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Description  |
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UTILITY
The present processes and resulting products which are chemically modified
immunotoxins have utility in animals and in human cells in vitro for
systematically differentiating and killing cancer cells. This utility has
been shown by standard biological tests.
BACKGROUND
Current approaches to cancer chemotherapy and other immunological therapies
focus on the use of cell-specific antibodies bonded to immunotoxins in
order to kill specific populations of human cells. Ideally, immunotoxins
should discriminate to a high degree between target and non-target cells.
The critical point, then, is the development of immunotoxins that are
highly toxic for specific populations of cells. The present invention
details a new class of immunotoxins employing a monoclonal antibody,
recognizing a specific human cell receptor, bonded to Pseudomonas toxin.
Pseudomonas exotoxin (PE) is modified with methyl-4-mercaptobutyrimidate
(MMB) so that, by itself, the toxin exhibits very little toxicity;
coupling the modified toxin to a monoclonal antibody, however, transforms
the toxin into a highly potent immunotoxin.
Other toxins have been modified in order to produce a suitable immunotoxin.
The two best known are ricin toxin and diptheria toxin. However, both of
these toxins must be cleaved and the A-chain subunits purified prior to
bonding with suitable antibodies. With Pseudomonas exotoxin, the cleavage
step is unnecessary. In addition, cleavage of ricin or diptheria toxins
into A and B chains removes the portion of the molecule containing
residues important for transport into the cytosol of the cell. In
contrast, when Psuedomonas exotoxin is modified, no part of the molecule
is removed; coupling the exotoxin to a suitable monoclonal antibody
produces a very potent cell-specific and easily internalized toxin.
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the inhibition of protein synthesis by PE immunotoxin when
added to HUT-102 (solid lines) or MOLT-4 (broken lines). Immunotoxins were
added to cells in small volumes from stock solutions to give the final
concentrations indicated in the specific disclosure. Cells were incubated
at 37.degree. C. for 20 hr and [.sup.3 H]leucine (3-4 .mu.Ci/ml, final
concentration) was added for a further 1 hr. Control dishes incorporated
about 70,000 cpm into trichloroacetic acid insoluble material.
FIG. 2 shows the enhancement of PE-anti-TAC toxicity by adenovirus.
PE-anti-TAC alone, adenovirus alone (2 .mu.g/ml) or PE-anti-TAC plus
adenovirus (2 .mu.g/ml) were added to HUT-102 cells for 4 hr at 37.degree.
C. At that time [.sup.3 H]leucine was added to a final concentration of
3-4 .mu.Ci/ml for a further 1 hr. Protein synthesis inhibition was then
determined by measuring the reduction in radioactivity incorporated into
trichloroacetic acid insoluble material from treated cells versus control
cells which received no additions of toxins or virus.
FIG. 3 shows the inhibition of protein synthesis due to PE conjugate.
Various concentrations of PE-hybrid toxins ( =PE-cys, .DELTA.=PE-HB21,
=PE-HB21 +8.times.10.sup.-8 M HB21, .quadrature.=PE-B3/25, and o=PE-EGF)
were added to KB cell monolayers for 16-18 hr at 37.degree. C.
Toxin-containing medium was then replaced for 1 hr with fresh medium
containing 2 .mu.Ci/ml [.sup.3 H]leucine. Protein synthesis levels were
determined and compared to untreated control cells. Control cells
incorporated into TCA insoluble material approximately 25,000 cpm/dish.
FIG. 4 shows the enhancement of toxicity due to adenovirus. Various
concentrations of PE hybrid toxins ( =PE-cys+adenovirus, =PE-cys,
.DELTA.=PE-HB21+adenovirus, =PE-HB21, .quadrature.=PE-B3/25+adenovirus,
=PE-B3/25, o=PE-EGF+adenovirus and =PE-EGF) were added to KB cell
monolayers for 1 hr at 37.degree. C. in the presence or absence of
3.times.10.sup.8 viral particles/ml. At the end of an hour, the medium was
replaced for a further hour with fresh medium containing 1 .mu.Ci/ml
[.sup.3 H]leucine. Protein synthesis levels were determined and compared
with the appropriate controls.
