Note: Descriptions are shown in the official language in which they were submitted.
W092/20~PCT/US92/~093
~! 3 2 5 8
CYTOKINE RECEPTOR TARGETED MOLECULES FOR
TREATMENT NEOPLASTIC CELL GROWTH
Backaround of the I~vention
5This invention relates to the treatment of
neoplastic cell growth, e.g., cancer.
There have been many attempts to limit neoplastic
cell growth using drugs targeted to neoplastic cells.
Both monoclonal antibodies and receptor ligands have been
10 suggested as targeting agents.
Murphy ~United States Patent 4,675,382) suggests
that a cytotoxin/interleukin-2 hybrid protein could be
used for treatment of cancers involving the immune
system.
LeMaistre et al. (Lancet 337:1124, 1991) describe
the treatment of a patient with chronic ly,mphocytic
leukemia using a diphtheria toxin/interleukin-2 hybrid
molecule.
ummary of the Invention
In general, the invention features a method for
treating neoplastic cell ,growth in a patient, wherein the
neoplastic cell is of non-lymphoid and non-monocytic
origin. The method includes administering to the patient
a molecule whirh is capable of specifically binding to a
25 cytokine receptor expressed on the neoplastic cell, which
molecule is capable of decreasing the viability of the
neoplastic cell. The cytokine receptor is a receptor
normally expressed on cells of lymphoid or monocytic
origin.
In various preferred embodiments, the neoplastic
cell is not of hematopoietic stem cell origin; the
neoplastic cell is a sarcoma; the neoplastic cell is a
W092/203~ PCT/US92~ ~3
r n
~J ~
-- 2
osteosarcoma; the neoplastic cell is a fibrosarcoma; the
neoplastic cell is a leiomyosarcoma; the neoplastic cell
is a carcinoma; and the neoplastic cell is a
rhabdomyosarcoma. In more preferred embodiments, the
5 carcinoma is a lung carcinoma; and the carcinoma is
hepatocellular aarcinoma.
In a preferred embodiment, the cytokine receptor
is an interleukin receptor. In more preferred
embodiments, the interleukin receptor is an interleukin-
10 2 receptor; the interleukin receptor is an interleukin-4
receptor; and the interleukin receptor is an interleukin-
6 receptor. In an even more preferred embodiment, the
interleukin-2 receptor is a high affinity interleukin-2
receptor. In another preferred embodiement, the cytokine
15 receptor is not expressed on non-neoplastic cells of non-
lyphoid and non-monocytic origin.
In still other preferred embodiments, the molecule
kills cells bearing the cytokine receptor; the molecule
is a hybrid molecule includes a first and a second
20 portion joined together covalently, the first portion
includes a molecule capable of decreasing cell viability
and the second portion includes a molecule capable of
specifically binding to the cytokine receptor. In more
preferred embodiments, the second portion includes all or
25 a binding portion of an antibody specific for the
cytokine receptor; the second portion includes all or a
binding portion of a ligand for the cytokine receptor;
and the first portion includes a cytotoxin; and the
antibody is a complement activating antibody. In an even
30 more preferred embodiment, the cytokine receptor is an
interleukin receptor. In a yet more preferred
embodiment, the ligand is an interleukin.
In another preferred embodiment, the cytotoxin is
a fragment of a peptide toxin which is enzymatically
~ 35 active but which does not possess generalized eukaryotic
~092/20~ 2 `1 0 3 2 5 8 PCT/US92/~093
receptor binding activity. In a more preferred
embodiment, the fragment of a peptide toxin includes
fragment A of diphtheria toxin and enough of fragment B
of diphtheria toxin to facilitate translocation into the
5 cytosol.
In other preferred embodiments, the molecule is
DAB389IL-2; the molecule is DAB389IL-4; the molecule is
DAB389IL-6; the molecule is DAB~86IL-2; the molecule is
DAB486IL-4; and the molecule is DAB~86IL-6.
By "non-lymphoid and non-monocytic origin" is
meant all cells that are not descended from lymphoid stem
cells or monocytes, e.g., cells other than T lymphocytes,
B lymphocytes, and macrophages. By " lymphoid and
monocytic origin" is meant cells descended from lymphoid
15 stem cells or monocytes, e.g., T lymphocyte~,
B lymphocytes, plasma cells, and macrophages. By
"specifically binding" is meant that the molecule does
not substantially bind to other cell surface receptors.
