Note: Descriptions are shown in the official language in which they were submitted.
.~-..
21 1 675;
NOVEL MONOCLONAL ANTIBODIh:S RECOGNIZING THE
EPIDERMAL GROWTH FACTOR-RECEPTOR, CELLS AND METHODS
FOR PRODUCING THEM AND COMPOSI7.'IONS CONTAINING THEM.
FIELD OF THE INVENTION
This invention is related to the field of monoclonal antibodies and provides a
method for selecting hybrids which produce monoclonal antibodies that
recognize
the Epidermal Growth Factor (EGF) receptor, an antigen present in normal cells
and tumor cells of epithelial origin. These antibodies exhibit superior
properties
and can be used for diagnosis, treatment and research, particularly of
malignant
neoplasms, and other diseases.
This invention is also related to therapeutic and diagnostic compositions
using
these antibodies.
DESCRIPTION OF THE PRIOR ART
The Epidermal Growth Factor (EGF), a 53 aminoacid polypeptide of 6 kD
molecular weight that stimulates epithelial and mesenchymal cell
proliferation,
has been considered one of the growth factors involved in malignant
transformations (Cohen S., Carpenter G., PNAS USA 72, 1317, 1975). This
action is mainly performed via its membrane receptors, a 1,186 amino acid
glycoprotein weighing 170 kD, the Epidermal Growth Factor Receptor (EGF-R)
2o has become object of growing interest in cancer research (Carpenter G.,
Cohen S.,
Receptor Regulation (B caries) vol. 13, 41, Lefkowitz, Ed. Chapman and Hall).
High levels of EGF-Receptor have been detected in malignant tumors of
epithelial
origin such as breast, bladder, ovarian, vulva, colonic, lung, brain and
esophagus
cancers. The role played by EGF and its receptor in regulating tumor growth is
unknown, but it has been suggested that the E(JF-Receptor expression in tumor
cells provides a mechanism for autocrine growth stimulation which leads to
#92o2s ~2
F
,
211 6'53
uncontrolled proliferation (Schlessinger J., Schreiber A.B., Levi A., Liberman
T.,
Yarden Y., Crit. Rev. Biochem, 1983, 14 (2) 93-111).
The presence of EGF-R in tumor cells has proven to be an indication of poor
prognosis in human breast cancer. It has also been considered that this growth
factor receptor could widen the concept of hornzone dependency in breast
cancer
(Perez R., Pascual M. R., Macias A., Lage .A., Breast Cancer Research and
Treatment 4, 189-193,1984).
There are reports of some studies with monoclonal antibodies (MAbs) obtained
against the EGF receptor, specifically selected for their antigenic
recognition
which block the EGF binding to the EGF-R. Preliminary studies with some of
these antibodies have shown that the EGF receptor presents preferential
accumulation in tumors of epidermoid origin. Therefore, potential value is
given
to the application of these monoclonal antibodies in the immunodetection and
inmunotherapy of these tumors (Mendelsohn J., :Masui H., Mac Lead C. Proc. Am.
Assoc. Can Res. 26, 287-294, 1985; Gullick W. J., Marsden J. J., Whitle N.,
Ward
B., Borrow, L. and Waterfield M. D. Cancer Research 46: 285-292, 1985.)
The use of biological response modifiers to design treatment schemes for
different
diseases constitutes a very attractive alternative. Monoclonal antibodies
constitute
a group of biomolecules being used for such purposes (Deusch K., Mauthe B.,
Reiter C., Endres N., Classen M., Riethmuller C., Proceeding of the
International
Congress of Immunology, Budapest Hungary, 11, 1992) Immunotherapy with
monoclonal antibodies is being studied by many researchers to test its
efficacy in
the treatment of different neoplastic localizations. (Frodin J. E., Philstedt
P.,
Lefvert A. K., Mellstedt H. Hybridoma, Vol 7, No 4, 1988). Many modalities
have been proposed, among them: native, conjugated with drugs, bispecific,
anti-
idiotype and human monoclonal antibodies.
#92025 v2 2
211 653
The high EGF receptor expression in poorly differentiated epidermoid tumors
offers the possibility of considering the antibodies as a route for onco-
specific
treatment whether native or associated with drugs, toxins or radionuclides;
(Vollmar A. M., Banker D. E., Mendelsohn J.; Herschman H-R. J Cell Physiol,
1987, 131, 418-425). Clinical trials are in progress for the treatment of
malignant
tumors with these monoclonal antibodies (Magdelenat H., Delarue J. Y., Mady
E.,
Faillot T., Vega F., Poisson M., Proceedings of VI Conference of MESTMO
International Journal of Tumor Markers, Nice, France Nov., 1991.)
The antibodies against the EGF receptor which have been used for treatment
have
been selected primarily for the antigenic recognition, producing tumor growth
inhibition through this action. This inhibition occurs through the mechanism
of
signal transduction in which the EGF receptor is involved. (Mendelsohn J.,
Kawamoto T., Sato G., Sato D., Schlesinger J. Givol D., Kris R., US Appl. No.
23988 EP 0359282).
On the other hand some gangliosides have been considered both as EGF receptor
phosphorylation inhibitors, and cell signal transduction inhibitors resulting
in cell
proliferation inhibition. This indicates that gangliosides could be considered
an
effective treatment for different neoplastic localization specially EGF
receptor
bearing tumors (Davis R. J., J. Biol. Chem. (1990) 265: 12059-12066).
2o Besides the possibility of an autocrine growth control in some tumors,
involving
their own growth factors on proliferation, has been described. This could
suggest
us the use of this mechanism to inhibit tumor l;rowth via monoclonal
antibodies
against growth factors, and growth factor receptors, and consider it as
another
possible therapy for malignant neoplasms (Todaro C. J. et al., Proc Natl Acad
Sci
USA 77; 5258-5262, 1980).
Up to now, no one has evaluated the possibi lity of selecting hybridomas that
produce monoclonal antibodies with the desired characteristics of: antigenic
#9zo25 ~z 3
F
2 '~ 1 6 7 5 3
recognition, complement-mediated or antibody dependent cellular cytotoxicity,
tumor growth inhibition and synergetic effect on proliferation inhibition of
certain
cell lines by the combination with gangliosides or monoclonal antibodies
against
EGF. The only aspect which has been evaluated is the possibility of tumor
growth
inhibition through inhibition of the transduction signal produced through the
EGF
receptor.