GENERAL DESCRIPTION OF THE INVENTION
A method has been developed to chemically modify Pseudomonas exotoxin so
that the exotoxin can be coupled with growth factors, antibodies, and
other biologically active molecules so that this toxin will selectively
kill target human tumor cells or other types of cells displaying specific
molecules on their cell surface.
PE has recently been conjugated to a variety of monoclonal antibodies
recognizing certain human tumors (Cetus Corporation) and to a monoclonal
antibody recognizing the human H Type 1 blood group substance [Richert et
al. (1983) J. Biol. Chem., 258:8902-8907, and Fredman et al. (1983) J.
Biol. Chem., 258:11206-11210]. The toxin-conjugates specifically kill
appropriate target cells. PE can now be coupled to a variety of peptides,
proteins and growth factors that react with specific receptors on cells.
These include sarcoma growth factors, malanocyte stimulating hormone
(MSH), somatostatin, glucogon, insulin, transferrin, low density
lipoprotein, calcitonin, .alpha..sub.2 -macroglobulin, and lysine
bradykinin. Conjugates with MSH and lysine bradykinin have already been
prepared and show some biological activity. Pseudomonas exotoxin is
particularly preferable to other toxins (such as ricin or diptheria toxin)
because it is easily prepared in large amounts and because humans do not
contain the antibodies to neutralize it (as is the case with diptheria
toxin) and because it does not have to be separated into subunits before
being conjugated.
SPECIFIC DISCLOSURE
Pseudomonas exotoxin (PE) is a known and readily available toxin isolated
from Pseudomonas aeruginosa. The particular exotoxin used in this
invention was the gift of Dr. S. A. Leppla, USAMRIID, Fort Detrick, Md.
PE was chosen for this invention because it acts in the cytosol of the cell
to inhibit protein synthesis by catalyzing the enzymatic
(ADP-ribosylation) inactivation of elongation Factor Two.
Monoclonal antibodies (Mabs) against the transferrin receptor (anti-TFR)
are known and available from the American Type Culture Collection
(labelled HB21). These Mabs are propagated as ascites in BALB/c mice and
purified by precipitation with 50% saturated ammonium sulfate and affinity
chromatography on a column containing S. aureus protein A.
Monoclonal antibodies against the T-cell growth factor, anti-TAC, are
purified in the same manner as described above for anti-TFR. Anti-TAC is a
known and available monoclonal antibody described by Uchiyama et al., J.
Immunol., Vol. 126, p. 1393 and p. 1398 (1981).
Epidermal growth factor (EGF) is a well known peptide growth factor,
readily available, and extensively studied [Carpenter et al., J. Cell
Biol., Vol. 71, p. 159 (1976); Maxfield et al., Cell, Vol. 14, p. 805
(1978); and Willingham et al., Cell, Vol. 21, p. 67 (1980)].
Pseudomonas exotoxin-monoclonal antibody conjugates (PE-Ab) are constructed
either using a disulfide exchange reaction or by forming a thioether bond.
For a particular regimen used for a specific Mab, see Examples 1-3.
Generally, however, PE is treated with methyl-4-mercaptobutyrimidate (MMB)
in order to introduce two thiol groups per molecule of toxin. This step is
optimally conducted in 10 mM KPO.sub.4 (pH 8.5). Derivatized PE from the
above step is then reacted with dithiobis(2-nitrobenzoic acid) (DTNB).
Purified antibody is also treated with MMB in order to introduce slightly
more than one thiol group per molecule. The treated antibody is then mixed
with excess treated PE and allowed to incubate for 2 hrs at 23.degree. C.
Alternatively the antibody can be modified with m-maleimidobenzoyl N
hydroxy-succinimide ester (MBS) and the resulting activated antibody
reacted with SH-PE-SH to produce a conjugate containing a thioether
bond--more stable in an animal environment since it cannot be inactivated
by reduction of a disulfide bond.
##STR1##
MMB was selected because it creates a sulfhydryl group when reacted with
the amino group present in lysines and at the amino-terminal of proteins.