By "not of hematopoietic stem cell origin" is meant all
;~; 20 cell are not descended from pluripotent stem~cells.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
Detailed Descri~tion
25 DAB389IL-4 and DAB486IL-2 Reduce the Viability of Several
Cancer Cell Ty~es
Our in vitro toxicity experiments have
demonstrated that certain cancer cells are sensitive to
interleukin-4 or interleukin-2 receptor targeted
30 cytotoxins. Accordingly these interleukin receptor-
targeted cytotoxins, described in detail below, can
provide a means by which to decrease the viability of
: ~
~ cancer cells.
~::
~.:
W092/~0~ PCT/US92/04093
2 5 ~ - 4 -
In the experiments described below various cancer
cell lines are shown to be sensitive to a fusion protein
(DAB389IL-4) in which the receptor binding domain of
diphtheria toxin (a single molecule of which, when
5 present intracellularly, can block protein synthesis) has
been replaced by a portion of human interleukin-4.
Various cell lines were exposed to DAB389IL-4 and
the level of protein synthesis was measured as described
below. The results of this analysis are presented in
10 Table 1 where the IC50 is defined as the concentration of
drug required to cause a 50% decrease in protein
synthesis. Cancer cell lines from striated muscle,
smooth muscle, liver, bone, and lung are sensitive to
DAB389IL-4; the IC50 for these cell lines was found to be
15 lO lOM to 10 8M.
T~ble 1: Sensitivity of Cancer Cells to DAB389IL-4
Ce~ ATCC No. C~sssi~ication IC50 ~ 2
Hep G2 ~ HB 8065 Human Hepaeocel~ular Carcinoma, 2.7 x 10 10
. Hepatitis B
NT-1080 CCL 121 Human F~brosarcoma 3.9 x 10-9
~673 CR~ 1598 Human Rhabdomyos~rcoma 2.7 ~ 10 9
A549 CCL 185 Human Lung Carcinoma 2.1 x 10
2 5 U-2 05 HTB 96 Human Ostec~enic Sarcoma, bone primary 2.6 x 10
SK-ES-1 HTB 86 Human ~nap~as~ic Osteo~arcoma, 2.2 x 10
bone primary
S~-LMS-1 HTB 88 Human Leiomyosarcoma, vulva 1.5 x 10
3 0 .. ~merican ~ype Culture Co~ection (Bethesda, YD) accession number.
The concentration of DAB3891L-4 ~hich leads to a 50X decreae in protein synthesis.
The sensitivity of these cancer cell lines is comparable
to that of cells, such as HTLV-1 transformed T-
lymphocytes and phytohemagglutinin activated T-
3 5 lymphocytes, which are expected to express high numbersof interleukin-4 receptors (Table 2). In contrast,
activated rat T-lymphocytes, which express an
interleukin-4 receptor that is not recognized by human
V092/20~ 2 l~a3,1~58 PCT/US92/04093
5 _
interleukin-4, are not nearly as sensitive to DAB389IL-4,
even though they are sensitive to diphtheria toxin (Table -
2). This result demonstrates that DAB389IL-4 selectively
intoxicates interleukin-4 receptor expressing cells.
S T~bl- 2: DAB389IL-4 Sensitivity of Normal and Neoplastic
Cells Derived from Lymphocytes and Monocytes
Celt or Cel~ Line Classification IC50 (M)
T r,ell origin
HUn 102/~TG Human, CTCL, HTLV I~ 2.9 X 10-11
C91~PL Human, HTLV-I~, transformed 6.3 X 10
1 5 ~ r,ell origin
~ji Human, 8urkitt's lymphoma E8V~ ?.2 X 10-1
~yel _ cle r r,ell
tr~37 Human, histiocytic lymphora 2.0 X 10 9
l ~C
2 0 PHA ctivated T cells Human 1.6 X 10 10
~onrpri _ te
Con A-activated norma~
splenic T cells Rat >10-7
'
2 5 This is the concentration of drug which leads to a 50X dbcresse in protein synthesis.
A similar set of experiments demonstrated that
human rhabdomyosarcoma cell line A673 (ATCC Accession No.
