Note: Descriptions are shown in the official language in which they were submitted.
1 3388 1 8
r rJN~
Field of the Invention
The present invention relates to cancer chemotherapy,
and, more particularly, to a reagent which selectively kills
- cancer cells and can be used to treat graft versus host disease.
Backqround of the Invention
The u~e of cytotoxic products in the treatment of
cancer i8 well known. The ~l;ff;~ ties associated with such
10 treatment are also well known. Of these difficulties, the lack
of cancer-specific cytotoxicity has received considerable
attention, ~1th~ h resolution of these difficulties has met with
marginal success. Cytotoxic products kill normal cells as well
as cancer cells. Such non-specificity results in a number of
15 undesirable side effects for patients undergoing cancer
chemotherapy with cytotoxic products, including nausea, vomiting,
diarrhea, h -Lllagic gastroenteritis, and hepatic and renal
damage. Due to normal cell toxicity, the therapeutic dosage of
cytotoxic products has been limited such that cancerous cells are
2 0 not killed to a 8uf f icient level that subsequently prevents or
delay~ new cancerous growth.
.
. - .
1338818
Current approaches to cancer chemotherapy and other
immunological therapies focus on the use of cell-specific
antibodies bonded to immunotoxins in order to kill specif ic
5 pop~ t; ~1n~ of cancer 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 popul~t;ons of
cells .
Monoclonal ~nt;ho~;es linked to toxic proteins
(immunotoxins) can selectively kill some tumor cells ln vitro and
ln v1vo. However, reagents that combine the full potency of the
native toxins with the high degree of cell-type selectivity of
15 monoclonal antibodies have not previously been designed. Two
heretofore inbeparable activities on one polypeptide chain of
h~h~ria toxin and ricin account for the failure to construct
optimal reagents. The B-chains facilitate entry of the A-chain
to the cytosol, allowing immunotoxins to kill target cells
20 efficiently and bind to receptors present on mobt cells,
imparting immunotoxins with a graft degree of non-target-cell
toxicity .
Some toxins have been modif ied to produce a ~uitable
25 immunotoxin. The two best known are ricin and ~l;phth~ria toxin.
Antibodies which bind cell surface antigens have been linked to
~liphth~ria toxin and ricin, forming a new pharmacologic class of
cell type-specific toxins. Ricin and diphtheria toxin are 60,000
to 65,000 dalton proteins with two subunits: the A-chain inhibits
30 protein synthesis when in the cytosol, and the B-chain binds cell
surface receptors and facilitates passage of the A subunit into
the cytosol. Two types of antibody-toxin conjugates
(immunotoxins) have been shown to kill antigen-pobitive cells in
vitro. Immunotoxins made by binding only the toxin A subunit to
35 an antibody have little non-target cell toxicity, but are often
only
~! 1 3388 1 8
m;n;m-lly toxic to antigen-positive cells. Another type
of immunotoxin is made by linking the whole toxin, A and
B subunits, to the antibody and blocking the binding of
the B subunit to prevent toxicity to non-target cells.
For ricin, the non-target cell binding and killing can
be blocked by adding lactose to the culture media or by
steric restraint imposed by linking ricin to the
antibody. Intact ricin immunotoxins may have only 30-
to 100-fold selectivity between antigen-positive and -
negative cells, but they are highly toxic, and the best
reagents can specifically kill a great many target
cells .
Intact ricin and ricin A-chain immunotoxins have
been found to deplete allogeneic bone marrow of T cells,
which can cause graft-versus-host diseases (GVHD), or to
deplete autologous marrow of tumor cells.
Diphtheria toxin is composed of two disulfide-
linked aubunits: the 21, 000 dalton A-chain inhibits
protein synthesis by catalyzing the ADP-ribosylation of
elongation factor 2, and the 37,000-dalton B-chain binds
cell surface receptors and ~;)r; 1; tat~q transport of the
A-chain to the cytosol. A single molecule of either a
diphtheria toxin A-chain or a ricin A-chain in the
cytosol is suf f icient to kill a cell . The combination
of these three activities, binding, translocation, and
catalysis, produces the extreme potency of these
proteins . The cell surf ace-binding domain and the
phosphate-binding site are located within the carboxyl-
terminal 8-kDa cyanogen bromide peptide of the B-chain.