Apparently, it is inactivation of these amino groups that alters PE so
that it no longer binds to the PE receptor on cells. Other reagents that
react with amino groups may be used in altering PE so that it can be
successfully derivitized to antibodies, growth factors, and hormones.
These include MBS, N-succinimidyl 3-(2-pyridylthio) proprionate (SPDP),
succinimidyl 4-(N-maleimido-(methyl) cyclohexane-1-carboxylate (SMCC) and
related agents.
The resulting PE-Ab conjugate is then purified in a multi-step procedure.
Typically, 1 ml of conjugate at 3-5 mg/ml is passed over a Sepharose 6B
column. Large aggregates in the void volume exhibited low activity and are
discarded. The material remaining on the Sepharose 6B column is further
purified and separated from unreacted PE by passing the conjugate over a
Sephadex G-200 column. The first pool, containing the PE-Ab conjugates
used in this invention, includes each PE molecule coupled to one or two
antibody molecules. This material reacted with DTNB and then cysteine to
derivatize all free-SH groups. Tracer amounts of .sup.125 I-PE may be used
to follow the separation procedure. The conjugate is assayed by adding it
to tumor cells bearing the appropriate cell surface recognition markers
and measuring inhibition of protein synthesis or cell death. The
ADP-ribosylating activity of the conjugates is also assayed in cell
extracts, usually reticulocyte lysates, using .sup.14 C-NAD as described
in Fitzgerald et al., Cell, Vol. 32, p. 607 (1983). All experiments were
conducted either in vivo on nude mice, or in vitro on human cells in
tissue culture.
Target Cells
As indicated in the Background section, this invention couples an exotoxin
to a MAB specific for certain cell receptor sites. The type of target
cell, then, depends upon the specificity of the monoclonal antibody. Three
receptor sites are described in the examples, and the corresponding target
cells are described here. However, the invention is not intended to be
limited thereby. Growth factor, hormones, monoclonal antibodies, and
target cells similar in description to those described herein are included
within the ambit of this invention.
KB and Swiss 3T3 cells are maintained as monolayers in Dulbecco's Modified
Eagle's Medium (DMEM), supplemented with 10% calf serum (for KB cells) or
10% fetal bovine serum (3T3 cells), and penicillin (50 IU/ml) and
streptomycin (50 .mu.g/ml).
HUT-102 and MOLT-4 cells are grown in suspension in RPMI 1640, 10% fetal
bovine serum, and penicillin-streptomycin (50 IU/ml--50 .mu.g/ml). The
HUT-102 cell line is derived from an adult T-cell leukemia patient and
expresses the T-cell growth factor receptor (TCGF). MOLT-4 is a long-term
T-cell line that does not bind TCGF and is non-reactive for anti-TAC. Both
cells are reactive, however, with antibodies that recognize the human
transferrin receptor. Human breast cancer cells (MCF7) and human ovarian
cancer cell lines express human transferrin receptors and some cancer
specific antigens recognized by monoclonal antibodies supplied by Cetus.
Toxicity Enhancement
The toxicity of the conjugates of this invention is enhanced by the
addition of adenovirus type 2 propagated in KB cells grown in suspension
culture. The virus is purified by the procedure of Green and Pina,
Virology, Vol. 20, p. 199 (1963) except that CsCl is used instead of RbCl.
Twice-banded virus is aliquoted in 10 ml of sterile tris-saline-30
glycerol and stored in this buffer at -20.degree. C.
The interaction of adenovirus with mammalian cells has been studied most
extensively using KB cells. To determine the generality of the adenovirus
effect, other human cell lines were assayed for their response to various
toxin conjugates in the presence and absence of adenovirus (Table I).
Three other human cell lines were sensitive to viral enhancement of
toxicity. Two of these were of epithelial origin (A431 and HeLa). The
other was a T cell leukemia line. The degree of enhancement and amount of
the toxin conjugate necessary to reduce protein synthesis below 50% of
normal varied from cell line to cell line. The reason for these variations
is not understood but is probably related to such properties as the number
of receptors for the conjugate, the number of viral receptors, and the
rate of uptake.