CRL 1598) is sensitive to a fusion protein (DAB4a6-IL-2) in
which the receptor binding domain of diphtheria toxin has
30 been replaced by a portion of human interleukin-2.
: DAB486IL-2 at 7.6 x 10 8M decreased protein synthesis by
50%.
Assay for Sensitivity of Neo~lastic Cells to Interleukin
Receptor Taraeted Cvtotoxins
Cytotoxicity was evaluated by measuring protein
synthesis~ tl~C]leucine incorporation in the presence and
absence of DAB389IL-4 (or DAB~86IL-2) as follows. Cells
were plated 96 well microtiter plates in growth medium
appropiate to the cell type. DAB389IL-4 ((or DABq86IL~2)
~: .
W092/20~ PCT/US92/~ ~3
r
,~ 3 ~
-- 6 --
was added at various concentrations and the cultures were
incubated for 20 hours prior to pulse labelling with
~l~C]leucine. Cells were then trypsinized, harvested onto
glass fiber filter mats and counted. The IC50 is the
5 concentration of cytotoxin which leads to a 50% decre~se
in tl~C]leucine incorporation.
The sensitivity of other cell types, including
cells isolated from a tissue sample (e.q., a biopsy)
obtained from a patient, can be evaluated as described
10 above. Viability can also be measured using any
standard viability assay appropriate to the cell type
being studied. For example, viability can be determined
using a trypan blue dye exclusion assay (Kruse et al.,
eds. Tissue Culture: Methods and Applications, Academic
; 15 Press, 1989).
Molecules Useful in the Method of the Invention
The molecules useful in the method of the
invention are tarageted to a cytokine receptor. In
general, there are three ways in which the molecules
20 useful in the invention can act: (1) the molecule can
kill a cell because the molecule has a cytotoxic domain;
(2) the molecule (an antibody) can cause cell lysis by
inducing complement fixation; and (3) the molecule; can
block binding or uptake of receptor's ligand. In all
25 three cases the molecule must be targeted to receptor
bearing cells; this is accomplished by including the
receptor's ligand (or a portion or derivative thereof~ or
an anti-receptor antibody as part of the molecule.
Interleukin-2 (IL-2) receptor targeted molecules
30 provide examples of each of these three approaches. A
fusion molecule which includes the IL-2 receptor binding
portion of IL-2 and a cytotoxin can be used to kill
neoplastic cells bearing the interleukin-2 receptor.
Likewise, the second type of molecule described above, a
35 complement fixing antibody, in this instance directed
~092/20~ 5 ~ PCT/US92/04093
against the IL-2 receptor, can eliminate IL-2 receptor-
bearing cells. In this example, the third type of
molecule could be a molecule that blocks binding of IL-2
to its receptor. This molecule would prevent neoplastic
5 cells that bear the interleukin-2 receptor from receiving
a proliferation signal from IL-2.
Molecules useful for treating neoplastic cell
growth can take a number of forms. When IL-2 itself is
the targeting agent, the molecule can be a cytotoxic
10 hybrid molecule in which IL-2 is fused to a toxin
molecule, preferably a polypeptide toxin. Derivatives of
IL-2 which bind to IL-2R, lack IL-2 activity and block
binding and/or uptake of bona fide IL-2 are useful in the
method of the invention because they will prevent IL-2-
15 induced proliferation of IL-2R bearing cells. When an
anti-IL-2R antibody is the targeting agent, a cytotoxic
hybrid molecule can be formed by fusing all or part of
the antibody to a cytotoxin. The effectiveness of such
an antibodyjtoxin hybrid, like that of an IL-2/toxin
20 hybrid, depends on the hybrid molecule being taken up by
cells to which it binds. Anti-IL-2R antibodies which
block binding and/or uptake of IL-2 are also useful in
- the method of the invention. Lytic anti-IL-2R antibodies
are useful in the invention because they can cause
25 complement-mediated lysis of IL-2R-bearing cells.
Some of the molecules can be hybrid molecules
formed by the fusion of all or part ~f two or more
molecules. The hybrid molecule can be a hybrid protein
encoded by a recombinant DNA molecule, in which case the
30 two domains are joined (directly or through an
intermediary domain) by a peptide bond. Alternatively,
two domains can be produced separately and joined by a
covalent bond in a separate chemical linkage step. In
some cases, the cytotoxic domain of a hybrid molecule may
35 itself be derived from two separate molecules.