Close to the C terminus region of the B-chain are
several hydrophobic domains that can insert into
membranes at low pH and appear to be important for
;rhthf~ria toxin entry.
Antibodies directed against cell surface antigens
have been linked to intact ~ hthf~ria toxin or its A
subunit to selectively kill antigen-bearing target
1338818
cells. Antibody toxin (immunotoxin~) or ligand toxin conjugates
crmt~;n;n~ only the ~l;rhtheria A-chain have relatively low
cytotoxic activity. Intact l;phth.-ria toxin conjugates can be
5 very potent, but can also have greater toxicity to normal cells.
Since the B-chain appears to facilitate entry of the A-chain to
the cytosol, it is possible that its presence in whole toxin
conjugates renders them more potent, although less specific.
Efforts have been made to construct more potent and specific
10 immunotoxins by separating the toxin B-chain domains involved in
cell binding from the domains involved in A-chain entry.
Target celL toxicity of immunotoxins can be increased
by including the toxin B-chain in the antibody-toxin complex or
15 by adding it separately. To achieve maximal 1 vitro target-cell
selectivity with immunotoxins c~nt~;n;n~ intact ricin, lactose
must be added to the medium to block non-target-cell binding and
toxicity of the immunotoxin via the ricin B-chain. This approach
is feasible in those clinical settings, such as bone marrow
20 transpl;lnt~t;~n, where the target cell population can be
incubated ~L vitro in the presence of lactose. Without blockage
of the B-chain binding domain, however, whole toxin conjugates
have a high degree of non-target-cell toxicity, thereby limiting
their usefulness in vlvo.
Construction of reagents that combine the potency of
intact toxin conjugates with the cell-type selectivity of toxin
A-chain conjugates may be possible if the binding site on the
toxin B-chain could be irreversibly blocked. Covalent and
30 noncovalent chemical modifications that block the binding
activity of ricin intracellularly also block its entry function,
suggesting that the binding and translocation functions may be
inseparable .
-- 4
.,
~_ .
~ 1 3388~ 8
Previou~ly, domain deletion was unsuccessfully used in
an attempt to separate the translocation and the binding
functions of ~l;rhthPria toxin B-chain. Immunotoxins made with the
A-chain, intact ~i;rhthPria toxin, and a cloned fragment of
5 ~l;rhthPria toxin (MspSA) that lacks the C-tprm;n~l 17 kDa region
of the B subunit were compared. The intact ~;rhthPria conjugate
was 100 times more toxic that the MspSA conjugate was, which, in
turn, was 100-fold more toxic than was the ~l;rhthPria toxin A-
chain conjugate. The C-terminal, 17-kDa region, which C-~nt;~;nR
10 the cell surface binding site, therefore potentiates immunotoxin
acidity 100-fold. It has not been possible to determine whether
this C-terminal translocation activity was distinct from the
binding activity.
Laird and Groman, J. Virol. 19, 220 (1976) mutagenized
Corynebacterium with nitrosoguanidine and ultraviolet radiation
and isolated several classes of mutants within the ~l;rhthPria
toxin structural gene. ~eppla and Laird further characterized
several of the mutant proteins and found that three of them,
CRM102, CRM103, and CRM107, retained full enzymatic activity but
had defective receptor binding.
Recombinan'c DNA technology has been used to improve
immunotoxin efficacy at the gene level. Greenfield et al. (1983)
in Proc. ~L. ~ Sci. U.S.A. 80, 6953-6857, reported that
they have cloned portions of ~i;rhthpria toxin and created a
modified toxin which c r)nt~;nR the N-tPrm;n~l hydrophobic region
Of ~l;rhthPria toxin but lacks the C-terminal cysteine for ease
of linking to antibodies. This LL _ t, lacks the cell surface-
binding site of ~irhthpria toxin but includes most of the
hydrophobic region thought to f acilitate ~ e transport .