In the methods used up to now, hybrids have been selected according to
antigenic
recognition and, moreover, the antibodies have been obtained through
immunizations with the antigen developed from tumor cell lines and have been
1o selected according to anti-tumor activity through this antigenic
recognition,
Researchers involved in this field have not taken into account the possible
antitumor effect that these monoclonal antibodies could elicit through
complement
mediated or antibody dependent cytotoxicity, nor the combination with other
biomolecules such as gangliosides or growth factors or other antibodies
against
growth factors which could induce a synergetic effect for inhibiting
proliferation
in cancer cell lines in combination with the monoclonal antibodies against the
EGF receptor.
SUMMARY OF THE INVENTION
This invention provides monoclonal antibodies and a selection method for
hybrids
2o producing them with qualitatively superior features that may be used to
develop
diagnostic systems, treatment and research.
According to the previous statement, the main goal of this invention is to
provide
monoclonal antibodies having superior properties, which recognize the EGF-
receptor in human placenta or A-431 cells with high affinity, are capable of
inhibiting EGF binding to the receptor, have the ability to produce antibody-
dependent cellular cytotoxicity, have the ability to develop complement-
mediated
cytotoxic activity, which also have the property of inhibiting tumor growth in
#92025 v2
211 653
certain cell lines and show a synergetic effect to inhibit proliferation of
these cell
lines when they are in combination with gangliosides or monoclonal antibodies
against EGF.
Another objective of this invention is to provide a selection method for new
hybrid cell lines that produce the monoclonal antibodies referred above.
Another objective is to provide monoclonal antibody-producing hybridomas which
meet the selection criteria.
One of the most important objectives of this invention is to provide a
pharmaceutical composition containing the monoclonal antibodies against the
EGF receptor and gangliosides or monoclonal antibodies against EGF, that
produces a synergetic effect on inhibiting cell growth.
The importance of this preparation could be of remarkable impact in the
treatment
of lung cancer, any other tumor of epidermoid origin, nervous system origin or
any
other EGF-Receptor bearing tumor.
Therefore, the invention provides the aforementioned selection method for
obtaining the monoclonal antibodies produced in this way and compositions for
diagnosis and treatment.
According to an aspect of the present invention, there is provided a
monoclonal
antibody which recognizes human Epidermal Growth Factor (EGF) receptor
which is capable of inhibiting EGF binding to the receptor, is capable of
inhibiting
growth of EGF-dependant tumor cells and in addition has the following
properties: a) has antibody-dependant cellular cytotoxicity, b) has complement-
mediated cytotoxicity activity, c)is capable of producing a synergetic effect
in
inhibiting proliferation of tumor cells if combined with N-acetyl GM3
ganglioside,
and d) is capable of producing a synergetic effect in inhibiting proliferation
of
tumor cells if combined with a monoclonal antibody against EGF.
#92025 v2
2~ 1 6 ~5 3
According to another aspect of the present invention, there is provided a
pharmaceutical composition for use in a therapeutical or prophylactic
treatment of
malignant neoplasms or preneoplastic diseases, comprising a therapeutically or
prophylactically effective amount of a monoclonal antibody according to any
one
of claims 1 to 9, together with a Garner, a diluent: or an excipient therefor.
According to a further aspect of the present invc,ntion, there is provided a
process
for preparing a monoclonal antibody which recognizes human Epidermal Growth
Factor (EGF) receptor, comprising the steps of immunizing a test animal with
human EGF receptor or an antigenic derivative or fragment thereof, isolating
1o antibody-producing cells from the immunized animal, immortalizing said
antibody-producing cells, selecting from said immortalized cells a cell which
produces a monoclonal antibody which recognizes human EGF receptor, is
capable of inhibiting EGF binding to the receptor, is capable of inhibiting
growth
of EGF dependent tumor cells, and in addition has the following properties: a)
has antibody-dependent cellular cytotoxicity, b) has complement mediated
cytotoxic activity, c)is capable of producing a synergetic effect in
inhibiting
proliferation of tumor cells if combined with N-acetyl GM3 ganglioside, and d)
is
capable of producing a synergetic effect in inhibiting proliferation of tumor
cells if
combined with a monoclonal.
2o According to yet another aspect of the present invention, there is provided
use of a
monoclonal antibody according to any one o~F claims 1 to 9 for preparing a
pharmaceutical composition for therapeutical a prophylactic treatment of
malignant neoplasm or pre-neoplastic diseases.
In a preferred embodiment, this invention consists of immunizing mice with a
partially purified fraction of human placenta epidermic growth factor
receptor;
removing their spleens and preparing a cell suspension; fusing the spleen
cells
with myeloma cells in the presence of a fusion promoter; diluting and
cultivating
the cells; evaluating the supernatant which contains the hybridoma; selecting
and
s~zozs ~z
21~6~53
cloning a hybridoma which produces antibodies with the following
characteristics:
recognition of the EGF receptor, with high affinity, and with the ability to
inhibit
EGF binding to the receptor, the capability of producing antibody-dependent
cellular cytotoxicity (ADCC) complement-mediated cytotoxic activity,
inhibition
of tumor growth in certain cell lines and show a synergetic effect in
proliferation
inhibition of these cell lines in combination with gangliosides or monoclonal
antibodies against EGF; recover the antibody from the supernatant liquid and
collect the malignant ascites or the serum which contains the desired
antibodies.
The selection, preparation and characterization oil the hybridomas and the
resulting
1o antibodies will be better understood with the following description and
examples.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1.- shows the saturation curves obtained during solid phase ELISA for
placental membrane and A-431 cell fractions. The axis of the
abscissa reflects the log of the antibody concentration. An
irrelevant antibody was used as the negative control (antibody
concentrations were 0.5 - 7500 ng/mL).
Figure 2.- shows the displacement curves obtained from Radio Receptor
Analysis. The competence assay was performed using microsomal
fractions of human placenta an<i different concentrations of the
2o antibody and a negative control (anti-cytokeratin monoclonal
antibody). The EGF displacement curve was used as positive
control.
Figure 3.- shows the monoclonal antibody immuno-reactivity assay (dot blot)
with the A-431 cell purified EGF'-R. Aliquots (3.0, 1.0, 0.1 I. pL)
of the purified EGF-R (30 ng/mL) were applied in the
nitrocellulose and incubated with different concentrations of the
antibody.
#92025 v2 '7
X11 6753
Figure 4.- shows a competitive assay among different monoclonal antibodies
against the EGF Receptor, labeled with 125-Iodine.