;
W092/20~ PCT/US92/~09~
~ 0 3.?58 - 8 -
Interleukin-2 as a Taraetina Aaent
Interleukin-2 (IL-2) or any IL-2 receptor binding
derivative thereof can be used as a targeting agent for a
cytotoxin. The DNA and amino acid sequences of IL-2 are
5 known (Tadatsugu et al., Nature 302:305, 1983), and its
structure has been predicted by x-ray crystallography
(Brandhuber et al., Science ~38 : 1707, 1987) . Analysis of
genetically engineered variants of IL-2 has provided some
information concerning which residues are important for
10 IL-2R binding (Collins et al., Proc. Natl. Acad. Sci. USA
85:7709, 1988) and bioactivity (Cohen et al. Science
234 : 349, 1989 ; Collins et al., supra) . Variants of IL-2
which are useful in the invention include deletion
mutants (Genbauffe et al., USSN 388,557, hereby
15 incorporated by reference) which lack one or more amino
acid residues in the region between residue 74 and
residue 79 (numbering according to Williams et al., Nucl.
Acids Res. 16:1045, 1988). These mutants effectively
target toxins to IL-2R-bearing cells (Genbauffe et al.,
20 supra). Generally, IL-2 variants useful for targeting a
cytotoxin must efficiently bind IL-2R and be endocytosed.
The ability of various derivatives to bind to the IL-2
receptor can be tested with an IL-2R binding assay
described below. ~
In designing molecules targeted to cells bearing
the IL-2 receptor it must be recognized that the IL-2
receptor, like other receptors, has several forms; and it
- may be desirable to target cells bearing one form and not
another. The human interleukin-2 receptor has a high-,
30 an intermediate-, and a low-affinity form. The high
affinity receptor has an apparent Kd of -10 lOM and is
composed of two subunits, p55 and p75 (also called p70).
When expressed on the cell surface, both the p75 and pS5
subunits are capable of binding IL-2. The p75 subunit
35 corresponds to the intermediate affinity receptor (Kd ~
-`'092/20~ PCT/US92/~093
8.2 x 10 lOM), and p55 subunit corresponds to the low
affinity receptor (Kd ~ 1-3 x 10 8M). The p75 subunit is
expressed on the surface of resting T cells, natural
killer cells, monocytes/macrophages, and lymphokine-
5 activated killer (LAK) cell precursors, while the highaffinity receptor is expressed on activated T- and B-
cells.
In the method of the invention it may be desirable
to target only cells bearing the high affinity receptor.
10 In these circumstances useful molecules will eliminate or
neutralize cells bearing the high affinity IL-2 receptor
at a concentration which leaves cells bearing the
intermediate or low affinity receptor largely unaffected.
When the molecule, like IL-2 itself, has affinity for all
15 three classes of IL-2 receptor, selectivity can be
accomplished by administering the molecule at a
concentration which does not permit significant binding
to cells bearing lower affinity receptors. A hybrid
molecule may have altered receptor affinities compared to
20 IL-2. Such hybrid molecules may be more or less
selective for cells bearing the high affinity IL-2
receptor. For example, cells bearing the high-affinity
receptor are 500-1000 times more sensitive to DAB~86IL-2,
a fusion protein consisting of part of diphtheria toxin
25 and part of IL-2, than are cells bearing the
intermediate- affinity receptor (Waters et al., Eur. J.
Immunol . ~0: 785, 1990).
A cytotoxin can be attached to an IL-2 derivative
in a number of ways. Preferably, an IL-2/toxin hybrid is
30 a hybrid protein produced by the expression of a fused
gene. Alternatively, the cytotoxin and the IL-2
derivative can be produced separately and later coupled
by means of a non-peptide covalent bond. Linkage methods
are described below.
W092/203~ PCT/US92/040
S ~3
-- 10 --
Useful cytotoxins are preferably significantly
cytotoxic only when present intracellularly and are
substantially excluded from any given cell in the absence
of a targeting domain. Peptide toxins fulfill both of
5 these criteria and are readily incorporated into hybrid
molecules. A mixed cytotoxin, a cytotoxin composed of
all or part of two or more toxins, can also be used.