It has been shown previously that adenovirus disrupts receptosomes
releasing the contents of the vesicle into the cytosol. When the contents
of the receptosome included adenovirus and PE-EGF, PE, PE-anti-TFR,
PE-anti-TAC, or other toxin conjugates, the inhibition of protein
synthesis due to these toxins was markedly enhanced.
TABLE 1
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Enhancement of Toxicity in Various Cell Lines
due to Adenovirus
Protein Synthesis
% Reduction
Without With
Cell Line
Conjugate Adenovirus
Adenovirus
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HUT-102 PE-HB21 (1 ug/ml)
17 65
HeLa S-3
PE-EGF (0.01 ug/ml)
0 88
A431 PE-EGF (0.01 ug/ml)
20 80
A431 PE-HB21 (1 ug/ml)
27 78
Swiss 3T3
PE-EGF (0.1 ug/ml)
58 67
KB Ricin A-HB21 (1 ug/ml)
15 86
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Binding Specificity Studies of PE-Ab Conjugates on HUT-102 and MOLT-4 Cells
Anti-TAC is labeled with tritium by reductive methylation to a high
specific activity (7.3 uCi/ug). Anti-TFR is radioiodinated by a modified
iodogen procedure [Fraker et al., Biochem. Biophys. Res. Commun., Vol. 80,
p. 849 (1978)]to a specific activity of 9 .mu.Ci/ug. Peripheral blood
mononuclear cells or T-cells are cultured with PHA and anti-TAC binding
assayed after 3-4 days. HUT-102 cells, taken directly from tissue culture,
are used to assess anti-TFR binding. Both cell types are washed twice in
binding medium (RPMI 1640, 1% BSA, 1 mg/ml human IgG, 0.1% azide) prior to
antibody additions. The presence of azide allows binding assessments to be
made at 23.degree. C. rather than in the cold. Binding experiments are
performed in 1.5 ml centrifuge tubes on a rocking platform. From
preliminary experiments, equilibrium binding is achieved for both
antibodies at approximately 45 min. The results presented represent the
average of triplicate samples with a binding time of 60 min. Standard
derivations for each point are less than 10% of the mean. From a starting
cell concentration of 2.times.10.sup.7 ml, 50 .mu.l is added to the
reaction tube, then 20-30 .mu.l of unlabeled antibody (or immunotoxin),
and finally 20 .mu.l of radiolabeled antibody. The radiolabeled antibody
is kept at a constant concentration (well below half saturation), while
the unlabeled antibody is varied over a wide range to determine the
concentration of each antibody species that displaces half the labeled
antibody. At the end of 60 min. the cells are transferred to a second tube
containing PBS, to which sucrose is added to a 1 M concentration. After
the appropriate washings, the cell pellet is collected and counted in an
appropriate beta or gamma counter.
Toxicity of PE-anti-TAC for HUT-102 and MOLT-4 Cells
PE-anti-TAC is added to cells at 37.degree. C. and the ability of this
conjugate to inhibit protein synthesis is determined after a 20 hr.
incubation (FIG. 1). The activity of PE-anti-TAC is compared with a
conjugate of PE coupled to an antibody against the transferrin receptor
(PE-anti-TFR) by assaying each conjugate on HUT-102 and MOLT-4 cells. Both
these cell types contain transferrin receptors, but only HUT-102 cells
contain TCGF receptors. Both cell types respond to the PE-anti-TFR
conjugate, with the MOLT-4 cell line being 2- to 3-fold less sensitive
(FIG. 1). Fifty percent inhibition of protein synthesis occurs at
2.5.times.10.sup.-10 M for HUT-102 and 6.5.times.10.sup.-10 M for MOLT-4.
In contrast, there is at least a 100-fold difference in the response of
HUT-102 and MOLT-4 cells to PE-anti-TAC. PE-anti-TAC causes a 50%
reduction in protein synthesis at 9.times.10.sup.-10 M with HUT-102 cells.
There is no detectable reduction of protein synthesis when PE-anti-TAC is
added to MOLT-4 cells at concentrations as high as 3.times.10.sup.-8 M.
Thus, PE-anti-TAC is cytotoxic for cells expressing the TCGF receptor but
inactive against receptor negative cells.
