Note: Descriptions are shown in the official language in which they were submitted.
~05001~
BACKGROUND OF THE INVENTION-
Several anti-cancer drugs have attained a certain degree of success
during recent years. Amongst these there may be specifically mentioned
compounds such as daunomycin, adriamycin, methotrexate, mithramycin, cytosine
arabinoside, 6-azauridine and the like. All these are low-molecular-weight
compounds and these can be conjugated to form novel drugs according to the
invention.
One of the drawbacks of the said anti-cancer drugs is a comparatively
high degree of toxicity of same. Thus it is hard in some cases to use
adequate dosages as these are too toxic to the mammal being treated.
During recent years various attempts have been made to prepare specific
an~ibodies towards tumors, but these have not given hitherto the desired
anti-cancer effects. Certain antibodies selective to tumors have been
complexed in a non-covalent manner to antitumor drugs in order to enhance
the effectiveness o~ such drugs against tumor cells. These too have not
given the desired results.
SUMMARY OF THE PRESENT INVENTION: :
The present invention relates to novel pharmaceutically active
compositions of matter. More particularly, the present invention relates
to novel compositions of matter comprising low-molecular-weight anti-cancer
drugs chemically linked to antibodies selective or specific to tumor antigens.
The present invention also relates to pharmaceutical compositions of matter
-for use in the treatment of ~ammals afflicted with various types of malig-
nant growths, comprising as active ingredient the novel compositions of
matter according to the present invention. The invention further relates
to a method of treatment of mammals afflicted with tumors, so as to alleviate
the said affllction or to cure same. Other and further aspects of the inventionwill become apparent hereinafter.
According to the present invention specific cytotoxic effects can be
attained by resorting to novel compositions of matter comprising anti-cancer
drugs bound covalently to antibodies selective or specific to tumor antigens.
The antibodies are chosen according to the specific cytotoxic effect
desired. Experiments have shown that a variety of tumor-specific anti-
bodies can be effectively chemically linked to anti-cancer drugs. The
latter are generally compounds of a comparatively low molecular weight.
The antlbod1es are of a much higher molecular weight, mostly of the order
of about 150,000, or a multiple (five units) of this molecular weight.
The effectiveness of the resul~ing composition of matter depends to a
large extent on the nature of the chemical bond used. This has to be
adjusted from case to case, and it is clear that in both components functional
groups have to be chosen which are not necessary for the activity of the
component. The ant~-cancer drug may be chemically bound to the antibody
either directly, or via a linking agent, such as a bivalent lin~i!"g agent
. ~
~ - 3 ~ 1 0 5 0 0 1 2
of the type of glutaraldehyde or the like.
~ hen the direct linkage is resorted to, this can be effected by various
chemical processes, such as the opening of a bond within the small molecule
(generally the anti-cancer drug), and linking it to the antibody. The
methods of linking desired chemical compounds to antibodies are quite well-
known from literature. It is emphasized that in the preparation of compo-
sitions of matter according to the present invention the method of chemical
linkage must be chosen in a careful manner, as great differences result
from varying p~ocedures. It has been fo~nd that the specificity of the
antibodies to tumor antigens can be maintained in the final product. Further-
more, the specificity of the antibodies results in a substantial concentra-
*ion of the novel products in the tumors or their immediate vicinity,
enhancing the resultant effects and substantially decreasing the overall
dosage necessary for cytotoxic effects. Thus, in certain cases where no
adequate dosage of antitumor drugs could hitherto be used, this becomes
possible by means of the novel products of the invention.
With daunomycin and with adriamycin, the method of choice of linkage
to specific immunoglobulins is the periodatc oxidation of the drug, followed
by the linking of the oxidized drug to the immunoglobulin, followed by the
stabili~ation of the resulting bond thrcugh the reduction of the product
with a suitable reducing agent, such as sodium borohydride. With metho-
trexate the method of choice was a linkage in the presence of l-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride with a suitable antibody.
The novel drugs according to the present invention can be used against
a wide variety of tumors, against which the anti-cancer drugs forming part
thereof are nowadays used. The novel drugs can also be used against other
tumors, against which the said drugs cannot be used because of the large
doses required when they are used as such, due to their comparative toxicity.