Several useful toxins are described in more detail below.
Interleukin-4 and Interleukin-6 as a Taraetina Aaents
Interleukin-4 (IL-4) is a cytokine which acts on a
variety of cell types. Its receptor is expressed on a
number of cell types, including CD4~ T cells and
monocytes. IL-4 can act as a T cell growth factor and it
is thought to have an influence on IL-2 induced
15 lymphocyte proliferation.
A cytotoxin directed against IL-4 receptor-bearing
cells or IL-6 receptor-bearing cells may enhance the
effectiveness of molecules directed against IL-2R-bearing
cells. The protein and DNA sequence of IL-4 and IL-6 are
20 known ~Lee et al., J. Biol . Chem. 263:10817,~1988; Hirano
et al., Nat~re 324:73, 1986). These lymphokines can be
used to create hybrid lymphokine/toxin molecules similar ,
to IL-2/toxin hybrid molecules.
Monoclonal Antibodies as Taraeting Agents
Monoclonal antibodies directed against the
lymphokine receptor of choice can be used to direct
toxins to cells bearing that receptor. These antibodies
or antibody fragments can be fused to a cytotoxin either
by virtue of the toxin and the antibody being encoded by
30 a fused gene which encodes a hybrid protein molecule, or
by means of a non-peptide covalent bond which is used to
join separately produced ligand and toxin molecules.
Several useful toxins are described below.
Antibody/toxin hybrids can be tested for their
35 ability to kill receptor bearing cells using a toxicity
-~092/203~ ~ 1~J 3 q ~ 8 PCT/US92/~093
assay similar to that which is described below for IL-2R
bearing cells.
Monoclonal antibodies useful in the method of the
invention can be made by immunizing mice with human IL-
5 2R or cultured T-lymphocytes, fusing the murine
splenocytes with appropriate myeloma cells, and screening
the antibodies produced by the resultant hybridoma lines
for the requisite IL-2R binding properties by means of an
ELISA assay. Antibody production and screening can be
10 performed according to Uchiyama et al. (J. Immunol.
126:1393, 1981). Alternatively, useful antibodies may be
isolated from a combinatorial library produced by the
method of Huse et al. (Science 246:1275, 1989).
The invention can employ not only intact
15 monoclonal or polyclonal antibodies, but also an
immunologically-active antibody ~ragment, for example, a
Fab or (Fab)2 fragment; an
antibody heavy chain, an antibody light chain; a
genetically engineered single-chain Fv molecule (Ladner
20 et al., U.S. Patent No. 4,94~,778); or a chimeric
antibody, for example, a "humanized" antibody which
contains the binding specificity of a murine antibody,
but in which most or all of the remaining portions are of
human origin (Reichman et al., Nature 332:323, 1988).
25 Toxins
The toxin molecules useful in the method of the
invention are preferably toxins, such as peptide toxins,
which are significantly cytotoxic only when present
intracellularly. Of course, under these circumstances
30 the molecule must be able to enter a cell bearing the
targeted receptor. This ability depends on the nature of
the molecule and the nature of the cell receptor. For
example, cell receptors which naturally allow uptake of a
ligand are likely to provide a means for a ~olecule which
35 includes a toxin to enter a cell bearing that receptor.
W092/20~ PCT/US92/04~3
~ - `J 3 ;~ S 8
- 12 -
Preferably, a peptide toxin is fused to an IL-2R binding
domain by producing a recombinant DNA molecule which
encodes a hybrid protein molecule. Such an approach
ensures consistency of composition.
Many peptide toxins have a generalized eukaryotic
receptor binding domain; in these instances the toxin
must be modified to prevent intoxication of non-receptor
bearing cells. Any such modifications must be made in a
manner which preserves the cytotoxic functions of the
10 molecule (see U.S. Department of Health and Human
Services, U.S. Serial No. 401,412). Potentially useful
toxins include, but are not limited to: cholera toxin,
ricin, 0-Shiga-like toxin (SLT-I, SLT-II, SLT IIV), LT
toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus
15 toxin, Pseudomonas exotoxin, alorin, saporin, modeccin,
and gelanin.