Figure 5.- shows a competitive assay with a microsomal fraction of human
placenta and different concentrations of the monoclonal antibodies
obtained against the EGF-R. EGF labeled with 125-Iodine was
used as the radio tracer.
Figure 6.- shows the monoclonal antibodies' effect on A-431 cells. The cells
were cultured with EGF (1 ~gr~mL); Gamma-IFN (100 ~g/mL);
EGF + G-IFN; and different concentrations of the monoclonal
1o antibody alone or combined with Gamma-IFN. Determination of
the proteins in the cellular monolayer was performed on the
seventh day of culture.
Figure 7.- reflects the monoclonal antibody's antagonist effect on EGFIs
biological activity. The cells were cultivated with EGF (1 ~g/mL);
Gamma-IF1V ( 100 NtlmL); EGF +G-IFN; and different
concentrations of monoclonal antibody in combination with
Gamma-IFN and EGF. Determination of the proteins in the
cellular monolayer was performed on the seventh day of culture.
Figure 8.- shows the inhibition of EGF-stimulated auto-phosphorylation
2o caused by the monoclonal antibody in placental membrane tissue.
The membranes were incubated with 3zP-ATP in the absence (band
1) or in the presence of EGF (bands 2-8) and were analyzed via
polyacrylamide gel electrophoresis and autoradiography. The
effects of increases in the concentration of the MAb is observed in
the incorporation of Phosphorus-32 in the EGF-R, Bands 2 and 3
without the MAb, band 4 : 1.8 p.g/mL, band 5 : 6 p,g/mL, band 6
#9zozs ~z g
216753
18 ~g/mL; band 7 : 60 ~.g/mL and band 8 : 180 ~ug/mL of the
MAb.
Figure 9.- demonstrates that the Fab Fragments retain the same capacity as the
monoclonal antibody in the A431 cells to inhibit EGF binding to its
receptor. A competitive assay was performed with a microsomal
fraction of human placenta and different concentrations of the
MAb, and its Fab fragment. EGF labeled with 125-Iodine was used
as the radio tracer.
Figure 10.- shows that the Fab Fragments retain the same capacity as the
native
monoclonal antibody in the EGFLs antagonist effect on A431 cells.
The cells were cultured in different concentrations of the MAb and
its fragments. Determination of the proteins in the cellular
monolayer was performed on the seventh day of culture.
Figure 11.- reports the results of the immunohistochemical analysis of breast
cancer. Epithelial cells of invasive ductal carcinoma are heavily
stained.
Figure 12.- refers to the monoclonal antibody's effect on the H-125 cellular
line; accelerated growth of the control group (which doubled its
growth several times) can be obsc;rved. The group treated with the
lowest concentration (10 ~g/mL) shows slightly slower growth than
the control group. Groups treated with higher concentrations
showed some cellular growth during days 1 and 2. Starting on Day
3, the culture entered a stable phase with no growth, by Day 5 there
is a 50 % inhibition in comparison to the control.
Figure 13.- shows the monoclonal antibody's effect on the U-1752 cellular
line.
The groups treated with the lowest concentration ( 10 ~g/mL)
showed no growth differences in comparison to the control. The
#92025 v2 9
F
2'~1s753
p..
groups treated with higher concentrations showed some cellular
growth during the first two days starting on Day 3, the culture
entered a stable phase during which there was no cellular growth by
Day 5, the inhibition was 50 % in comparison to the control.
Figure 14.- shows the cytotoxic cellular effect in the presence of rabbit
complement of the monoclonal antibodies in the H-125 cellular
line.
Figure 15.- describes the effect of the combination of the monoclonal
antibodies at a fixed concentration with two different
concentrations of N-acetyl GM3 in relation to the controls.
Control group No 1 contains an irrelevant antibody with the same
concentration as the monoclonal antibody against EGF Receptor.
Control groups No 2 and No 3 contain the same irrelevant antibody
at the same concentration together with N-acetyl GM3 in two
different concentrations.
Control group No 4 contains the: monoclonal antibody alone at a
concentration of 30 ~g/mL.
Control groups No 5 and 6 contains monoclonal antibody against
EGF Receptor at a concentration of 30 pg/mL and N-acetyl GM3 at
2o concentrations of 2.5 and 5 pg/mL, respectively.
TABLE 1 and 2.- show the monoclonal antibody recognition pattern in
normal and tumor tissue performed in fresh tissue in cryostatic sections.
#92025 v2 10
F
211675
DETAILED DESCRIPTION OF THE INVENTION.
The hybridoma preparation method generally includes the stages described
below,
which includes the methods used for the selection of hybridomas.
A. Mice immunization with a partially purified fraction of human placental
EGF-R. Although it has been verified that female Balb/c mice are best,
other breeds of mice may be used. The immunization program and antigen
concentration must be carefully measured to produce useful amounts of
adequately stimulated splenocytes. One method found to work well was:
4 immunizations at 0, 14, 21 and 28 day intervals with 25 ~g of the
to immunogen per mouse in 0.2 mL of phosphate buffered saline solution,
with complete adjuvant on day 0 and in<;omplete adjuvant on days 14, 21
and 28. The last inoculation was 50 pg applied intravenously, previous to
cellular fusion.
B. Removal of the spleen of each immunized mouse and preparation of a
spleen cell suspension using the adequate: medium. Approximately 50 mL
of medium per spleen is adequate according to known experimental
techniques.
C. Fusion of the spleen cells suspension with murine myeloma cells from an
adequate line under the effect of a fusion agent. The preferred ratio is 10
2o spleen cells per myeloma cell. An approximate total volume of 0.5-1 mL
of fusion medium to every 108 cells is appropriate. Many murine myeloma
cell lines are recognized and available i:rom the academic community or
specialized banks. The cell line used, however, should be of the so-called
"drug-resistant" type so that the fused myeloma cells will not survive in a
selected medium, but the hybrids do. The most commonly used classes are
the cell lines resistant to 8-azaguanine, that lack the hypoxanthine-
guaninephosphor-ilosyl transferase enzyme and, therefore, will not survive
#92025 v2 11
F
2116753
in a HAT medium (hypoxanthine-aminopterin and thymidine). It is also
generally preferable to use a myeloma line of the class known as "non-
secreting", as these do not produce antibodies on their own. If necessary,
however, classes that secrete may also be used.
