of dithiothreitol and
are incubated for 1 hour at room temperature. The
protein thus reduced is then purified by chromatography
on a Sephadex~ G-25 column equilibrated in PBS buffer,
or dialysed against the same buffer. The reduced toxin
is placed in the presence of the modified
a-foetoprotein and the solution of the two proteins is
concentrated by ultrafiltration on a cellulose membrane
having a cutoff threshold of 10,000 daltons. The
mixture is then left to incubate for 18 hours at room
temperature.
The product obtained is then purified by
chromatography on a Sephacryl~ S-3.0 column
(Pharmacia).
The final yield for the conjugation is 34~.
- a-foetoprotein (AFP) and ricin A (R).
1 mg of a-foetoprotein is dissolved in 1 ml of
PBS buffer and mixed with 6 mg of SPDP (N-succinimidyl
3-(2-pyridyldithio)propionate) in ethanolic solution to
a proportion of 1 mg/ml. The mixture is incubated for
minutes, at room temperature, with constant
stirring. The a-foetoprotein thus modified is dialysed
against a 0.05 M borate buffer, pH 8.5.
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In parallel, ricin A is treated under the same
conditions and in the same amounts as the
~-foetoprotein.
The two modified proteins are then placed
together, concentrated by ultrafiltration on a
cellulose membrane with a cutoff threshold of 10,000
daltons down to a final volume of 5 ml, and are
incubated for 18 hours at room temperature.
The resulting product from the conjugation thus
performed is then purified by filtration chromatography
on Sephacryl~ S-300 gel. The conjugate is recovered in
its pH 8.5 buffer, at the molecular weight
corresponding to the sum of the molecular weights of
the two proteins, i.e. about 102,000 daltons.
The final yield for the conjugation is 42~.
b) ~-Foetoprotein-enzyme conjugates
- ~-foetoprotein (AFP) and asparaginase (Asp).
In order to perform the said conjugation,
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) is
used. Human AFP (Asp:AFP molar ratio of 1:1) is added
to a solution of asparaginase, activated by an excess,
of a factor of the order of 2000, of CMC, at pH 5.4.
The mixture obtained is incubated for 16 hours at room
temperature and dialysed against PBS.
c) ~-Foetoprotein - low molecular weight
biologically active substance conjugates
- ~-foetoprotein (AFP) and carboxyphosphamide
(CFA).
Carboxyphosphamide is the metabolite of
cyclophosphamide, a widely used anti-cancer molecule.
Its metabolite, carboxyphosphamide, cannot penetrate
into cells on account of its high negative charge, and
thus exhibits a low toxicity. If, by means of
conjugation, carboxyphosphamide penetrates into the
cells, it will be cleaved into acrolein and
phosphoramide by phosphoamidases which are particularly
active in cells during proliferation. The phosphoramide
thus obtained is highly cytotoxic.
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The conjugation between ~-foetoprotein and
carboxyphosphamide is carried out at +4C in pyridine
containing 0.01 M HCl and EDC (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide) in an EDC:AFP
proportion of 2500:1. Furthermore, hexamethylene-
diamine (HMDA) is used as intercalating agent. The
final a-foetoprotein-hexamethylene diamine-CFA pro-
portions are 1:10:120. The conjugate which results
therefrom is dialysed against PBS and then stored at
-70C.
The final yield for the conjugation is 72%.
- ~-foetoprotein (AFP) and amboclorine (AC) .
Amboclorine, of formula HOOC-(CH2)3-C6H4-N(CH2-
CH2-Cl)2, may be conjugated with ~-foetoprotein in the
same manner as carboxyphosphamide, in a yield of about
65%.
- ~-foetoprotein (AFP) and methotrexate (MT).
Methotrexate (MT) is dissolved in a pyridine-
HCl buffer, pH 5.0 and the ~-foetoprotein (AFP)
solution is prepared in the same buffer. EDC is added
and the whole mixture is stirred. The MT:AFP:EDC ratio
is 10:1:2500. After stirring for 3 hours, the
preparation is dialysed against a PBS buffer and
diluted in PBS until an AFP concentration of 20 mg/ml
is obtained.
a-foetoprotein (AFP) and doxorubicin (DR).