Diphtheria Toxin-based Molecules
Diphtheria toxin can be used to produce molecules
useful in the method of the in~ention. Diphtheria toxin,
20 whose sequence is known, is described in detail in Murphy
U.S. Patent 4,675,382, hereby incorporated by reference.
The natural diphtheria toxin molecule secreted by
Corynebacterium diphtheriae consists of several
functional domains which can be characteri~ed, starting
25 at the amino terminal end of the molecule, as
enzymatically-active Fragment A (amino acids Glyl -
Arg~93) and Fragment B (amino acids Serlg~ - Ser535), which
includes a translocation domain and a generalized cell
binding domain (amino acid residues 475 through 535).
The process by which diphtheria toxin intoxicates
sensitive eukaryotic cells involves at least the
following steps: (i) the binding domain of diphtheria
toxin binds to specific receptors on the surface of a
sensitive cell; (ii) while bound to its receptor, the
35 toxin molecule is internalized into an endocytic vesicle;
~0 92/20364 PCI /IJS92/04093
~r~
~ ~ J ~ h~
-- 13 --
(iii) either prior to internalization, or within the
endocytic vesicle, the toxin molecule undergoes a
proteolytic cleavage between fragments A and B; (iv) as
the pH of the endocytic vesicle decreases to below 6, the
5 toxin crosses the endosomal membrane, facilitating the
delivery of Fragment A into the cytosol; (v) the
catalytic activity of Fragment A (i.e., the nicotinamide
adenine dinucleotide - dependent adenosine diphosphate
(ADP) ribosylation of thè eukaryotic protein synthesis
10 factor termed "Elongation Factor 2") causes the death of
the intoxicated cell. It is apparent that a single
molecule of Fragment A introduced into the cytosol is
sufficient to block down the cell's protein synthesis
machinery and kill the cell. The mechanism of cell
15 killing by Pseudomonas exotoxin A, and possibly by
certain other naturally-occurring toxins, is very
similar.
DAB486IL-2, a fusion protein in which the receptor
binding domain of diphtheria toxin has been replaced by a
20 portion of human IL-2 (Williams et al., J. Biol. Chem.
35:20673, 1990; see also Williams et al., Protein Eng.
1:493, 1987), is an example of a molecule useful in the
method of the invention. This molecule selectively kills
IL-2R-expressing tumor cells and lymphocytes (Waters et
25 al., Eur. J. Immunol. 20:785, 1990; Kiyokawa et al.,
Cancer Res. 49:4042, 1989). Because of its ability to
kill activated lymphocytes, DAB486IL-2 has been used to
control graft rejection (Pankewycz et al.,
Transplantation 47:318, 1989; Kickman et al.,
30 Transplantation 47:327, 1989) and to treat certain
autoimmune disorders (Forte et al., 2nd International
Symposium on ~mmunotoxins, 1990).
DAB~86IL-2 is a chimeric molecule consisting of
Met followed by amino acid residues 1 throuqh 485 of the
35 mature diphtheria toxin fused to amino acid residues 2
WO 92/20364 PCI/US92/04~3
iJ ~ 2 ~ 8
-- 14 --
through 133 of IL-2. Thus, DAB~86IL-2 includes all of
diphtheria toxin fraqment A, which encodes the
enzymatically active portion of the molecule, and a
portion of fragment B. The portion of fragment B present
5 in DAB~86IL-2 does not include the generalized receptor
binding domain but does include the translocation domain
which facilitates delivery of the enzymatically active
portion into the cytosol.
Pre~aration of DAB486IL-2 and DAB389IL-2
DAB~86IL-2 was produced in E. coli harboring the
DA8~,86IL-2 encoding plasmid, pDW24 (Williams et al., J.
Biol. Chem. 265:20673, 1990, except ampr is replaced by
kanr). The protein was purified by in~nunoaffinity
chromatoqraphy and high pressure liquid chromatography
15 (Williams et al., supra) . DAB389IL-2 can be prepared as
described below for DAB389IL-4 by substituting IL-2 for
IL-4.
Preparation of DAB389IL-4 and DAB486IL-2
A synthetic gene encoding human interleukin-4 was
io synthesized (Milligen/Biosearch i500 DNA sy~thesizer).
The IL-4 sequence (Yodota et al., Proc Nat ' l Acad Sci .