Adenovirus-Mediated Enhancement of PE-anti-TAC Toxicity
Human adenovirus type 2 enters cells by receptor-mediated endocytosis and
begins to escape into the cell cytosol 2-5 min. after it is delivered from
coated pits into receptosomes. In human fibroblasts and human KB cells,
the virus allows ligands cointernalized with the virus to be released into
the cytosol. When the ligand is a toxin or a toxin conjugate, there is an
enhancement of toxicity compared with the addition of toxin alone. To
determine if the phenomenon occurred in lymphoid cells, adenovirus (2
ug/ml) and PE-anti-TAC are incubated with HUT-102 cells (FIG. 2). As
observed with other human cell types, adenovirus enhanced toxicity.
Toxicity of PE-anti-TAC is enhanced approximately 50-fold in the presence
of adenovirus (2 ug/ml) when compared to the addition of PE-anti-TAC
alone. The ability of adenovirus to enhance toxicity also is observed with
adenovirus capsids lacking DNA and with U-V treated adenovirus which
cannot replicate in human cells.
Toxicity of PE-TFR Conjugate
Toxicity of PE-TFR conjugate, due to entry via the transferrin receptor, is
enhanced 100- to 300-fold in the presence of adenovirus.
Conjugates of PE coupled to the anti-TFR (HB21) antibody are assessed for
toxicity on monolayers of KB cells by measuring levels of protein
synthesis (FIG. 3). To do this, various concentrations of the toxin
conjugates are added to cell monolayers at 37.degree. C. and incubated for
16-18 hr. To determine levels of protein synthesis, the toxin-containing
medium is replaced with fresh medium containing 2 uCi/ml [.sup.3
H]-leucine and the amount of [.sup.3 H]leucine incorporated into protein
determined after a further incubation of 1 hr. For purposes of comparison,
the two conjugates were assayed in parallel with PE-EGF, a conjugate that
was previously shown to inhibit protein synthesis in KB cells. the
PE-anti-TFR and the PE-EGF conjugates were of similar potency with a
TCD.sub.50 of approximately 3.times.10.sup.-10 M.
The specificity of toxicity of the conjugates with the transferrin
antibodies was tested in two ways. In the presence of excess unconjugated
antibody (2.8.times. 10.sup.-8 M), the toxicity of PE-anti-TFR is reduced
approximately 10-fold (FIG. 3). Presumably this reduction is due to
competition for antibody binding sites. To determine how much activity is
due to toxin entry separate from that mediated by antibody binding, PE
coupled to cysteine in place of the antibody molecule was assayed for its
potency on cells. PE-cysteine (PE-cys) was 500- to 1000-fold less active
than PE-anti-TFR or PE-EGF (FIG. 3).
Mouse cells are very sensitive to PE. To evaluate the toxicity of PE-cys
more stringently, Swiss 3T3 mouse fibroblasts were used. PE-cys or native
PE is added to Swiss 3T3 cells at 4.degree. for 2 hr, the cells are washed
extensively, warmed to 37.degree. C. and incubated overnight. PE-cys at
1.4.times.10.sup.-7 M is not toxic, whereas PE at 1.4.times.10.sup.-10 M
reduces protein synthesis by 40%. Thus, when a binding step at 4.degree.
C. is included in the assay, PE-cys is at least a 1,000-fold less active
than native PE.
Toxicity of PE-anti-TFR in the Presence of Adenovirus
PE-anti-TFR is added to KB cells at 37.degree. C. for 1 hr in the presence
of human adenovirus type 2. After 1 hr. the medium is removed from the
cells and replaced with fresh medium containing 2 uCi/ml [.sup.3
H]leucine. Adenovirus alone at 1 ug/ml viral protein does not reduce the
level of protein synthesis. During this short assay period neither
PE-anti-TFR at 2.8.times.10.sup.-9 M nor PE-EGF at 1.2.times.10.sup.-8 M
affected protein synthesis. However, when adenovirus and PE-TFR
(2.8.times.10.sup.-9 M) are added together, 70 to 80% inhibition of
protein synthesis is noted. In various experiments adenovirus at 1 ug/ml
enhanced the toxicity of PE-anti-TFR 100- to 300-fold. In comparison,
addition of PE-cys (1.4.times.10.sup.-8 M) and adenovirus does not result
in a significant reduction in protein synthesis.