Although the preferred fusion promoting agent is polyethylene glycol with
an average molecular weight of approximately 1000 to 4000, other
promoters may also be used.
D. Dilute and cultivate in the plate wells, the mixture of nonfused spleen
cells, non-fused myeloma cells and fused cells in a selective medium that
l0 does not maintain the non-fused myeloma cells (i.e. HAT). After a week,
the non-fused spleen cells cease to reproduce. The fused cells, on the other
hand, continue to reproduce as a result of the malignant characteristics
inherited from the parental myeloma and the survival capacity of parental
spleen cells in the selective environment.
E. METHODS OF HYBRIDOMA SELECTION
1. Evaluate the hybridoma supernatant in each well to determine the presence
of
the antibodies which selectively recognize with high affinity the human
placenta
EGF receptor, and the receptor present in the A-431 tumor cell line and these
monoclonal antibodies are also capable of inhibiting EGF binding to the
receptor.
2o This evaluation can be made according to the preferred techniques of either
radio-
receptor or immunoassay using as antigens the cell lines rich in EGF receptor
and
the receptor from human placenta which has been partially purified according
to
affinity purification methods described below.
2. Evaluate the hybridoma supernatant in each well, to determine the presence
of
the antibodies which have the properties of inhibiting tumor growth of lung
cancer
cell lines. Different concentrations of the monoclonal antibody are added to
the
cells in suspension and a comparison is made with control groups, one without
an
#92025 v2 12
2'~ 1 653
antibody and other with an irrelevant antibody, Measurements of protein
concentration are performed with the Lowry Rosebrough method, but any other
method for measuring total protein concentration could be used.
3. Evaluate the hybridoma supernatant in each well, to determine the presence
of
the antibodies which have the properties to produce antibody-dependent
cellular
cytotoxicity, using the standard measurement method for this activity, which
is
carned out through target cells labeled with -'~lCr, effector cells and
different
concentrations of the monoclonal antibody.
4. Evaluate the hybridoma supernatant in each well to determine the presence
of
to the antibodies which have the properties to produce complement-mediated
cytotoxic activity. This is done with standard methods for measuring this
activity,
using target cells labeled with SICr, rabbit complement and different
concentrations of the monoclonal antibody.
5. Evaluate the hybridoma supernatant in each well to determine the presence
of
the antibodies which have the property to produce synergetic effects in cell
proliferation inhibition when they are combined with gangliosides.
A cellular suspension is obtained and resuspended in culture medium and the
gangliosides added at a concentration range of 0.5 -50 microMoles; then the
monoclonal antibodies (supernatant) are added in a concentration range of 10
to
100 micrograms per mL. At the third and sixth day total protein concentration
is
measured. The Lowry Rosebrough method is preferably used, but any other
method could be used.
6. Evaluate the hybridoma supernatant in each well to determine the presence
of
the antibodies which have the property to produce synergetic effects in cell
proliferation inhibition when they are combined with a monoclonal antibody
against EGF.
#92025 v2 13
2116753
A cellular suspension can be obtained and resuspended in culture medium and
the
MAb against EGF is added at an adequate concentration range and the monoclonal
antibodies are added in a concentration range of 1.0 to 100 micrograms per mL.
At
the third and sixth day total protein concentration is measured. The Lowry
Rosebrough method is preferably used, but any other method for measuring total
protein concentration could be used.
F. Select (i.e. via limiting dilution) and clone the hybridoma that produces
the
antibodies.
Once the desired hybridoma has been selected and cloned at least twice, the
l0 antibodies may be produced using either of two methods:
a) The monoclonal antibodies may be produced through in vitro cultivation of
the hybridoma in an adequate medium for the indicated time, followed by
the recovery of the desired antibody from the supernatant. The proper
medium and time are known or easily determined. This in vitro technique
produces an essentially monospecific antibody that is fundamentally
exempt of other anti-human specific immunoglobulins. There is a small
quantity of other immunoglobulins as the medium contains xenogenic
serum (fetal calf or newborn calf serum). This in vitro method can be
scaled up in bio-reactors to obtain the appropriate amounts of the desired
2o monoclonal antibodies.
b) The other production method involves injecting the hybridoma in
preferably syngenic mice. Following an appropriate incubation period the
hybridoma will cause the formation of non-solid tumors that produce
antibodies. These will provide a hi~;h concentration of the desired
antibodies (5 -20 mg/mL) in the bloodstream and the peritoneal exudate
(ascites) of the murine host. Although these hosts also present normal
antibodies in blood and ascites, the concentration of monoclonal antibodies
#92025 ~2 14
".,.,
2'~ 1 653
is higher. Furthermore, as the normal antibodies are not anti-human
specific, the monoclonal antibodies obtained are essentially free of any
anti-human immunoglobulin contaminant. The immunoglobulins
produced by the incorporation of light myeloma chains are non-specific
irrelevant peptides that merely dilute the monoclonal antibodies without
affecting their specificity.
In agreement with this aspect, the invention provides hybridoma cell lines
capable
of producing monoclonal antibodies which recognize the EGF receptor with high
affinity. The hybridoma cell lines are capable of inhibiting EGF binding to
the
1o receptor, and also are capable of producing antibody-dependent cellular
cytotoxicity. The hybridoma cell lines are cal>able of developing complement-
mediated cytotoxic activity, that can inhibit tumor growth in some cell lines
and in
combination with gangliosides or monoclonal antibodies against EGF, produce a
synergetic effect in the inhibition of proliferation.
Said antibodies could be used to develop immuno-detection techniques, develop
new treatments with bio-molecules, study the antigen's molecular topography,
develop anti-idiotypical and humanized antibodies, study their use in
idiotypical
regulation of the immune network and in immuno-purification and structural
characterization of the antigen they recognize.
2o G. OBTENTION OF A THERAPEUTIC COMPOSITION WITH THE
MONOCLONAL ANTIBODIES WHICH HAVE BEEN OBTAINED
WHICH ELICIT INMUNOLOGICAL EFFECT.
Once the purified antibodies have been obtained, a therapeutic composition is
developed which contains an effective quantity of any monoclonal antibodies
produced by the hybridoma obtained through the Hybridoma Selection Method,
and which elicit immunological effect. The antibodies selected for this
condition
were characterized by the complement mediated cytotoxic activity and antibody
dependent cellular cytotoxicity. This was demonstrated using the standard
#92025 v2 15
F
2116753
techniques. The A-431 cell line labeled with SICr was used as target cells.