USA, 83:58994, 1986) was modified to incorporate E. coli-
preferred codon usage (deBoer et al., in Maximizing Gene
Expression , Reznikioff et al., eds., 1986, Butterworths,
25 Boston), and restriction endonuclease cleavage sites were
added to facilitate subsequent cloning steps. IL-4
coding sequence (Hisl to Serl29) was inserted into pDW27
plasmid. pDW27 is derived from pDW24 (Williams et al.,
J. Biol. Chem. 265:11885, 1990) by deleting DNA
30 corresponding to amino acids 388 to 485 OI native
diphtheria toxin. DAB~86IL-4 can be prepared as described
above for DAB~86IL-2 by substituting IL-4 for IL-2.
CYtotoxicitY of DAB389IL-4
~092/203~ ,~ A~ r 8 PCT/US92/04093
~_ J~
- 15 -
The ability of DAB389IL-4 to reduce viability of
various cell types was measured using an inhibition of
protein synthesis-assay; the results of this assay are
presented in Table 3. ICso (M) is the concentration of
5 DAB389IL-4 required for a 50% decrease in protein
synthesis. The rat, Con A-activated, normal splenic
lymphocytes were far less sensitive to DAB389IL-4 than any
of the other cells or cell lines. Since the rat
interleukin-4 receptor does not bind human interleukin-
10 4, this result demonstrates the specificity of DAB389IL-
4. These rat cells are sensitive to a diphtheria
toxin/rat interleukin-2 hybrid molecule.
Preparation of DAB389IL-6 and DAB~86IL-6
A synthetic gene encoding human interleukin-6 was
15 synthesized (Nilligen/Biosearch 7500 DNA synthesizer).
The IL-6 sequence (Revel et al., EPA 8611404.9) was
modified to incorporate E. Coli preferred codon usage
(deBoer et al., supra ), and restriction endonuclease
cleavage sites were added to facilitate subsequent
20 cloning steps. The entire IL-6 coding sequence was
inserted into pDW27 plasmid as described above for
DA8389IL-4. DAB~86IL-6 can be produced as described above~
for DABq86IL~2 by substituting IL-6 for IL-2.
Mixed Toxins
2S The cytotoxic portion of some molecules useful in
the invention can be provided by a mixed toxin molecule.
A mixed toxin molecule is a molecule derived from two
different polypeptide toxins. Generally, as discussed
above in connection with diphtheria toxin, polypeptide
30 toxins have, in addition to the domain responsible for
generalized eukaryotic cell binding, an enzymatically
active domain and a translocation domain. The binding
and translocation domains are required for cell
recognition and toxin entry respectively. The
W092/20~ PCT/US92/04~93
~ ~ f~ r~ ~
d
- 16 -
enzymatically active domain is the domain responsible for
cytotoxic activity once the molecule is inside a cell.
Naturally-occurring proteins which are known to
have a translocation domain include diphtheria toxin,
5 Pseudomonas exotoxin A, and possibly other peptide
toxins. The translocation domains of diphtheria toxin
and Pseudomonas exotoxin A are well characterized (see,
e.g., Hoch ~t al., Proc. Natl. Acad. Sci. USA 82:1692,
1985; Colombatti et al., J. Biol. Chem. 261:3030, 1986;
10 and Deleers et al., FEBS Le~t. 160:82, 1983), and the
existence and location of such a domain in other
molecules may be determined by methods such as those
employed by Hwang et al., Cell 48:129, 1987; and Gray et
al., Proc. Natl. Acad. Sci. USA 81:2645, 1984).
One useful IL-2/mixed toxin hybrid molecule is
formed by ~using the enzymatically active A subunit of E.
coli Shiga-like toxin (Calderwood et al.j Proc. Natl.