DR is dissolved in a pyridine-HCl buffer,
pH 5.0 and the ~-foetoprotein (AFP) solution is
prepared in the same buffer. EDC and adipic acid are
added. Adipic acid acts as an intercalating agent
between the AFP and the DR; the DR:adipic acid:AFP:EDC
ratio is 10:50:1:2500.
- Conjugation of ~-foetoprotein with
daunomycin.
Daunomycin has a free amino radical, which
allows direct conjugation with ~-foetoprotein.
Daunomycin is dissolved in pyridine to a
concentration of 10%. A solution of ~-foetoprotein in a
pH 5.0 hydrochloric buffer is added, followed by
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addition of adipic acid as intercalating agent so that
the proportions between the various products are
variable but the ratio between ~-foetoprotein and EDC
is still 1 to 2500.
The yield for the conjugation very much depends
on the various proportions between the products and
ranges from 22~ to 57~.
- ~-foetoprotein (AFP) and rubomycin (RM).
The AFP-RM conjugates are prepared according to
the method used for doxorubicin.
- a-foetoprotein (AFP) and bleomycin (BM).
The AFP-BM conjugates are prepared according to
a method similar to that used for doxorubicin (no
addition of adipic acid); the DM:AFP:EDC ratio is
100:1:2500.
- ~-foetoprotein (AFP) and cisplatin (CP).
AFP is added to a solution of cisplatin and EDC
in a 0.1 M pyridine - HCl buffer, pH 5Ø The reaction
mixture is stirred overnight at 4C or for 2 hours at
20C. The conjugate obtained is dialysed 3 times
against a PBS buffer at 4C, for 24 hours. Samples of
conjugate may be stored in a freezer. The EDC:AFP molar
ratio should not be less than 2000:1.
- ~-foetoprotein (AFP) and vinblastine (VB).
Vinblastine has no free amino group;
consequently, it is necessary to esterify it before
performing a conjugation with AFP.
The esterification is performed, at 37C, by
dropwise addition of 0.1 M KOH to a solution of
vinblastine, until a pH of 9.5 is obtained. The
reaction mixture is neutralized with 0.1 M pyridine -
HCl solution at pH 5Ø The vinblastine thus modified
is conjugated with AFP, according to a method similar
to that described above for cisplatin; however, for the
application of this method to vinblastine, the EDC is
introduced last, since vinblastine interacts with the
amino groups of AFP.
REPLACEMENT SHEET (RULE 26)
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- a-foetoprotein (AFP) and calichemicine (CCh).
In order to obtain better conjugation
conditions, calichemicine is first cleaved, by addition
of dithiothreitol (up to 1 M) and the AFP is modified
with SPDP in PBS, pH 7.2, at 4C, for 12 hours. The
solutions of calichemicine, of EDC and of modified AFP
are mixed together with stirring overnight. The
conjugate obtained is dialysed against a PBS buffer,
for 24 hours. The SPDP:AFP molar ratio depends on the
desired calichemicine:AFP ratio, and should be greater
than the latter ratio by a factor of 8.
EXAMPLE 3: Effects of the various conjugates in
accordance with the invention on h~an cancer cell
lines in culture.
The action of the conjugates was tested on the
following lines;
lymphoma T QOS (QOS), lymphoma T CEM (CEM),
lymphoma B Raji (Raji), lymphoma B Namalva (Nam),
hepatoma HepG2a (Hep), astrocytoma (Ast), melanoma Bro
(Bro), and neuroblastoma IMR - 32 (IMR).
The experimental procedure, common to all the
tests carried out, is as follows: the cells are
cultured in plates containing 96 wells (Linbro~) in an
RPMI medium complemented with 10~ foetal calf serum,
with penicillin (100 units/ml) and with streptomycin
(100 mg/ml), at 37C and under an atmosphere containing
5~ of CO2. The conjugate tested is added to a
concentration of 5 mg/ml and, after 48 to 72 hours, the
proliferating activity is checked.