Amongst types of tumors which can be treated with the novel drugs of the
present invention, there may be mentioned: acute ly~phatic leukemia, acute
myelocytic leukemia, lymphom&, carcinoma of the breast, carcinoma of the
bladder, carcinoma of the testis, osteogenic sarcoma, soft tissue sa~coma
and similar malignant growth types. - -
-3(a)
lOSOO~
Any suitable warm-blooded animal can be used for the production
of the antibodies. For example, there may be used any of the conven-
tional laboratory animals, such as rabbits, rats, dogs, etc. There
may be used larger animals, such as rabbits, horses, goats and the like.
For the treatment of human malignancies, tumor antigens from humans have
to be used; such antibodies being formed in hetero-species such as horse,
goat or rabbit. Such antibodies are chemically bonded to the desired
specific drug and formulated into pharmaceutical preparations by
conventional techniques. The production of such antibodies is carried
out according to conventional techniques, and these are well known to
persons versed in the art.
Various types of bonding may be utilized. The preferred are chemical
bonds of the covalent bond type. These may be either direct bonds, or
they may be effected via suTtable bivalent of other linking agents. The
covalent bonds are stable ones, and such bonds are also stable inside
the body of the patient treated.
The novel drugs are advantageously administered by injection. The
dosage has to be varied according to the type of drug used and according
to the specific use. With various laboratory animals it was found that
an average dosage of about 1 to 5 mg of drug-antibody conjugate was
necessary for achieving significant curative results. The dosage to
be used for humans is thus of an order of about 50 mg to about 500 mg
per unlt dosage. The drug-antibody conjugates are prepared in the
form of conventional injectable pharmaceutical compositions of matter,
wlth suitable adjuvants and/o- carr;ers.
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~ 4 ~ 1 0 ~ 0 1 Z
DESCRIPT_ON OF THE PREFE~RED EMBODIMENTS:
Daunomycin and adriamycin were covalently bound to im~nunoglobulin
isolated from specific antitumor sera by periodate oxidation, ~resumably
cleaving the bond between C3 and C4 of the amino-sugar moiety of the
molecule, resulting in the formation of carbonyl ~roups capable of reacting
with free amino groups of the protein, and the resulting Schiff base l;nk-
age was reduced with sodium borohydride. About 40 mg/ml of the drug, in
1 ml PBS, was mixed with a slight molar excess of 0.1 M sodium periodate
and incubated for about 1 hour in the dark at ambient temperature. 1
glycerol was added t~ a final concentrat~on of 0.05 M to consume excess
of periodate. The solution of the oxidized material was mixed with 1 ml of
tumor-specific immunoglobulin, 20-25 mg/ml of 0.15 M potassium carbonate
buffer, pH 9.5 and incubated again for 1 hour at ambient temperature.
Sodium borohydride was added to a final concentration of 0.3 mg/ml, and the
reaction was allowed to proceed for 2 hours at 37C. Free and bound drugs
were separated by gel filtration chromatography, using Bio-Gel P-100 or
Sepharose 6B. Small quantities of the free drug were removed from the ~ro-
tein fractions by adsorption chromatography on Poropak Q. The protein-
bound drugs passed through the column unretarded. About 2 to 5 moles drug
per mole of antibody were bonded covalently.
Conjugates were prepared with various immunoglobulins, specific to
various tumors. Amongst these were three mouse lymphoid tumors. The
conjugates were tested for their toxic effects on various tumor target cells,
measured by the inhibition of RNA synthesis or by their reduction of the
growth of the tumor cells after transplantation. The novel drugs according
to the present invention preferentially attack target cells recognisable
by the antibody part of the drug conjugate.
Tumors. Several murine lymphoid tumors were used in this study. These
include a carcinogen-induced B cell leukemia In SJL/J mice (Nature 241
(1973) 396), a Maloney virus ~nduced lymphoma (YAC) in A/J mice (J. Nat.
Canc. Inst. 32 (1964) 547) and a mineral oil induced plasmacytoma (PC5) in
BALB/c mice (J. Nat. Canc. Inst. 25 (1960) 847). In addition a lymphoma
induced in Lewis rats by the intrathymic injection of murine radiation
leukemia virus was also used. This rat lymphoma shares viral related cell
surface antigens with the PC5 plasmacytoma but not with the other mouse
tumors employed here. All tumors were maintained ~y passage in their
respective inbred animal strains.