Although cleavage of ricin or ~l;rhthPria toxin into A
and B-chains had been thought to improve the specificity of the
-- 5 --
1 3388 ~ 8
. ~
immunotoxin~ produced from the A-chain, cleavage of ricin or
tl;rh~h~ria toxins into A and s-chains removes the portion of the
molecule cr~nt~;n;ng residues important for transport into the
cytosol of the cell. Specific cytotoxic reagents made by coupling
toxin A subunits to ~nt;hs~;~5 have low systemic toxicity but
also very low tumor toxicity. More potent reagents can be made
by coupling intact toxins to monoclonal ~nt;ho~l;.o~, as detailed
in J. T ~1l. 136: 93-98 and Proc. Natl. Acad. Sci. U.S.A. 77:
5483-5486. These reagents, however, have a high systemic toxicity
due to the toxin binding to normal cells, although they can have
applications n vitro in bone marrow tr~n~pl~nt~tion (cf. Science
~: 512-515).
It was found by Youle et al., as reported in ~our.
~Lm~, o~. cit., that monoclonal antibody-intact tl;rh~h~ria cell
conjugates reacted ~uite differently from the intact ricin
immunotoxi~s. Of the four reagents eY~m;n~tl, a monoclonal
antibody against the T3 antigen linked to tl;rh~heria toxin
(UCHTl-DT) had unique properties. This reagent showed greater
selectivity in its toxicity to T cells as compared to stem cells
than UCHTl-ricin. UCHTl-DT was found to be 10 to 100 times more
selective than any previously reported immunotoxin.
Neville et al., in U.S. Patent Nos. 4,359,457 and
4,440,747, disclose that the receptor specificity of toxins can
be altered by collrl ;ng the intact toxin to monoclonal antibodies
directed to the cell surface antigen Thy 1.2. However, the only
toxin specif ically disclosed to be treated in this manner is
ricin. The same inventor~ in U.S. Patent No. 4,500,637, disclose
the covalent linkage of a monoclonal antibody known as TA-l
directed against human T-cells for use in treating human donor
-- 6 --
~'
1 3388 ~ 8
bone marrow before the marrow is fused into a human recipient.
Thus, this reagent has been found to be useful in preventing
graft ver~us host disease.
Another method of treating ricin to increase the rate
of protein ~ynthesis inhibition is by adding excess ricin B-chain
to target cells ;n~ n~nt of the amount of ricin A-chain bound
to the cell surface membrane. The ricin A-chains used in this
10 procedure are conjugated to anti-Thy 1.1 monoclonal antibodie~.
This process is disclosed in Neville et al ., U. S . Patent No .
4 , 52 0 , 0 11 .
Yet another method of treating graf t versus host
15 disease i8 disclosed in Neville et al ., U. S . Patent No .
4,520,226. In this method, monoclonal antibodies specific for T-
lymphocytes in human donor bone marrow are covalently linked to
separate ricin toxin, combined in a mixture to form a treatment
reagent, and ,~ ;n~f~ with bone marrow removed from a human
20 donor. The bone marrow-reagent mixture is then infused into an
irradiated recipient, which virtually eliminates T-lymphocyte
activity .
However, none of the prior art has shown effective
25 immunotoxins prepared from ~ hth.-ria toxin which have the
de~ired specif icity and activity.
Summary of the Invention
It is an object of the present invention to overcome
the above-described deficiencies in the prior art.
Further objects are to improve anti-cancer therapy and
35 to reduce ~-~rh.oYin in cancer patients.
It is another object of the present invention to
provide an immunotoxin with greater potency against cancer cells
than previous immunotoxins, that at the same time is safer and
4 0 less toxic to normal cells .
-- 7 --
3~
13388~8
It i~ yet a further object of the present invention to
provide an immunotoxin with greater selectivity between antigen
positive and antigen negative cells than any previously described
5 reagent.
It is another object of the present invention to
provide a reagent which aelectively kills cancer cells.
It is yet another object of this invention to provide
a reagent for treating graft-versus-host-disease.
According to the present invention, a binding moiety,
which can be a monoclonal antibody such as UCHT1 or transferrin
15 or epidermal growth factor or any other binding agent which binds
specifically to a cell, cell type or specific receptor, is
coupled to a toxin in which the native toxin binding site has
been inactivated. This providea extremely potent and specif ic
agents against cancer cells, and is particularly effective in
20 treatment of graft versus host disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a comparison of the toxicity of
~l;phth~ria toxin, CRM102, CRM103, and CRM107 ln vitro, as
compared to native diphtheria toxin using a sixteen hour protein
synthesis assay. Panel A shows the in vitro toxicity tested on
Jurkat cells. Panel B shows i.IL vitro toxicity tested on Vero
3 0 cells .