The abovementioned culture medium may, in
addition, be adapted to each specific culture.
The results obtained are illustrated in
Tables I to XI below, which give the percentage of
surviving cells after 72 hours of incubation with the
cytotoxic substance alone (control) or with a conjugate
in accordance with the invention (test).
- ~-foetoprotein (AFP) and diphtheria toxin
(DT).
The results are illustrated in Table I below:
REPLACEMENT SHEET (RULE 26)
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TA~3LE I
Effect of the AFP-TD conjugate on human T QOS lymphoma
cell lines (~ of surviving cells)
Test Control
20 ~g/ml AFP and 48 58
12 ~g/ml DT
20 ~g/ml AFP and 44 34
24 ~g/ml DT
Control: test performed with DT alone, at the same
concentrations.
- a-foetoprotein (AFP) and ricin A (R).
The results are illustrated in Table II below:
TABLE II
Effect of the AFP-R conjugate on human T QOS lymphomas
(~ of surviving cells)
Test Control
20 ~g/ml AFP and 51 62
10 ~g/ml R
20 ~g/ml AFP and 14 20
20 ~g/ml R
Control: test performed with R alone, at the same
concentrations.
- ~-foetoprotein (AFP) and carboxyphosphamide
(CFA).
A) The cytotoxicity of ~-foetoprotein coupled
to carboxyphosphamide is determined with variable
HMDA-~-foetoprotein proportions of between 0 and 120.
This determination is performed on QOS cells, belonging
to a lymphoblastoid line, by measuring the amount of
tritiated thymidine incorporated relative to the
controls (~-foetoprotein (AFP) alone,
~_ ~15595~
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carboxyphosphamide (CPA) and HMDA alone or as a mixture
in the same proportions as in the conjugate).
1. Without HMDA (% of tritiated thymidine
incorporated):
Concentration CPA:AFP molar Effect Effect
of CPA in the proportion CPA-AFP CPA
medium
0.75 ~M 10:1 25% 0%
1.50 ~M 20:1 98% 0~
3.75 ~M 50:1 100% 0%
9.00 ~M 120:1 100% 2%
2. With HMDA (% of tritiated thymidine
incorporated):
Concentration CPA:HMDA:AFP Effect Effect
of CPA in the molar CPA-HMDA CPA+AFP
medium proportion AFP
0.75 ~M 120:20:1 97~ 0%
1.50 ~M 50:20:1 100% 0%
3.75 ~M 20:50:1 100% 0%
9.00 ~M 10:120:1 100% 0%
b) The following results are obtained under the
same conditions as those illustrated in Tables I and II
(% of surviving cells):
TABLE III
AFP-CFA CFA AFP
1:120 (0. 8 ~M)
Effect 1 87 144
- ~-foetoprotein (AFP) and amboclorine (AC).
The results are illustrated in Table IV below:
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TABLE IV
Effect of the AFP-AC conjugate on various human cell
lines (~ of surviving cells)
Type AC AFP:AC AC AFP:AC AC AFP:AC
of (60 nM) = 1:5 (120 nM) = 1:10 (240 nM) = 1:20
cell (60 nM) (120 nM) (240 nM)
QOS 95 90 87 70 53 12
Hep 98 97 89 81 80 61
Bro 89 87 89 65 73 48
CEM 90 74 78 60 43 22
- ~-foetoprotein (AFP) and methotrexate (MT).
a) Comparative tests are conducted on QOS cells
(lymphoblastoid line) and normal human lymphocytes. The
conjugates, comprising various proportions of
methotrexate, are added to cultures of QOS cells and to
cultures of normal human lymphocytes in boxes
containing 96 wells (200 ~l of medium). The cultures
are incubated for 72 hours. The living cells are
detected and counted by staining with thiazolyl blue
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide).