Antisera. Antiserum to bovine serum albumin (anti-B~A) was produced
in rabbits by weekly subcutaneous injection of 2 mg BSA emulsified in
complete Freund's adiuvant.
Rabbit antisera to the B leukemia cells and to the PC5 cells were
prepared by 4-5 intravenous injections of 108 tumor cells at 5-day intervals.
-- 5 --
lOSOOlZ
Antibody activity was measured by complement~dep2ndent cytotoxicity as
previously described tCancer Research 35, 1175 (1975). Titers of 1/100 to
1/200 were obtained for these antisera against their respective immunizing
cells. The anti-B-leukemia antisera showed similar cytotoxicity against the
YAC tumor cells. The anti-PC5 antisera were used after absorption with nor-
mal BALB/c thymus and spleen cells, and in the absorbed form were cytotoxic
against both the immunizing PC5 cells and the rat lymphoma cells, but not
against the YAC cells.
The immunoglobulin fractions of these antisera were prepared by
precipitation with ammonium sulfate at 33% saturation, and used for the
preparation of drug conjugates.
Drug Activity. The pharmacologic activity of daunomycin and adriamycin
was measured primarily by their inhibition of cellular RNA synthesis. The
assays were carried out in microtiter plates (Cooke, V bottom plates) in
Eagle's minimal essential medium containing penicillin and streptomycin.
Cells were suspended in medium at a concentration of 2 x 107 cells/ml and
dispensed into the wells of the plates in 50 ~l aliquots. Drugs were
diluted in PBS (0.15 M NaCl - 0.01 M P04, pH 7.2) and then added to the cells
in 50 ~l amounts. The plates were incubated for 2 hours (unless otherwise
stated in the text) at 37C in a humidified atmosphere of 5% C02 in air.
At that time 10 ~l containing 1 ~c of 5-[3H] uridine was added to each well
and, after another 1-2 hours of incubation, 25 yl of 25% trichloroacetic
acid (TCA~ was added and the plates were placed at 4C overnight. TCA
precipitates were washed, solubilized in NaOH, and transferred to vials for
counting as descrlbed in Transplantation 13 (1972) 541-5. The scintillation
mixture consisted of a toluene based scintillation solution, triton x-100,
0.1 N HCl in the proportions of 6/3/1. The HCl was included to counteract
chemiluminescence. Assays were performed in triplicate, which generally
had less than 10% varîation. The experiments demonstrated that drug act~vity
was retained after the conjugation to the antibody molecules, as shown in
Table 1.
Pharmacologic Effects of the Druq Con~u~ates. The specific cytotoxicity
of drug-immunoglobulin conjugates was tested after allowing them to attach
to target cells during a short incubation in vitro, washing to remove non-
spec~fic proteins and the~r drug conjugates, and examining the cells for
residual drug effects. Tumor cells were washed and suspended in Eag7e's
medium at a concentration of 2 x 107 cells/ml, and dispensed into the wells
of microtiter plates (Cook, V bottom plates) in 50 ~l volumes. Various
concentrations of free drug, immunoglobulins or drug-immunoglobulin conjugates
were added in 50 ~1 volumes, and after agitation (Cook AM 69 microshaker)
the plates were incubated for 5 minutes at 37C. One hundred ~1 of medium
, . ;- ~. . . . .
- 6 - 10~012
TABLE 1
~ytotoxic activity of drug-conjugates compared to ~ree-drug
.
I b ti ti % Inhibition o~ [3H]:uridine incorporation
(min) Free daunomycin : :.... :Bound daunomycin . . . . . . . .
-
Daunomycin-anti BSA Daunomycin-anti B
leukemia
............... : ... :..... :... :.. :.. .... ::. .. ...... .. . .... .
43 20 25
58 - 35 . 35
61 39 48
. 120 66 53 57
240 83 . 76 . 89
....... '...... :.. :... :.. .. :..... .. ....... .. ... ... . . .'
107 B le~kemia cells were incubated at 37C in the presence of 4 ~glml of
daunomycin, either free or protein-bound. Incorporation of~3H~uridine into
ce~lular material precipitable by TCA was measured and expressed as percent
inhibition ~100-percent of control culture, containing no drug).
'. .
.
:. ~ , , .