Figure 2 shows the binding activity of native
~;~hth~ria toxin and the three CRM mutants to Vero cells.
Figure 3 shows the location of the CRM point mutations
within the diphtheria structural gene.
Figure 4 shows a comparison of the toxicities of
immunotoxins made by conjugating UCHT1 with CRM102, CRM103,
CRM107, and native diphtheria toxin.
-- 8
~- ~ 1 3388 1 8
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides mutants of ~l;rhth~ria
5 toxin which are conjugated to a binding moiety which is a binding
agent which binds specifically to a cell, cell type, or specific
receptor. The binding agent may be a monoclonal antibody,
transferrin or epidermal growth factor, for which receptors are
found in a great variety of human tumor cells. The conjugates of
10 the present invention can be used to prepare formulations for
treating a great variety of tumors without the undesirable
effects of native diphtheria toxins on the patients.
Immunotoxins were made by conjugating three forms of
~1;rhthf~ria toxin, CRM102, CRM103, and CRM107 (cf. J. of Virology
220-227, 1976) that differ in only one or two amino acids
from native diphtheria toxin in the C region, with UCHTI, a
monoclonal antibody to the T3 antigen receptor found on human T-
cells, or to transferrin, or to any one of a number of known
20 binding agents such as ~r; ~lPrr-l growth factor and polyclonal
sera of certain types. The conjugation used a slight modification
of previously published procedures (PNAS 77: 5483-5486, 1980).
The phenotypic designation CRM is used to designate the
25 protein product of a tox gene that is serologically identical
with diphtheria toxin. The number following the CRM designation
indicates the molecular weight of the protein.
The nnnt~;nf~enic mutants of coryn~ha-tF~riophage beta
30 have been classified into four major classe~. Class I consists
of ten mutants, each of which produces a protein that forms a
line of identity with ~l;rhth~ria toxin when tested by
immunodiffusion against ~l;rhth~ria antitoxin. These mutants
probably represent nonsense mutations in the structural gene for
~5 toxin. The mutant! in ~ubcl~ give a po~iti~ el~in te~t but
388 1 8
,
a negative tiE3E3ue culture test. Two po~ible explanation~ are
that the mutant protein has a low level of activity or i5
produced in smaller amounts.
Class II mutants produce proteins that form lines of
partial identity with toxin when tested against antitoxin by
r-~; ffu3ion. On glab gel electrophoresis, only one of these
proteins was detected, but, based on immunodiffusion tests, all
10 appear to be smaller than purified toxin. 33ither a deletion, a
nonsense mutation in the structural gene for toxin, or
preferential proteolysis could account for the shortened
polypeptide.
Class III mutants produce two proteins serologically
related to toxin, two lines being l~terted in the immunodiffusion
test. One line shows full identity with purified toxin, and the
other shows only partial identity.
Class IV mutants do not produce a protein serologically
related to ~;rhth-~ria toxin, nor are they capable of eliciting
a positive guinea pig skin test. The phenotype of these mutants
has been designated CRM-. This would indicate that the intact
toxin molecule is either not produced or is produced or excreted
in very small amounts. This CRM phenotype likely results from
such mutational events as a deletion, a very early nonsen~e
mutation in the toxin structural gene leading to the production
of small fragments of toxin, or a mutation in a regulatory site
or gene.
The CRM102, CRM103, and CRM107 have not been classified
in one of the four major classes of mutants of r~;rh~h~ria toxin,
although; ~1; ffu~ion shows complete antigenic homology with
diph~h~r; ~ toxin. The molecular weight of these three CRM' 8 was
35 ~lptf~rm;n~d by electrophesis to be in the range of about 62,000.
.
- 10 -
13388~8
Dlphtheria toxin and CRM102, CRM103, and CRM107
were conjugated to m-maleimidobenzoyl ~-
hydroxysucr;n;m;rl~ ester (MBS) by incubating the toxins
with a 10-fold excess of MBS for thirty minutes at room
5 temperature. The mixture was then applied to a G-25
column to remove f ree MBS f rom the toxin . UCHTl was
reduced with 10 mM dithiothreitol for 30 minute8 at room
temperature, and f ree DTT was separated f rom the
antibody on a G-25 column. M~3S-conjugated toxin was
10 mixed with reduced antibody and incubated at room
temperature f or three hours . Immunotoxins were
separated from unconjugated antibody and toxin by gel
filtration on a TSK-3000 HPI-C column.