A solution of thiazolyl blue at a concentration
of 1 mg/ml is added at the end of the incubation, to a
proportion of 50 ~l per well. Incubation is again
carried out for 16 hours, followed by centrifugation
for 5 minutes at 5000 rev/min. The supernatant is
removed (150 ~l accurately measured) and 150 ~l of DMSO
are added. After complete dissolution of the crystals
of formazan, the O.D. is read at 540 nm. The conjugate-
free cultured control is assigned a value of 100~.
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Concentration Methotrexate- % of % of
of methotrexate AFP surviving surviving
in the mediumProportion QOS cells lymphocytes
51.50 ~M 50:1 60 87
3.00 ~M 100:1 53 82
6.00 ~M 200:1 48 91
12.00 ~M 400:1 45 90
b) The effect of the AFP-MT conjugate is also
studied on other cell lines. The results are
illustrated in Table V below:
TABLE V
Effect of the AFP-MT conjugate on human cell lines
(~ of surviving cells)
TypeMT AFP:MT MT AFP:MT MT AFP:MT
of(24 nM) = 1:2(60 nM) = 1:5 (240 nM) = 1:20
cell (24 nM) (60 nM) (240 nM)
QOS 94 7589 24
Raji91 7680 72
CEM 89 33 65 31 53 40
Nam 88 29 75 56 57 52
Bro 98 81 90 55 61 50
Ast 93 90 83 41 40 35
- ~-foetoprotein (AFP) and doxorubicin (DR).
a) Comparative tests are conducted on QOS cells
(lymphoblastoid line) and normal human lymphocytes
exactly as for methotrexate.
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Concentration Doxorubicin- ~ of ~ of
of methotrexate AFP surviving surviving
in the mediumProportion QOS cells lymphocytes
51.50 ~M 50:1 22 66
3.00 ~M 100:1 6 74
6.00 ~M 200:1 3 56
12.00 ~M 400:1 2 71
b) The effects of the AFP-DR conjugate have
also been studied on other cell lines; the results are
illustrated in Table VI below:
TABLE VI
Effect of the AFP-DR conjugate on human cell lines
(~ of surviving cells)
TypeDR AFP:DR DR AFP:DR DR AFP:DR
of(24 nM) = 1:2(48 nM) = 1:4 (60 nM) = 1:5
cell (24 nM) (48 nM) (60 nM)
QOS 87 1785 10 80 4
Raji99 23
CEM 95 4982 27
Nam 90 2581 6 65 3
IMR 95 8385 53 60 42
Bro 91 5595 80 71 36
Hep 104 8195 80 71 30
Ast - - 87 70 55 14
- ~-foetoprotein (AFP) and daunomycin (DN).
The test is performed on the same cells and
under the same conditions as for ~-foetoprotein
conjugated with carboxyphosphamide (Test a).
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1. Without intercalatinq aqent:
Concentration DN:AFP Effect Effect
of DN in the molar DN-AFP DN
medium proportion
0.15 ~M 10:1 75~ 5
0.30 ~M 20:1 75~ 26
0.75 ~M 50:1 100~ 37~
1.50 ~M 100:1 100~ 100%
1.80 ~M 120:1 100~ 100
2. With adipic acid as intercalatinq aqent:
Concentration DN:AFP Effect Effect
of DN in the molar DN-AFP DN
medium proportion
0.15 ~M 10:50:1 75~ 5
0.75 ~M 50:120:1100~ 37
1.80 ~M 120:20:1100~ 100
1.80 ~M 120:50:1100~ 100
1.80 ~M 120:120:1100~ 100~
1.80 ~M 120:250:1100~ 100%
- ~-foetoprotein (AFP) and rubomycin (RM).
The effects obtained with the AFP-RM conjugate
on human T QOS lymphoma cells are illustrated in
Table VII below:
TA~3LE VII
Effect of the AFP-RM conjugate on human T QOS lymphomas
~ of surviving cells)
3 0 Type RM AFP: RM RM AFP: RM RM AFP: RM
of (24 nM) = 1:2 (60 nM) = 1:5 (120 nM) = 1:10
cell (24 nM) (60 nM) (120 nM)
QOS 106 64 64 44 74 20
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- ~-foetoprotein (AFP) and bleomycin (BM).