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- 7- 105001Z
was then added to each well, and the plates were centrifuged at 4 C at
1800 rpm ~International P~-J centrifuge equipped with Cook plate carriers)
for 10 minutes. Supernatants wcre removed by a single shake of the inverted
plates, and the wells were refilled with 200 ~1 of fresh medium. This
washing procedure was repeated one more time, and the cells were finally
resuspended in Eagle's medium, 100 ~l~well, and incuba$ed for 2 hours at
37C in a humidified atmosphere of 5% C02 in air. Ten ~1 containing 1 ~Ci
of [3H3 uridine was then added to each well, and after a further 1 hour
incubation 25 ~1 of 25% TCA was added. TCA precipitates were washed, solu-
bilized in NaOH, and counted for radioactivity as described by Rosenberg
et al., Transplantation 13 ~1972~ 541-5~. The results are expressed as
percent inhibition of [3H]uridine incorporation, compared to the control
which contained either saline or free antibody at a concentration equivalent
to the antibody concentration of the corresponding conjugate. The variation
of triplicates in this assay was generally less than 10%.
Tn addition to the [3H]uridine incorporation assay, the target tumor
cells were tested fortheir ability to grow after transplantation. After
exposure to the drug conjugates in vitro and washing, the cells were
transplanted into their respective syngeneic strains, and the survival of
recipients was followed-
Specifîc Cy~o~oxicity of Daunomycin Anti-B-leukemia Conjugates.
I
Daunomycin was conjugated both to anti-B-leukemia and anti-BSA immunoglobulins,
and tested for cytotoxicity against the B leukemia cells as well as several
other tumors'în vitro. These conjugates retained approximately 50% of the
activity of the free drug. The different tumors used here had similar sen-
sitivities to the drug immunoglobulin conjugates. For the present experi-
ments a concentration of daunomycin-immunoglobulin conjugate was used which
gave 4~-60% inhibition of ~3H]uridine incorporation in test cells when it
was left in contact with the target cells for the enti~e period of the
incubation. To re~eal the'specificity of the conjugates, the test cells
were exposed to them for only 5 minutes, to allow attachment of specific
antibody, then washed to remove non-specific immunoglobulins, and the toxicity
bf the daunomycin remaining in contact with the cells was assessed as des-
cribed above.
~ rom the results shown in Table 2, it can be seen that the daunomycin-
anti-B-leukemia conjugate showed significant residual inhibition of ~3H~
uridine incorporation in the B-leukemia cells after this brief exposure
and washing. When different target cells were tested in this assay we'found
that their sensitiYity to the'specific daunomycin-anti-B-leukemia conjugate
followed the specificity of the antibody. That is, this conjugate was toxic
to the cross-reacting YAC cells but not b the non-cross-reacting PC5 or
rat lymphoma cells tline' l~, e~en though the sensitivity of these cells to
lOSOO~Z
TABLE 2
Specific cytotoxicity of daunomycin linked to anti-B-leukemia
.... .. ................................. . .. . . ..
~ . . .
...% Inhibition.of [ H]uridine incorporationa
Incubated with .. : ................ Test.cells. .. . . ........ Mean survival
tilne
... . ..... :..... B.leukemia... YAC.... ....PC5....... .Rat.lymphoma (days)b
....
Daunomycin-anti-
B-leukemia 38a 42 . 17 gb 19.4
Daunomycin--anti-
BSA lb ob ~ - 12.2
Daunomycin-anti-
BSA
anti-B-leukemia 18c od N,D. N.D. 12.2
Free daunomycin17 49 33 41 10.7
PBS 11.. 3
aO.6 ~g drug either as the protein conjugate or free drug was incubated with 106cells in a total volume of 100 ~l for 5 minutes at 37C. Medium was then
removed, cells were washed and resuspended in fresh medium and pulsed with
~ 3H~uridine at the end of two hours of further incubation.
Differences between a and b p < .001 by the Student'sTTest.
Differencebetween a and c p < .05.
M.fference between a and d p < .001.
bAnimals were injected with 107 cells which were previously trea~ed by brief
exposure to the various drug-conjugates. : -
.