The
target cells in an adequate concentration were pipetted on microplates with
the
monoclonal antibody. The complement source could be human or rabbit serum in
culture medium. The incubation of H-125 cell lines with the MAb showed in the
presence of complement, a cytotoxic effect . The cell cytotoxicity mediated by
antibodies was carried out using Balb/c mouse peritoneal macrophages as
effector
cells, which allowed the obtention of a cytolytic effect.
In this preparation a pharmaceutical vehicle should be used, which may be any
of
those used in conventional pharmacopeia, or maltose, or trehalose, saccharose
or
polyethylene glycol 800 or the combination of these with the Zn cation, in
different concentrations. This preparation could be from said antibody or from
its
fragments (Fv, Fab, Fab', F(ab')2), or from the humanized, chimeric,
"reshaped",
bispecific antibody or any of the aforementioned antibodies conjugated with
toxins, radionuclides, or any other available drug, or it may be from the anti-
iodiotypic antibodies (AB2, AB-) obtained against said antibody; for the
treatment
or prevention of malignant neoplasms or pre-neoplastic diseases, especially in
central nervous system tumors, tumors of the head and neck, and breast and
lung
adenocarcinomas.
H. OBTENTION OF A THERAPEUTIC COMPOSITION FROM THE
2o MONOCLONAL ANTIBODIES WHICH RAVE BEEN OBTAINED IN
COMBINATION WITH A GANGLIOSIDE.
Once the purified antibodies have been obtained, a therapeutic composition is
developed which contains an effective quantity of any monoclonal antibodies
produced by the hybridoma obtained through the Hybridoma Selection Method,
including an adequate concentration of a ganglioside.
The synergetic effect demonstrated by the combination of these monoclonal
antibodies and different gangliosides has been shown. Different concentrations
of
#92025 ~2 16
~'-v
21 ~ 6~5 3
gangliosides produced growth inhibition of cell proliferation when they are
combined with monoclonal antibodies against the EGF receptor.
A cellular suspension is obtained from H-125 and U-1752 cell lines,
respectively
and is resuspended in culture medium. Gangliosides, in a concentration range
of
0.5 -50 microMoles are added and the monoclonal antibodies against the EGF
receptor are added in a concentration range of 10 to 100 micrograms per mL.
At the third and sixth day total protein concentration is measured by the
Lowry
Rosebrough method (Lowry D.H. 1951 J Biol. Chem. 193:265), but any other
method could be used. A synergetic effect of the combination ganglioside -
t0 monoclonal antibody against EGF receptor is observed in inhibiting
proliferation
of a lung cancer cell line in relation to the controls used in this test.
There was almost 100°Io inhibition of proliferation in relation to the
control; the
effect produced by the combination is greater than the simple additive effect
of
both molecules independently.
Is In this preparation a pharmaceutical vehicle should be used, which may be
any of
those used, in conventional pharmacopeia, or maltose, trehalose, saccharose or
polyethylene glycol 800 or the combination of these with the Zn cation, in
different concentrations. This preparation could. be from said antibody or
from its
fragments (Fv, Fab, Fab', F(ab')2), or from the humanized, chimeric,
"reshaped",
20 bispecific antibody or any of the aforementioned antibody conjugated with
toxins,
radionuclides, or any other available drug, or :it may be from the anti-
idiotypic
antibodies (AB2, AB-) obtained against said antibodies; for the treatment or
prevention of malignant neoplasms or pre-neoplastic diseases, especially in
central
nervous system tumors, tumors of the head and neck, and breast and lung
25 adenocarcinomas.
#92025 v2
F
2116'53
I. OBTENTION OF A THERAPEUTIC COMPOSITION FROM THE
MONOCLONAL ANTIBODIES WHICH HAVE BEEN OBTAINED IN
COMBINATION WITH MONOCLONAL ANTIBODIES AGAINST EGF.
Once the purified antibodies have been obtained, a therapeutic composition is
developed which contains an effective quantity of any monoclonal antibodies
produced by the hybridoma obtained by the Hybridoma Selection Method,
including an adequate concentration of a monoclonal antibody against EGF, and
a
pharmaceutical vehicle, which may be any of those used in conventional
pharmacopeia, or maltose, trehalose, saccharose or polyethylene glycol 800 or
the
1o combination of these with the Zn canon, in different concentrations. This
preparation could be from said antibody or from its fragments (Fv, Fab, Fab',
F(ab')2), or from the humanized, chimerical, "reshaped", bispecific antibody
or
any of the aforementioned antibodies conjugated with toxins, radionuclides, or
any
other available drug, or it may be from the anti-iodidtypic antibodies (AB2,
AB-)
obtained against said antibodies; for the treatment-r prevention of malignant
neoplasms or pre-neoplastic diseases, especially in central nervous system
tumors,
tumors of the head and neck, and breast and lung adenocarcinomas.
EXAMPLE ONE
SELECTION METHODS FOR HYBRIDOMAS.
a) Selection of the Hybridoma According to the Antigenic Recognition
Method.
The hybridoma supernatant in each well was evaluated, to determine the
presence
of antibodies which selectively recognize with high affinity the human
placenta
EGF receptor, and the receptor from the A-431 <:ell line of epidermoid origin
and
is also capable of inhibiting EGF union with the receptor.
After the cellular fusion, the cells were cultured in selective HAT medium
(hypoxanthine, aminopterin and thymidine) at 37%, with a 5% C02, moist
atmosphere.
#92o2s ~2 1 g
211 s~53
Three weeks later, 10 pL of supernatant from the cultures which contain
hybridomas were added to the wells of polyvinyl chloride plates which had been
previously coated with extracts of placental merrabrane or from the A-431 cell
line,
depositated at ATCC- CRL-1555, which contain EGF receptor. Detection of the
antibodies which react with the antigen expressed in these membrane extracts
was
made through a solid phase, indirect, immune-enzymatic assay. The antigen was
immobilized by coating the polyvinyl chloride plates with partially purified,
soluble EGF receptor. HEPES 10 Mm, pH 7.:5 buffer solution was used. The
probe used as a second antibody was peroxidase conjugated anti-mouse antibody
to (Amersham).
Hybridoma cultures which contain antibodies that react to membrane extracts
containing the receptor, are selected and they ~~re cloned twice according to
the
limiting dilution method, in the presence of conditioning cells.