The results are illustrated in Table VIII
below:
TABLE VIII
Effect of the AFP-BM conjugate on human cell lines
(~ of surviving cells)
Type of cell AFP:BM = 1:100 AFP BM
(120 nM)
QOS 37 137 67
Hep 45 103 56
Ast 29 124 44
- ~-foetoprotein (AFP) and cisplatin (CP).
The results are illustrated in Table IX below:
TABLE IX
Effect of the AFP-CP conjugate on human cell lines
(~ of surviving cells)
TypeCP AFP: CP CP AFP: CP CP AFP: CP
of(24 nM) = 1:2(48 nM) = 1:4 (60 nM) = 1:5
cell (24 nM) (48 nM) (60 nM)
Ast120 76108 60 98 48
IMR99 36 65 42 65 40
QOS108 56 98 50 83 42
Bro97 78 90 72 65 51
CEM99 58103 49 80 40
- ~-foetoprotein (AFP) and vinblastine (VB).
The results obtained on astrocytoma cells are
illustrated in Table X below:
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TABLE X
Effect of the AFP-VB conjugate on human astrocytoma
cell lines (~ of surviving cells)
Type VB AFP: VB VB AFP: VB VB AFP: VB
of(24 nM) = 1:2 (48 nM) = 1:4 (60 nM) = 1:5
cell (24 nM) (48 nM) (60 nM)
Ast42 24 41 26 38 20
- ~-foetoprotein (AFP) and calichemicine (CCh).
The results obtained on various human cell
lines are illustrated in Table XI below:
TABLE XI
Effect of the AFP-CCh conjugate on human cell lines
(~ of surviving cells)
Type CCh AFP CCh: AFP: CCh AFP: CCh AFP: CCh AFP:
of ~24 CCh (48 CCh (60 CCh (240 CCh(24 CCh
cell pM) 1:2 pM) 1:4 pM) 1:5 pM) 1:2nM) 1:2
(24 (48 (60 (240 (24
2 0 pM) pM) Pm) pM) nM)
QOS 99 38 98 40 100 37 15 919 9
Raji - - - - - - 58 4121 10
CEM - - - - - - 60 35 21 17
Bro 100 55 95 38 90 30
Incubation for 48 hours.
EXAMPLE 4: Inhibitory effects of the various conjugate~
in accordance with the invention on the proliferation
of various leukaemia cells.
I - Effects of the AFP-DR and AFP-VB coniuqates
on various leukaemias in man.
a) Equipment and method:
After withdrawing the blood (5-7 ml), the
samples obtained are incubated at 37C for 30-45 min,
in order to allow erythrocytes to sediment out. The
plasma is removed and the amount of lymphocytes is
evaluated. The plasma is transferred to an RPMI medium
-~ 2~55~53
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in the presence of various antibiotics (penicillin,
gentamycin). The amount of lymphocytes should not
exceed 2 x 106/ml. When there are large amounts of
leukaemia cells in the sample, bovine foetal serum is
added.
The proliferative activity of the cells is
measured by a standard method on plates using tritiated
thymidine after 24, 48 and 72 hours.
b) Patients:
Patient 1: chronic myeloid leukaemia,
Philadelphia chromosome + (9-year-old child),
Patient 2: chronic myeloid leukaemia,
Philadelphia chromosome - (69-year-old woman),
Patient 3: acute leukaemia (10-year-old girl),
Patient 4: primary lymphosarcoma.
c) Results:
The results obtained with the AFP-DR and AFP-VB
conjugates, in the four abovementioned patients, are
illustrated in Tables XII-XIV below:
TABLE XII: Patient 1
The tests, with Patient 1, were performed on
lymphocytes and on plasma, after 24 and 48 hours.
Total Lymphocytes Total Lymphocytes
plasma (24 h) plasma (48 h)
(24 h) (48 h)
DR 5.3~ 6.1% 7.0~ 8.1%
AFP:DR 1:50 2.9~ 2.2~ 1.8~ 3.9
VB 65.5~ 52.1
AFP:VB 1:10 48.3~ 43.8~
These results show that after incubation for
24 hours with the 1:50 AFP-DR conjugate, a decrease by
a factor of the order of 35 is observed in the
proliferative activity, whereas DR alone only results
in a decrease by a factor of the order of 20 (total
plasma).