Peak fractions cr~nt~;n;n~ the immunotoxins were
15 collected and tested for toxicity to an antigen positive
human leukemic T-cell line. Protein synthesis was
assayed by incubating 105 cells in 100 microliters of
leucine-free RPMI 1640 rrntFl;n;nrJ 29~ fetal calf serum in
96 well microtiter dishes. Toxins, immunotoxins, and
20 control buffers (11 microliters) were incubated with the
cells for sixteen hours at 37C. Twenty microliters of
phosphate buffered saline c~nt~;n;nrJ 0.1 microCurie of
l4C-leucine was then added for ~0 minutes. Cells were
harvested onto glass f iber f ilters using a PHD cell
25 harvester, washed with water, dried, and counted. The
results are expressed as percentage of l4C incorporation
in mock-treated control cultures.
Figure 1 shows the toxiclty of CRM102, CRM103,
CRM104, and native diphtheria toxin to Jurkat cells (A)
30 and Vero cells (B). Protein synthesis was a8sayed by
incubating 5 x 104 Jurkat cells in 100 microliters
leucine-free RPMI 1640 medium cont~;n;ng 2g6 FCS in 96-
well microliter plates.
DT (-), CRM102 (X), CRM103 (O), or CRM107 (~) were
35 added in 11 microliters buffer and incubated with cells
for 16 hours at 37C. The cells were then
c
1338818
.
pulsed with 20 microliters of PBS ~cnt~;n;n~ 0.1 microCurie of
14c-leucine, incubated for one hour at 37 C, harvested onto glass
fiber filters by means of a PHD cell harvester, washed with
5 water, dried, and counted. The results are expressed as a
percentage of the 14c-leucine incorporation in mock-treated
control cultures.
Vero cells have a higher number of ~1;rhthpria toxin
10 receptors than do Jurkat cells, and are thus more sensitive to
diphtheria toxin inhibition of protein synthesis than are Jurkat
cells. CRM102 and CRM103 are 1000-fold less toxic than native
r~;rhth.oria toxin ig to both Vero cells and Jurkat cells.
Figure 2 shows the binding activity of native
rl;rhth~ria toxin and the three CRM mutants to Vero cells. While
most cell types, including lymphoid cells such as Jurkat, have
undetectable levels Of ~ h~h~ria toxin receptors, Vero cells
contain 105 diphtheria toxin receptors per cell and have been
used extensively to study tlirhth,oria toxin binding. At 4 C the
affinity of both CRM102 and CRM103 is 100-fold less than that of
native ~l;rhth~ria toxin, and the affinity of CRM107 is 800-fold
less than that of native ~lirhth.oria toxin.
The reduced af f inity correlates with the reduced
toxicity for CRM107 but differs by 10-fold for CRM102 and CRM103.
sinding was determined after six hours at 4 C, while toxicity was
determined after 24 hours at 37 C. The discrepancy between
binding and toxicity for CRM102 and CRM103 may reflect
3 0 dif f erences in temperature and time in the two assays . Binding
cannot be determined at 37 C, since ener~y inhibitors commonly
used to block intf~rn:ll; 7ation decrease the number of surface
fl;rhth~ria toxin receptors. Alternatively, the mutations within
CRM102 and CRM103 may inhibit toxin activities other than binding
-- 12 -
~ 1 3388 1 8
that may account for the 10-fold difference between toxicity and
binding .
Figure 3 shows the location of the amino acid changes
5 within the B-chain for each of the three mutations. CRM103
c-mti~; nc a gingle mutation at position 508 (Ser-Phe) . CRM102
contains a similar mutation at position 508, but has an
1;t;~n;l1 mutation at position 308 (Pro-Ser) . CRM107 C~-nt:~;n~
a single mutation at position 525 (Ser-Phe). That CRM102 has two
10 mutations while CRM103 contains only one indicates that the two
mutants are independent isolate~. The presence of multiple GC-AT
transitions is consi3tent with nitrosoguanidine-induced
mutagene~is .