- g - lOSOO~Z
the free drug was, if anything, greater than that Qf the B leukemia cells
~line 4). The specific effect observed here depended upon the antibody
activity since no effect was seen with the daunomycin-'anti-BSA conjugate
tline 2). In the case of the B leukemia test cells, the effect of the
daunomycin-antibody conjugate was even greater than that of the free drug
(line 4, column 1). Free antibody did not render the cell more sensitive
to the effects of the free drug (data not shown), but increased slightly
the effect of the non-specific daunomycin-anti-BSA conjugate on these
cells (column 1, line 3). The heavy agglutination of the cells caused by
the antibody may have resulted in some trapping of the daunomycin anti-BSA
conjugate making it more difficult to remove by washing. YAC cells, which
were not strongly agglutinated by the anti-B-leukemia antibodies were not
affected by the mixture of anti-B-leukemia and daunomycin-ant;-8SA (lir.e 3,
column 2).
In addition to their effects on RNA synthesis, the conjugates were
tested for their effects on tumor cell growth. After brief exposure of
the B leukemia cells in vitro to the conjugates, free antibodies or free
drug, t'hey were washed and transplanted in the syngeneic SJL/J host (Table 2,
last column). The mean survival of animals receiving untreated cells was
11 days. None of the other control groups, which received cells exposed
to free drug, free antibody or the mixture of anti-B-leukemia antibody
and daunomycin-anti-BSA, differed in their survival from those receiving
untreated cells. Only the group receiving cells exposed to the specific
taunomycin-anti-B-leukemia conjugate showed prolonged survival. Their
survival was equivalent to that of animals receiving only 103 untrea~ed
cells.
''Specific Cytotoxic Effects of Daunomycin-anti-PC5 Conjugates. Similar
experiments we~e performed using daunomycin conjugated to anti-PC5 imlluno-
globulins. Once again it can be seen CTable 3) that the specific conjugate
showed toxicity against the homologous PC5 target cell and against the
cross-reacting rat lymphoma cells, but much less against the non-cross-reacting
YAC cells. In this series of experiments the daunomycin-anti-B-leukemia
conjugate was included as a specificity control, showing the converse pattern
of toxic effects. Again, the homologous system, daunomycin-anti-PC5
against the PC5 test cell, was even superior to the effect of free d~ug on
the same cells.
When the growth of the treated PC5 cells was examined in syngeneic
BALB/c animals, we found a slight effect of free drug as well as of free
antibody, but the specific daunomycin-anti-PC5 conjugates resulted in an
even greater effect, including failure of tumor take in over 50% of the
animals (last column of Table 3}. The survi~al of ~his group of animals
was equi~alent to animals receiving only 102 untreated tumor cells. The
,,,,~ .
-- 10 --
~osoo~z
TABLE 3
Specific cytotoxicity of daunomycin linked to anti-PC5 immunoglobulms
% Inhibition of [3H~uridine incorporationa
Mean survival
Incubated with ;Test cells time
PC5 Rat lymphoma YAC
Daunomycin-anti- a b 3 mice ~6~
RPC5 60 63a 20 2 mice 27.5
Daunomycin-anti-
BSA 7b 14b N.D. 19.8
Daunomycin-anti- b
B-leukemia 16 14b ~2a
Free daunomycin 32c 53 67 25.6
PBS 21
al.5 ~g of drug was used, other conditions were identical to those in Table 1.
All a tifferent from all b by p < .001, Student's T test.
a different from c by p < .001
bAnimals we-:e injected with 107 cells which were previously treated by brief
exposure to the various drug conjugates.
.
, .
.. .
~0012
long term survi~ors were resistant to subsequent challenge with 103 tumor
cells.
In an additional series of experiments, the cells were treated briefly
in vitro as before, but transplanted without washing, relying on dissipation
of drug and non-specific drug conjugates in the animal. The results are
similar. An additional control group in which cells were exposed to-a
mixture of free drug and anti-PC5 antibody, resulted in no apparent effect
over that of either the free drug or the free antibody alone.
In the present series of experiments it was shown that daunomycin
covalently bound to antibodies directed against individual t~nors showed
preferential cytotoxicity against these specific tumor cells. When the drug
w~s bound to anti-B-leukemia antibodies, the conjugates were toxic to the
homologous B leukemia cells as well as to the cross-reacting YAC cells
~Table 2), but were not significantly toxic to the non cross-reacting PC5
or rat Iymphoma cells tTables 2 and 3).
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