The method has the special characteristic of simultaneously selecting the
antibodies for both antigens: the receptor from human placenta, which
possesses
normal epitopes, and the receptor from the A-431 cell line, which epitopes
appears
during the malignant progression, which mean;> that the epitope selected in
the
antibody by the Selection Method is preserved in the receptor molecules
present in
normal tissues and also appears in most of the receptor molecules present in
tumor
tissue of epidermoid origin.
b) Selection of the Hybridoma According to Anti-Tumor Activity.
The hybridoma supernatant in each well was evaluated to determine the presence
of the antibodies which inhibit tumoral growth in lung cancer cell lines U-
1752
and H-125 (NCI, Bethesda, Maryland). These cell lines-show an EGF dependence
for their proliferation. The culture medium used was RPMI-1640 (Gibco, USA)
supplemented with 5-10% calf serum. The cells were kept in incubators at
37° C
in a humid atmosphere and carbon-dioxide air (95%-5%).
#92025 v2 19
F
~'~'~ 6~5 3
The monoclonal antibody's inhibitory effect was measured in the cells in
trypsin
suspension and re-suspended at concentrations of 2.5 x 104 cells/mL of culture
medium to which different concentrations of monoclonal antibody were added:
10, 30 and 100 ~g~mL. A control group with an irrelevant antibody was also
studied in concentrations of 100 ~g/mL. The cellular suspensions were placed
in
24-well culture plates (1 mL per well), and each sample was tested three
times.
Plates were prepared for each cellular line and analyzed at different exposure
times (1, 3, 5 and 7 days). The determination of the total number of cells was
made according to the quantitative method of total protein determination (D.H.
l0 Lowry).
Inhibition of the H-125 and U-1752 lung cancer cell line proliferation was
produced in 50% in relation to the control, this result can be observed in
Figure
12.
This part of the selection method has the :particular characteristic that the
hybridomas selected produce monoclonal antibodies with a specific recognition
of
the antigen and simultaneously present the ability to inhibit tumor growth.
c) Selection of the Hybridoma According to the Capacity to Produce
Antibody-Dependent Cellular Cytotoxicity.
The hybridoma supernatant in each well was evaluated to determine the presence
of the antibodies which produce antibody-dependent cellular cytotoxicity. A
standard measurement technique for this activity was used. The A-431 cell line
labeled with SICr (100 ~Ci) was used as targets.
The effector cells were blood cells from normal adults prepared with a Ficoll-
Hypaque gradient in which the mononuclear cells from the peripheric blood were
separated, washed three times and adjusted in a effector cell/target cell
proportion
of 200:1.
#92025 v2 2~
z~ls~~3
The target cells in 104 concentration were pi;petted into 96-well plates which
contain 25 ~L of monoclonal antibody (ascitic liquid diluted 1/10). The plates
with the target cells and the antibody were incubated at 4°C for 30
minutes. The
effector cells (100 p,L) were added to the plate and centrifuged immediately
100
xg for 2 minutes. They were then incubated during four hours in a humid
chamber
at 37°C with 5-95°Io COz air. From this experiment it could be
demonstrated an
antibody dependent inhibitory effect of 22%.
In this part of the selection method, monoclonal antibodies producing
hybridomas
have already been obtained which simultaneously possess three properties:
1o antigenic recognition, inhibition of tumor growth and at the same time,
antibody-
dependent cellular cytotoxicity.
d) Selection of the Hybridoma According to the Ability to Produce
Complement-Mediated Cytotoxic Activity.
The supernatant in each well was evaluated to determined the presence of the
antibodies which produce complement-mediated cytotoxic activity, using the
standard measurement method. The A-431 cell line labeled with 5lCr (100
pCi)was used as target cells.
The target cells in a concentration of 104 were pipetted into 96-well plates
which
contain 25 ~L of monoclonal antibody (ascitic liquid diluted 1/10).
2o The complement source consisted of 100 ~L of rabbit serum at 25% in culture
medium. It was incubated for four hours in a humid chamber at 37°C with
5-95°Io
C02 air. The plates were later centrifuged and t:he counts per minute (cpm)
were
measured in 100 ~I. of the supernatant in a gamma counter.
Several working solutions of the antibodies were tested to verify what was the
maximum toxicity value.
#9zozs ~z 21
~~~6~5~
In this experiment a 60% complement mediated inhibitory effect was
demonstrated as it is shown in Figure 14.
e) Selection of the Hybridoma according to the Ability to Produce a
Synergetic Effect in Combination with N-acetyl GM3.
The H-125 cell line expressing 104-105 EGF receptor binding sites and
classified
as lung adenocarcinoma was used as the experimental model for the screening. A
cellular suspension was obtained with 104-105 cells per mL and was resuspended
in culture medium. N-acetyl GM3 is added then at a concentration of 5
microMoles and a monoclonal antibody against the ECF Receptor is added at a
to concentration of 30 micrograms per mL.
At the third and sixth day total protein concentration was measured by the
Lowry
Rosebrough method.
A synergetic effect of the combination N-acetyl GM3-monoclonal antibody
against the EGF receptor was observed in inhibiting proliferation of a lung
cancer
cell line in relation to the controls used in this test. There was almost 100%
inhibition of proliferation in relation to the control. Therefore, the effect
produced
by the combination is much more than the simple additive effect of both
molecules independently.
This result is observed in Figure 15.
2o fj Selection of the Hybridoma According to the Ability to Produce a
Synergetic Effect in Combination With Monoclonal Antibodies Against EGF.
The H-125 cell line expressing 104-105 EGF receptor binding sites and
classified
as lung adenocarcinoma was used as the experimental model for the screening. A
cellular suspension was obtained with 104-105 cells per mL and was resuspended
in culture medium. The MAbs against EGF we added at a concentration of 5
~g/mL and monoclonal antibodies against the EGF Receptor are added at a
#92025 v2 22
211675
concentration of 30 micrograms per mL. At the third and sixth day total
protein
concentration was measured by the Lowry Rosebrough method.
A synergetic effect of the combination MAbs against EGF-monoclonal antibody
against the EGF receptor was observed in inhibiting proliferation of a lung
cancer
cell line in relation to the controls used in this test.
The antibodies produced by the hybridomas based on the selection method used
have superior properties which give them the distinction of: high affinity
antigenic recognition for the EGF Receptor; antibody-dependent cellular
cytotoxicity; complement-mediated cytotoxic activity; the capacity of
inhibiting
the tumor growth in cell lines and a synergetic effect in combination with N
acetyl
GM3 or MAbs against EGF.