Line 1 is the restriction map of the ~ tl~f~ria toxin
structural gene, indicating the location of the sites used for
sequencing. Line 2 is the expansion of the B-chain structural
region, indicating the native amino acid and DNA sequence
corresponding to the point mutations found within the CRM' 8 .
Mutations found within the B-chain of CRM102 (line 3), CRM103
(line 4), and CRM107 (line 5) are shown. Line 6 shows the end of
the MspRT clone previou~ly described. The sequences were obtained
by cloning the two MboI-ClaI fragments into M13MP and M13MP19 and
sequencing by the method of Sanger et al., J. Mol. Biol. 162, 729
(1982), or by cloning the two MspI fragments into pBR322 and
sequencing by the method of Gilbert and Maxam, Methods EnzYmol.
65, 499 (1980).
The 100-fold decreased binding affinity of CRM103 and
CRM102 demonstrates that the serine at position 508 is important
for toxin binding. The alteration at position 525 causes the
8000-fold decrease in binding activity. The mutations
-- 13 --
~,
t 338 8 1 8
at positions 508 and 525 are consistent with data which
suggest that the ~;r)hthPria toxin binding domain lies
within the carboxyl 17-kDa portion of the molecule.
Both mutations exchange a phenylalanine for a serine.
The relationship of binding to translocation in
~l;rhthPria toxin was P~m;nF~rl by linking each of the
CRM's and native ~;~hth~ria toxin to a new binding
domain, the monoclonal antibody UCHT1, which is ~3pecific
for the T3 antigen on human T-cells.
Figure 4 shows that, unlike the unconjugated CRM's,
all three CRM immunotoxins are highly toxic. Excess
antibody blocks toxicity, demonstrating that the
toxicity is antibody-mediated. The immunotoxins
prepared with CRM103 and CRM107 are equally toxic as the
immunotoxin prepared with native A;rhthPria toxin,
whereas the immunotoxin prepared from CRM102 is
approximately 10-fold less toxic. The 10-fold decrease
in UCHT1-CRM102 toxicity relative to UCHT1-CRM103,
despite identical binding activity of CRM102 and C~M103,
suggests that the amino acid at position 303 contributes
to the translocation activity of (l;rhth~ria toxin. That
the conjugates prepared with CRM103 and CRM107 are as
toxic as are conjugates prepared with native ~l;rhthPria
toxin indicates that binding of the toxin to its
receptor is not necessary for efficient translocation of
the toxin-A fragment to the cytosol. Therefore, the
~l;rhth~ria toxin binding and translocation functions can
be separated.
Figure 4 shows the comparison of the toxicities of
immunotoxins made by conjugating UCHT1 with CRM102,
CRM103, CRM107, and native ~;rhthPria toxin. The
antibody wa3 linked to the toxins via a thioether bond
as described previously. Immunotoxins were separated
from unconjugated antibody and toxin by gel filtration
on a TSK-3000 HP~C column. The immunotoxin peak was
collected, and toxicity was
- 14 -
~ 3388 1 8
evaluated with the protein synthesis assay as described
in Figure 1. ~CHT1-DT (O), UCHT1-CRM102 (V), UCHT1-
CRM103 (~), and UCHT1-CRM107 (C) were incubated with 5
x 10~ Jurkat cells for sixteen hours, followed by a one
5 hour pulse with l4C-leucine. Incubation with excess free
UCHT1 (100 micrograms/ml) blocked toxicity.
As shown in both Figures 1 and 4, native diphtheria
toxin and UCHT1-A;rhth~ria toxin inhibit Jurkat cell
protein synthesis 50% at 3 x 10-llM. The selective
toxicity of UCHT1-DT to T3 bearing cells is 100-fold,
and exists 801ely becau~e cro~l ;nk;n~ diphtheria toxin
to antibody inhibits tl;rh~h~ria toxicity 100-fold. The
mutant toxins, CRM102, CRM103, and CRM107, inhibit
Jurkat cell protein synthesis 5096 at 1 x 10-7M to 4 x lO~M
15 (Figure 1), whereas the IJCHT1-CRM immunotoxins act at 3
x 10-llM to 3 x 10-lM (Figure 4) . This 1000-10, ooo-fOld
difference in c~1n~pntration between the CRM's and the
UCHT1-CRM' 8 required to inhibit protein synthesis
represents a three to four order of magnitude increase
20 in CRM immunotoxin selectivity over the native
diphtheria immunotoxin.