Toxicity of PE-EGF Conjugate
PE and EGF are covalently coupled in the ratio of PE:EGF/1:2. When measured
against a standard curve of known toxin concentrations, PE-EGF is found to
have retained full enzymatic (that is, ADP-ribosylation) activity (Table
2). As with the native PE, dithiothreitol (DTT) is required for full
enzyme activity. However, even in the absence of DTT, the conjugate is
partially in an active state. The conjugate is assayed for toxicity on KB
cells and compared with native PE (FIG. 4). The hybrid toxin is fivefold
more toxic than native toxin. The increased toxicity is abolished in the
presence of excess native EGF, indicating that hybrid entry is mediated by
EGF receptors.
TABLE 2
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ADP-Ribosylating
Activity of PE, PE-EGF and PE-Au
NAD Transferred
(pmoles/.mu.g/30 min)
.mu.g/ml +DTT -DTT
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PE 50 140 .+-. 7.4
7.8 .+-. 0.6
25 98 .+-. 4.9
5.0 .+-. 0.2
5 43 .+-. 1.9
1.1 .+-. 0.8
PE-EGF 25 98 .+-. 1.8
24 .+-. 3.6
PE-AU 25 92 .+-. 6.8
41 .+-. 4.5
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Results are expressed as pmoles of NAD transferred (in 30 min.) per
microgram of EF.sub.2.
Protein Synthesis Assay
Inhibition of protein synthesis was used as the index of toxicity. The
number of counts per minute in TCA-insoluble material from each dish or,
alternatively, the number of counts per minute per microgram of cell
protein was determined. Each data point was derived from duplicate dishes
with the standard deviation being routinely less than 5% of the mean.
Samples were compared with the appropriate controls and results were
expressed as percent of control. Each experiment was performed 3 to 4
times.
Adenovirus Enhancement of PE-EGF-Mediated Toxicity
Adenovirus also enhances the toxicity of the hybrid toxin. KB cells have
3.times.10.sup.4 -10.sup.5 receptor sites per cell and 1-2.times.10.sup.3
PE receptors per cell (unpublished results). Because of the high number of
EGF receptors, the uptake of the conjugate, but not necessarily its escape
to the cytosol in the absence of adenovirus, should be more efficient than
that of native toxin. As seen in FIG. 6, the presence of adenovirus
increased the toxicity of the hybrid toxin 10,000-fold. When tested in the
presence of excess EGF, this enhancement is substantially diminished,
indicating that the hybrid toxin is entering the cell via the EGF
receptor. The rate of protein synthesis after incubation with adenovirus
plus PE is determined in the absence and presence of excess EGF (10
ug/ml). The same level of inhibition was noted regardless of the presence
or absence of EGF. These results further support the case for
adenovirus-induced disruption of receptosomes.
EXAMPLES
Examples 1-3, as described in the specific disclosure, incorporate actual
values used in the laboratory during synthesis of the desired PE-AB
conjugates. The values provided should not be regarded as absolute, but
may be varied within experimentally acceptable ranges.
Example 1--PE-anti-TFR Conjugates
PE-anti-TFR conjugates were constructed using a disulfide exchange reaction
as described above. Two mg PE was treated with 26 mg
methyl-4-mercaptobutytrimidate (MMB) at 37.degree. C. for 2 hr to
introduce two thiol groups per molecule of toxin. Derivatized PE was then
reacted with 1 mM dithiobis(2-nitrobenzoic acid (DTNB). Purified antibody
(2 mg) was derivatized with 1.3 mg MMB for 10 min at 37.degree. C.,
thereby introducing slightly more than one thiol group per molecule. The
derivatized antibody was mixed with a 3-fold molar excess of DTNB-treated
toxin and allowed to incubate for 2 hr at room temperature prior to
purification and storage or use as a PE-anti-TFR conjugate.