EXAMPLE TWO
BIOCHEMICAL CHARACTERIZATION OF THE MONOCLONAL
ANTIBODIES OBTAINED BY THE SELECTION PROCEDURE.
Monoclonal antibodies obtained are IgG2a -type immunoglobulins secreted by the
hybridomas obtained from the fusion of SP2/Agl4 mouse myeloma with Balb/c
mouse spleen lymphocytes immunized with a partially purified fraction of Human
placenta ECF receptor.
The purification of solubilized EGF receptor was performed by affinity
chromatography. Briefly: the placenta crude membrane fractions were
solubilized in Tris-HCl 20 mM buffer, pH 7.4, glycerin 10% and Triton
X-100TM 1% solution during 1 hour at room temperature. The
homogenate was centrifuged at 10 000 g.. The supernatant was applied to
an EGF affinity matrix. The EGF enriched fraction was eluted with
ethanolamine 5 mM buffer, pH 9.7 with glycerin 10% and Triton X-
100TM 0.1% solution. This solubilized EGF receptor-enriched fraction
has the capacity to bind 125I_EGF, and showed a major 170 kD band in
#92025 v2 23
21 1 67~i 3
SDS-PAGE. One hundred grams were processed each time. Average
yield was 15-25 pg of partially purified EGF receptor per gram of wet
tissue.
These monoclonal antibodies possess the following characteristics:
A high binding affinity for the receptor with Kd values of 10-9 M calculated
through the semi-saturated concentration curve obtained, as it is shown in
Figure
1, this affinity is similar to the receptor affinity to EGF.
They are capable of inhibiting the 1251_EGF's binding to the membrane receptor
using a microsomal fraction of human placenta (Figure 2 and Figure 4) it is
1o possible to deduce from its 10-g M Ki values, that the binding site is not
the same
as that of Epidermal Growth Factor, and that the inhibition is provoked by a
conformational or steric effect. These monoclonal antibodies suppress the
EGF's
effect on the A431 cell line, even in the presence of synergetic agents such
as
gamma-IFN (Figure 6 and 7). These MAbs also inhibit EGF-dependent
clonogenic growth of NRK cells and inhibit EGF- receptor dependent auto-
phosphorylation (Figure 8), considered to be a crucial step in the
transduction of
the cellular proliferation signals, which demonstrates the EGF-antagonistic
role
played by these monoclonal antibodies.
Studies that corroborate this effect were carried out with the MAb and its Fab
2o fragment obtained by papain digestion (Figure 9 and 10). This effect proved
to be
dose-dependent. The fragment retains the same; ability to suppress EGF
cellular
proliferation.
The capacity of these monoclonal antibodies for inhibition of the
proliferation of
different cellular lines has also been shown. In the lung cancer cell lines
studied
(H-125 and U-1752) it was found that in concentrations of 10 ~ug/mL of these
antibodies, growth was slightly decreased and that there was no significant
difference in comparison to the control, however, when it was applied in
~zozs ~2 24
'~
_ ~.* 2116753
concentrations of 30 ~g/ml, growth inhibition was achieved in the culture by
day 3
which remained stationary and on the fifth day, an inhibition of 50% compared
to
the control was observed. (Figures 12 and 13)
These monoclonal antibodies are also characterized by complement-mediated
cytotoxic activity and antibody-dependent cellular cytotoxicity.
The incubation of the H-125 cellular line with the monoclonal antibodies
showed,
in the presence of rabbit complement, a cytotoxic effect of 60% at a
concentration
of 12.5 pg/mL (Figure 14). The cellular cytotoxicity mediated by antibodies
carried out using Balb/c mouse peritoneal macrophages as effector cells with
110:1 ratio (effector : target) produced a cytolytic: effect of 22%.
The synergetic effect demonstrated by the combination of these monoclonal
antibodies and different concentrations of gang;liosides, was also shown. This
combination can produce a growth inhibition of cell proliferation. N-acetyl
GM3
was added in a concentration of 5 micromoles and the MAb was added in
concentration of 30 micrograms per mL.
A synergetic effect of the combination N-acetyl GM3-monoclonal antibody
against EGF receptor is observed in inhibiting proliferation of a lung cancer
cell
line in relation to the controls used in this test.
There was almost 100% inhibition of proliferation in relation to control; the
effect
2o produced by the combination is greater than the simple additive effect of
both
molecules independently (Figure 15).
A study of the sequence of the variable regions of the heavy chain of the
immunoglobulins obtained through the Polymerase Chain Reaction (PCR) was
performed: amplifications of at least 2 independent PCR samples and at least
two
independent PUC19 clones were used to determine the sequence. Once the
variable regions of the light and heavy chain were amplified, they were cloned
in
#92025 v2 25
2116~~i
the PUC19 vector sequence and the heavy chain sequence was identified.
According to the Kabat Database, this sequence has been classified of the
miscellaneous group. The re-ordering of the variable regions of the heavy
chain
defines the epitope of the EGF receptor which is recognized by these
monoclonal
antibodies.
EXAMPLE THREE
CHARACTERIZATION OF THE REACTIVITY OF THE
MONOCLONAL ANTIBODIES OBTAINED BY THE SELECTION
PROCEDURE.
l0 Fresh tissues were selected for the study, which included samples of heart,
prostate, liver, lung, kidney, spleen, ovary, pancreas, brain, testis, adrenal
glands,
hypophysis, lymph nodes, medulla, thyroid, tonsils, stomach, small intestine,
colon, skin, submaxillary gland, placenta and cf;rebellum. Several nerve
tissues,
hematopoietic and sarcomatous tumors were also studied.
The fresh tissues were frozen in liquid nitrogen and stored at -
20°.
Histopathological study was evaluated using hematoxylin-eosin stained
sections.
Consecutive sections based on the histopathologically evaluated blocks were
used
for staining with the immunoperoxidase technique.
The tissue reactivity was demonstrated using the biotin-strepto-avidin-
peroxidase
2o complex (Amersham, U.K.).
The study includes sections with positive (skin sections) and negative (tris
buffered saline solution) controls, The reaction with the enzyme produces a
brown
precipitate and is classified as follows:
*no reaction (-);
*mild reaction (+);
*moderate reaction (++);
*intense reaction (+++).
#92025 v2 26
F
211675
Fresh normal tissue was taken from recently performed autopsies and tissue
reactivity
was shown intensively according to the established pattern in pancreas and
testis.