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In the case of the lymphocytes, the inhibitory
effects are respectively of a factor of the order of 45
and 16.
After incubation for 48 hours, the effect is
even more pronounced.
The inhibitory effect of the AFP-VB conjugate
proved to be less pronounced, but the test was
performed with very low concentrations of VB.
The tests performed with Patient 2 were carried
out solely on plasma.
TABLE XIII: Patient 2
Total plasma
24 h 48 h 72 h
DR 58~ 70~ 33
AFP:DR 1:5047~ 55~ 21
VB 68~ 55~ 31%
AFP:VB 1:1053~ 37~ 17~
The inhibitory effect of the conjugates is less
pronounced than for Patient 1; this is due to the type
of leukaemia and to the patient's age and previous
treatments.
The tests with Patients 3 and 4 were performed
on whole blood, after incubating for 72 hours.
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TABLE XIV
Patient 3 Patient 4
DR 14~ 78
AFP:DR = 1:50 7~ 48
CCh 29~ 92
AFP:CCh = 1:10 8~ 45
VB 10% 88
AFP:VB = 1:10 7~ 62~
For Patient 3, the inhibitory effect of the
conjugates is significantly more pronounced than that
obtained with DR or CCh, alone.
II - Effects of the AFP-CCh coniuqates on mouse
leukaemias.
A P388 or L1210 leukaemia is transmitted by
injection of 107 suitable cells to male DBA mice
weighing about 18 g. On the second day after the
injection, when the tumour has a size of about
0.01 cm3, the treatment is established and comprises
the SC injection of an AFP-CCh conjugate (AFP:CCh ratio
of 1:4), in accordance with the invention or of
calichemicine alone (CCh at a dose of 0.6 ~g/kg).
The anti-cancer preparations are introduced,
subcutaneously into the region of the tumour, or
intravenously (once/day for 7 days). A first group of
animals serving as a control received no preparation; a
second group of animals also serving as a control
received 0.2 ml of 0.9~ NaCl solution subcutaneously.
Each group is made up of 6 animals. The increase in
lifespan is calculated according to the following
formula: ~ILS=100. (T/C-1), in which T represents the
average survival time in days of the experimental group
and C represents the average survival time in days of
the control group. The size of the tumour is also
monitored. The volume of the tumour is calculated
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during the experiment, according to the following
formula: V=1/2 L.W2, in which L represents the length
of the tumour and W represents the width of the tumour.
The results are illustrated in Tables XV, XVI and XVII
below:
TABLE XV
Type Agent Route
of Of Volume cm3
tumour admin.
0 3rd 5th 7th 9th
day day day day day
Control - 0.01 0.18 0.61 1.05 3.1
0.9~ s.c. 0.01 0.10 0.30 0.83 2.81
NaCl
CCh i.v. 0.01 0.12 0.74 1.14 3.42
P388 CCh s.c. 0.01 0.08 0.19 0.42 1.21
AFP- i.v. 0.01 0.09 0.34 1.13 2.6
CCh
AFP- s.c. 0.01 0.08 0.14 0.27 0.81
CCh
control - 0.01 0.05 0.19 0.22 0.6
0.9% s.c. 0.01 0.09 0.15 0.31 0.56
NaCl
CCh i.v. 0.01 0.02 O.lS 0.30 0.52
L1210 CCh s.c. 0.01 0.03 0.08 0.12 0.29
AFP- i.v. 0.01 0.04 0.11 0.20 0.28
CCh
AFP- s.c. 0.01 0.03 0.10 0.09 0.16
CCh
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TABLE XVI
Leukaemia Agent Route of Life ~ ILS
admin. span
Control - 20 0
O.9t NaCl s.c. 21 7
CCh i.v. 21.5 7.5
CCh s.c. 24.5 22.5
P388 AFP-CCh i.v. 19 -5
AFP-CCh s.c. 27.8 39
control - 11.5 0
PBS s.c. 13 13
CCh i.v. 12.5 8.7
L1210 CCh s.c. 17 48
AFP-CCh i.v. 14 21.7
AFP-CCh s.c. 19.5 69.5
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TABLE XVII
Agent Dose number of Life ~ ILS
~g/kg mice span
Control0.9~ NaCl 30 7 0
CCh 0.9 6 9.2 31.4
AFP-CCh 0.9 6 10.5 51.8
CCh 2.7 6 9.7 38.6
AFP-CCh 2.7 6 8.5 21
CCh 8 6 9.5 35.7
AFP-CCh 8 6 11.5 60.7
CCh 24 6 6 -14
AFP-CCh 24 6 5.7 -18.6
Table XV shows that, both in leukaemia P388 and
in leukaemia L1210, a significant decrease in the
volume of the tumours is observed in the animals
treated. These results confirm the value of the
conjugates in accordance with the invention. Table XVI
shows the value of the SC administration of a conjugate
in accordance with the invention.