The toxicities of the different immunotoxins were
compared on non-target Vero cells, which lack antibody-
binding sites but express a high number of diphtheria
25 toxin cell-surface binding sites. UCHT1-DT inhibits
Vero protein synthesis 90% at 6 x 10-lM, because of
toxicity via the ~ h~h~ria toxin binding site. In
contrast, all three CRM immunotoxins had no effect on
protein synthesis at this concentration. Thus, the 1088
30 of toxicity of the CRM's, as shown in Figure 1, is
exhibited also by the CRM immunotoxins on non-target
cells .
The immunotoxins as described herein can also be
conjugated with human transferrin (Tfn) . Transferrin is
35 highly conserved across species, and, as a result, human
transferrin exhibits species cross-reactivity that
-- 15 --
1 3388 1 8
enables the comparison of the toxicity of transLerrin-
toxin conjugates on cell~ derived from human (Jurkat,
K562), monkey (Vero), and mou~e (Wehi, E:~-4), as shown
in Table l.
-- 16 --
t338818
.
~- ~ 11 l ~ Q' C~ R
,10 X X X X ~
U a) r~l 11i N N
c æE ~ ~ ~ r
o o ~ o ~ o o o -
x x O O x o X X X ~ ,,, ~a
X X X ,,~ ' 'C ' '
'.C 'C ~ o ~
O - ~ ~ o
:~: X,~:_ m ~ ,1 m
m~ X X 'D 'D ~ ~ ,~ u
I' ~i ~ N ~ o
X ~ ~ o o~ ~ O O O '
O X X X X X X X X ~ ` (d ~,
E~a al ~ ~ N N ~i ~) ~i ~ JJ 3 S~
3 ~
r ~q O
H ' ' ~ - ' rt U
r r r~ r~ ,, rr 3
~ ~ ~ ~ ~ h r- o
O O ~ ~ O O ~ U ~ C
~,
on
r-l C) O N . ~ r~ 1:` ~1
b 3 ~ æ ~ ~ Q
~338818
.
Before conjugating the toxin with transferrin,
human transferrin (Tfn) was loaded with iron according
to the method of .~h;n~lr~n et al., (1981), Int. J.
Cancer 27: 329 . The conjugation of Tfn was ~ hf~
5 by first generating free sulfhydryl groups on Tfn with
2-iminothiolane. The 2-iminothiolane was dissolved in
0.8M boric acid, pH 8.5, and incubated with Tfn in an
8:1 molar ratio. After a one hour ;n-llh~t;on at room
temperature, the modified Tfn was separated from the 2-
10 iminothiolane by gel filtration on a G-25 column. M-
maleimidobenzoyl N-llydL~y~ ;n;m;de (MBS) was
dissolved in dimethylformamide and added in five-fold
molar exces:3 to the toxin, which was either native
diphtheria toxin, CRM102, CRM103, or CRM107. This
15 mixture wa~ incubated for thirty minute~3 at room
temperature, and free MBS waE3 removed from the toxin by
chromatographic separation using a G-25 column. The
MBS-conjugated toxin waa mixed with thiolated
transferrin in 1:1:3 molar ratio and incubated for three
20 hours at room temperature. Immunotoxins were purified
by gel filtration on a TSK-3000 HPLC column.
Species cross-reactivity enables one to evaluate
the toxicity and the effectivene3s of the toxin
conjugate in animal models as well as to examine the
25 effectiveness of the Tfn-toxin conjugate in a wide
variety of human tumors~. Tfn-CRM107, when assayed on
Jurkat cells, exhibited a 400, 000-fold differential in
toxicity over CRM107 alone. ~ This repre3ents a
con3iderable il..,~L-JV~ t in the 10, 000-fold selectivity
previously observed between UCHT1-CRM107 and CRM107 on
Jurkat cells.
It has also been found that K562, a human
erythroleukemia cell line characterized by high levels
of transferrin receptors, is also sensitive to Tfn-
CRM107. Assuming that the binding and toxicity of
CRM107 is reduced by 10, 000-fold relative to diphtheria
-- 18 -
~3~
1 ~38 8 ~ 8
toxin on K562 cells, as it is on Vero and Jur~cat cells,
then the differential in toxicity between CRM103 and
Tfn-CRM107 for K562 cells i~ greater than one million.