Fresh normal tissue was taken from recently performed autopsies and tissue
reactivity
was shown moderately intense, according to the established pattern, in:
placenta, skin, prostate, liver, kidney, ovary, thyroid, intestine and non-
lymphoid
tonsil tissue. (see Table 1 )
TABLE 1
MONOCLONAL ANTIBODY RECOGNITION IN NORMAL ORGANS
1 O ORGAN REACTIVTY
I
- -/+ + ++ +++
Heart 3/3
Prostate 2/2
Liver 3/3
Lung 3/3
Kidney 2/2-
Spleen 2/2
Ovary 2/2
Pancreas 1I2 1I2
Testis 1 /2 1/2
Adrenal Glands l/1
Hypophysis 2/2
Lymph Nodes 2I2
Medulla 1/1
Thyroid 2/2
Tonsils 3/3
Stomach 2/2
Small Intestine 2/2
Large Intestine 2/2
Skin 2/2
Submaxillary 2/2
Gland
Placenta 111
Brain 1 /
1
Cerebellum 1 /
1
3 5 Legend
no reaction (-);
weak reaction (+);
moderate reaction ( + +);
strong reaction (+ + +).
27
211 67~i3
Epidermoid tumors, and central nervous system tumors such as glioma,
meningioma, malignant histiocytoma, neuro-fibrosarcoma, as well as mammary
and nasopharyngeal tumors have shown an intense reaction, according to the
established pattern, and in much higher concentration than in normal tissues
of the
same origin. (see Table 2)
TABLE 2
MONOCLONAL ANTIBODY RECOGNITION IN TUMORS
ORGAN REACTIVITY
- -/+ ++ +++
Glioma 1/1
Menin ioma 1/1
Mali pant Histioc toma 1/1
Neurofibrosarcoma 1 / 1
Breast cancer 4/14 5/14 5/14
Lun Cancer 3/3
Tumors of the Head and 5I5
Neck
Basal Carcinoma 1/1
Legend
no reaction (-);
weak reaction (+);
moderate reaction (+ +);
strong reaction (+ + +)
Detection only occurs in tissue containing the receptor, whichch demonstrates
that
the recognition is specific to the molecule's epitope, and therefore its
private
nature, as well as the absence of related epitope;s in other antigens.
EXAMPLE FOUR
DEVELOPMENT OF A THERAPEUTIC (:OMPOUND WHICH ELICIT
IMMUNOLOGICAL EFFECT.
A therapeutic compound is developed containing an effective quantity of
monoclonal
antibody produced by the hybridoma obtained through the Hybridoma Selection
Method that produces antibody-dependent cellul~~r cytotoxicity. To do this, we
used
the standard measurement technique for this activity, A-431 cell line labeled
with
5lCr (100 pCi) was used as targets. The effector cells were blood cells from
normal
adults prepared with a Ficoll-Hypaque gradient in which the mononuclear cells
from
the peripheric blood were separated, washed three times and adjusted in a
effector
cell/target cell proportion of 200:1.
27a
F
21~6~~3
The plates with the target cells and the antibody were incubated at 4°C
for 30
minutes. The effector cells (100 pL) were added to the plate and centrifuged
immediately (100 x g) for 2 minutes. They were then incubated for four hours
in a
humid chamber at 37°C with 5-95% C02 air. The counts per minute (cpm)
were
measured in 100 ~tL of supernatant in a gamma counter.
The complement-mediated cytotoxic activity vas measured using the standard
measurement method and using A-431 cell line labeled with 5lCr radioactive
(100
~Ci) as target cells. The target cells in a concentration of 104 were pipetted
on 96
well plates which contain 25 pL of monoclonal antibody (ascitic liquid)
diluted
1/10.
The complement source consisted of 100 p,L of rabbit serum at 25% in culture
medium. It was incubated for four hours in a humid chamber at 37° C
with 5-95%
C02 air. The plates were later centrifuged and the counts per minute were
measured in 100 ~L of the supernatant in a gamma counter. Several working
solutions of the antibody were tested to verify what was the maximum toxicity.
These experiments showed that in the presence of rabbit complement, a
cytotoxic
activity of 59 % at a concentration of 12.5 ~g/mL , and the cellular cytotoxic
effect
mediated by antibodies carried out with peritoneal macrophages produced a
cytolytic effect of 22 %.
2o This preparation includes a pharmaceutical vehicle which contains 2.55 mg
of
monobasic sodium phosphate, 9.00 mg of dibasic sodium phosphate, 43.00 mg of
sodium chloride, 1 mg of polysorbate 80, 5.00 mL of injection water.
EXAMPLE FIVE
DEVELOPMENT OF A THERAPEUTIC COMPOUND WITH N-ACETYL
GM3
#92025 v2 2g
,~ . ;~ 1 ~ 6 ~' 5 3
A therapeutic compound is developed containing an effective quantity of
monoclonal antibody produced by the hybridoma obtained through the Hybridoma
Selection method, together with N-acetyl GM3.
A synergetic effect of the combination N-acetyl GM3 with the monoclonal
antibodies was observed in inhibiting proliferation of a lung cancer cell
line.
To perform the experiment, a cellular suspension was obtained and resuspended
in
culture medium. N-acetyl GM3 was added at a concentration of 5 micromoles and
the MAb was added at a concentration of 30 ~g/mL. At the third and sixth days,
total protein was measured by the Lowry method.
1o There was almost 100 % inhibition of proliferation in relation to the
control but
the effect produced was greater than the simple additive effect.
This preparation includes a pharmaceutical vehicle which contains 2.55 mg of
monobasic sodium phosphate, 9.00 mg of dibasic sodium phosphate, 43.00 mg of
sodium chloride, l mg of polysorbate 80, and 5.00 mL of injection water.
EXAMPLE SIX
DEVELOPMENT OF A THERAPEUTIC'. COMPOUND WITH MAB
AGAINST EGF.
A therapeutic compound is developed containing an effective quantity of
monoclonal antibody produced by the hybridoma obtained through the Hybridoma
2o Selection Method, together with anti EGF MA,b, plus a pharmaceutical
vehicle
which contains 2.55 mg of monobasic sodium phosphate, 9.00 mg of dibasic
sodium phosphate, 43.00 mg of sodium chloride, 1 mg of polysorbate 80, and
5.00
mL of injection water.
#92025 v2 29
F