AS regards leukaemia L1210, the effect of
various concentrations of calichemicine alone or in the
form of a conjugate was tested (concentrations from
O.9 llg to 24 ~g); Table XVI shows that the best results
are obtained using low concentrations of cytotoxic
agent.
III - Effects of the AFP-VB coniuqates on mouse
leukaemia.
A P388 or L1210 leukaemia is transmitted by
injection of 107 suitable cells to male DBA mice
weighing about 18 g. On the second day after the
injection, when the tumour is about 0.01 cm3 in size,
the treatment is established and comprises the SC
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injection of V~3 or of AFP-V~3 conjugate (VL at a dose of
200 ~g/kg, the AFP:V~3 ratio being 1:10) in the region
of the tumour (once/day for 7 days). A first group of
animals serving as a control received no preparation; a
second group of animals also serving as a control
received 0.2 ml of 0.9~ NaCl solution subcutaneously.
Each group is made up of 6 animals. The increase in
lifespan is calculated using the equation
~ILS=100. (T/C-1), in which T represents the average
survival time in days of the experimental group and C
represents the average survival time in days of the
control group.
The results obtained are illustrated in Table
XVII below:
TALLE XVII
LeukaemiaAgent Route Life ~ ILS
span
Control - 20 0
o.s~ NaCl s.c. 21 7
P388 VB s.c. 20 0
AFP-VB s.c. 23.8 19
control - 11.5 0
0.9~ NaCl s.c. 13 13
L1210 VB s.c. 18.8 63.5
AFP-V~3 s.c. 22 91.3
This Table XVII shows that the two types of
leukaemia are treated both with ~3 alone and with the
conjugate in accordance with the invention; however,
the ~ILS is significantly greater for the AFP-~3
conjugate than for V~3 alone.
As regards leukaemia L1210, the effect of
various concentrations of ~3 alone or of AFP 3
~ . 21559~3
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conjugate were tested. The treatment procedure is
similar to that described above; however,
concentrations of between 7.5 ~g and 200 ~g/kg were
tested, as was the peritoneal route.
TABLE XVIII
Agent Dose Number Life % ILS
~g/kg of mice span
Control0.9% NaCl 30 7 0
VB 7.5 6 8.8 25.7
AFP-VB 7.5 6 10.5 50
vn3 22 6 8.5 21.4
AFP-VB 22 6 11.3 68.6
vn3 67 6 9.2 31.4
AFP-VB 67 6 12 71.4
VB 200 6 9.7 38.6
AFP-VB 200 6 13.2 88.6
This Table XVIII shows the significantly better
results obtained with the conjugates in accordance with
the invention, especially at doses of 200 ~g/kg.
As emerges from the preceding text, the
invention is in no way limited to that of its modes of
implementation, of production and of application which
have just been described in greater detail; on the
contrary, it encompasses all the variants thereof which
may occur to those skilled in the art, without
departing from the context or the scope of the present
lnventlon .