Binding of the toxin conjugates was Tfn-mediated as
5 shown by the fact that free Tfn completely inhibits
toxicity, as shown in Table 2.
- 19 -
1 3388 ~ 8
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a~ ~ o ~ o
1- r~ o ~ ~ o ~I N ~1
~;
O ,~
U
0~
o
-- o
O ~ ~ o
O
h ~ E~
+ + a~
+
X X X X ~
~ o o
,,, ~ ~` ---- X X
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o o N N
U
I I C)
P ~
- 20 -
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13388~8
Immunotoxins made with antibodies and whole toxins that
are genetically altered in their binding domain possess several
advantages over antibody-toxin-A-chain conjugates. First, as
5 shown with ~;rhthPria toxin, the B-chain translocation activity
can be used in the absence of its binding function to increa~e
reagent potency 10, 000-fold over that of A-chain conjugate~.
Reduction of the disulfide linkage leads to rapid 1088 of
immunotoxin ln vivo, and the release of free antibody that can
10 bind more cells and compete with intact immunotoxins. Use of
whole toxins permits construction of noncleavable thioether
linkages between toxin and antibody. Intact toxins are less
susceptible to proteolytic inactivation than are toxin A
fragments, and may survive longer i vivo.
The immunotoxins of the present invention have full A-
chain activity and full B-chain translocation activity, but they
lack the binding for native ~irhthPria toxin and possess a new
binding domain, which is covalently attached. The immunotoxins
2 0 of the present invention have a greater potency than any
previously ~ sed immunotoxin, and have greater selectivity
between antigen po~itive and antigen negative cells.
Conjugates of the binding moiety and the inactivated
25 f1;rhthPria toxin may be made uging a variety of bif-lnrti~n~l
protein coupling agents. Examples of such reagents are SPDP,
iminoth;ol~nP (IT), bifunctional derivatives of imidoesters such
as dimethyl adipimidate HC1, active esters such as
cin;m;dyl suberate, aldehydes such as glutaraldehyde, bis-
30 azido compound such as bis(~-~7;~ hPn7oyl)-ethylPnP~ m;nP~
diisocyanates such as tolylene 2, 6-diisocyanate, and bis-active
fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene. The
binding moiety, if it ~r~nt~; nçl a carbohydrate moiety, may be
linked to the ~l;rhthPria toxin by means of a covalent bond to
-- 21 -
1 338~ ~
an oxidized carbohydrate moiety on the antibody aa disclo~ed by
U.S. Patent No. 4,671,958.
The immunotoxins of the present invention are useful
in the treatment of any condition requiring bone marrow
transplantation. That i8, T-cell activity from peripheral blood
which r-mt~m;n~tes human bone marrow transplants can be largely
eliminated by prior treatment with the immunotoxins of the
present invention by preventing the reaction of T-cells in the
donor marrow against the host ce}ls, causing graf t -versus-host
disease. Therefore, this reagent is particularly useful in the
treatment of aplastic anemia or leukemia patients who receive
bone marrow tran~plants.
In treating such conditions, human bone marrow and
peripheral blood r~nll~ r cells are treated with varying
c~,n,--~n~rations of an immunotoxin prepared according to the
present invention. The T-lymphocyte cell activity can be reduced
by the immunotoxins at ~ nron~rations which have very little
effect on the activity of the stem cells necessary to rPpoplll~e
the patient' 8 marrow.
The protocol used for the actual treatment of human
donor bone marrow is as follows: the bone marrow is removed from
the human donor, treated i rl vitrQ with an immunotoxin according
to the present invention, and then infused into the irradiated
recipient .
The foregoing description of the specific ~ l;r- ~
will 80 fully reveal the general nature of the invention that
others can, by applying current knowledge, readily modify and/or
adapt for various applications such specific embodiments without
departing from the generic concept, and therefore such
adaptations and modifications are ;nt-on-lP~ to be comprehended
within the meaning and range of equivalents of the disclosed
-- 22 --
,t.
133~8 1 ~
embodiments. It is to be understood that the
phraseology or terminology employed herein is for the
purpose of description and not of limitation.
- 23 -
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