Example 2--PE-anti-TAC Conjugates
PE-anti-TAC conjugates were constructed using the same procedure described
in Example 1. Two mg PE were treated with 26 mg MMB at 37.degree. C. for 2
hr. Derivatized PE was then reacted with 1 mM DTNB. Purified anti-TAC
antibody (2 mg) was derivatized with 1.3 mg MMB for 10 minutes at
37.degree. C., and then mixed with a 3-fold molar excess of DTNB-treated
PE and incubated for 2 hr at room temperature. The conjugate immunotoxin
was then stored or used in the toxicity studies described above.
Example 3--PE-EGF Conjugate
PE (0.5 mg) was reacted with 6.5 .mu.g of methyl-4-mercaptobutyrimidate
(MMB) at 37.degree. C. for 2 hr in 0.55 ml of 10 mM KPO.sub.4 (pH 8.5).
NAD was added to a final concentration of 1 mM. (NAD was present to
protect the active site of the enzyme.) Derivatized PE was separated from
the reaction mixture on a PD10 column, previously equilibrated with 10 mM
KPO.sub.4 (pH 8.5) and 1 mM NAD. The number of thiol groups introduced by
this procedure was assayed by the addition of 20 .mu.l 0.1 M
dithionitrobenzoate (DTNB). It was determined from OD absorbance at 412 nM
that 2 moles of the thiol group had been introduced per mole of PE. EGF
was reacted with MMB to introduce one SH group. Finally, derivatized PE
and EGF (in excess) were mixed together and the disulfide exchange
reaction allowed to go to completion. The conjugate was separated from
free EGF by fractionation on a G-75 column.
EXAMPLE 4
Pseudomonas exotoxin modified with MMB need not be coupled to a monoclonal
antibody in order to be biologically effective. At this point, no other
toxin can be so modified and remain effective without killing the host
animal. For example, less than 1 .mu.g of unmodified toxin is lethal I.P.
When Pseudomonas exotoxin was modified and coupled to EGF, nude mice were
killed by 20-50 .mu.g of the conjugate. Autopsy showed that they died from
liver failure. This is because the liver has a large number of EGF
receptors. On the other hand, when Pseudomonas exotoxin was coupled to
cysteine by the same chemical reaction, animals given 0.3-0.5 mg of toxin
exhibited no signs of toxicity. These experiments show the low toxicity of
the chemically modified toxin when coupled to an inert molecule and how it
can be coupled to a growth factor to kill receptors containing cells of
the liver.
Example 5--Residual Immunotoxin Cell-Binding Activity
Following the coupling of PE to either anti-TAC or anti-TFR, the residual
cell-binding activity of each immunotoxin was determined. Only antibody
binding was assessed since it has been shown that the chemical coupling of
PE to EGF and various antibodies destroyed the toxin's ability to bind to
its own receptor. The relative binding activity of PE-anti-TAC compared
with anti-TAC was assessed on PHA-stimulated lymphocytes and PE-anti-TFR
with anti-TFR on HUT-102 cells. PE-anti-TAC was not assayed for binding
activity of HUT-102 cells for technical reasons related to the very large
number of anti-TAC binding sites. .sup.3 H-anti-TAC (3.5.times.10.sup.-10
M) was added to PHA lymphocytes in the presence of various concentrations
of either anti-TAC or PE-anti-TAC. The binding of .sup.3 H-anti-TAC was
reduced to a 50% level in the presence of 1.2.times.10.sup.-9 M anti-TAC
or 2.8.times.10.sup.-8 M PE-anti-TAC. In a similar experiment
7.4.times.10.sup.-10 M .sup.125 I-anti-TFR was added to HUT-102 cells and
displaced to a 50% level by 1.6.times.10.sup.-7 M anti-TFR or
1.4.times.10.sup.-7 M PE-anti-TFR. The coupling of PE to anti-TAC reduced
the binding activity of the antibody by 20-fold, whereas the coupling of
PE to anti-TFR had very little effect on its cellular binding.
"Cell binding protein" as used in the specification and claims is defined
to mean and include anti-TAC monoclonal antibody, anti-TFR monoclonal
antibody, epidermal growth factor, and cysteine.
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