Acad. Sci. USA 84:4364, 1987) to the translocation domain
(amino acid residues 202 through 460) of diphtheria
20 toxin, and to IL-2. This three-part bybrid molecule,
SLT-A/DTB'/IL-2, is useful in the method of the invention
in the same way as DAB~86IL-2 described above. The IL-2
portion of the three-part hybrid causes the molecule to
attach specifically to IL-2R-bearing cells, and the
25 diphtheria toxin translocation portion acts to insert the
enzymatically active A subunit of the Shiga-like toxin
into the targeted cell. The enzymatically active portion
of Shiga-like toxin, like diphtheria toxin, acts on the
protein synthesis machinery of the cell to prevent
30 protein synthesis, thus killing the cell. The difference
between these two types of hybrid toxins is the nature of
their enzymatic activities: the enzymatic portion of
DAB486IL-2 catalyzes the ADP-ribosylation by nicotinamide
adenine dinucleotide of Elongation Factor 2, thereby
35 inactivating this factor which is necessary for protein
W092/203~ 2 1 0 ~ 2 5 ~ PCT/US92/~093
synthesis, while the enzymatic portion of SLT-A/DTB'/IL-
2 is a ribonuclease capable of cleaving ribosomal RNA at
a critical site, thereby inactivating t~e ribosome. SLT-
A/DTB'/IL-2 hybrid would therefore be useful as a
5 treatment for the same indications as DAB~86IL-2, and
could be substituted or used in conjunction with it.
Linkaae of Toxins to Bindina Liaands
The binding ligand and the cytotoxin of useful
hybrid molecules can be linked in several ways. If the
10 hybrid molecule is produced by expression of a fused
gene, a peptide bond serves as the link between the
cytotoxin and the binding ligand. Alternatively, the
toxin and the binding ligand can be produced separately
and later coupled by means of a non-peptide covalent
15 bond.
For example, the covalent linkage may take the
form of a disulfide ~ond. In this case, if the IL-2R
binding ligand is a protein, e.g., IL-2, the DNA encoding
IL-2 can be engineered to contain an extra cysteine codon
20 as described in Murphy et al. U.S. Serial No. 313,599,
hereby incorporated by reference~ The cysteine must be
positioned so as to not interfere with the IL-2R binding
activity of the molecule. For example, the cysteine
codon can be inserted just upstream of the DNA encoding
25 pro2 of the mature form of IL-2. The toxin molecule must
be derivatized with a sulfhydryl group reactive with the
cysteine the modified IL-2. In the case of a peptide
toxin this can be accomplished by inserting a cysteine
codon into the DNA sequence encoding the toxin.
30 Alternatively, a sulfhydryl group, either by itself or as
part of a cysteine residue, can be introduced using solid
phase polypeptide techniques. For example, the
introduction of sulfhydryl groups into peptides is
described in Hiskey (Peptides 3:137, 1981).
35 Derivatization can also be carried out according to the
~ .
W092/20~ PCT/US92/~093
~lJ~258 18 -
method described for the derivatization of a peptide
hormone in Bacha et al. U.S. Patent No. 4,468,382, hereby
incorporated by reference. Similarly, proteins can be
derivatized at the DNA or protein chemistry level. The
5 introduction of sulfhydryl groups into proteins is
described in Maasen et al. (Eur. J. Bioch~m. 134:32,
1983). The cytotoxin and the IL-2R binding ligand are
then produced and purified, and the disulfide bond
between the purified molecules formed by reducing both
10 sulfur groups, mixing toxin and ligand, in a ratio of
about 1:5 to 1:20, and allowing disulfide bond formation
to proceed to completion (generally 20 to 30 minutes) at
room temperature. The mixture is then dialyzed against
phosphate buffered saline to remove unreacted ligand and
15 toxin molecules. Sephadex chromatography or the like is
then carried out to separate on the basis of size the
desired toxin-ligand conju~ates from toxin-toxin and
ligand-ligand conjugates.
~ssays for IL-2 Receptor Bindina and IL-4 Receptor
20 Bindina
The IL-2R binding ability of various molecules can
be measured using an IL-2R assay for high affinity (Ju et
al., J. Biol . Chem. 262:5723, 1987) or intermediate
affinity receptors (Rob et al., Proc. Natl . Acad.- Sci .
25 USA 84:2002, 1987~. The IL-4R binding activity of
various molecules can be measured using the assay
described by Park et al. (J. Exp. ~ed. 166:176, 1984) or
the assay described by Foxwell et al. (Eur. J. Immunol.
19:1637, 1989).
30 Assays for ToxicitY
Molecules of the invention (both antibodies and
hybrid molecules) can be screened for the ability to
decrease viability of cells bearing the targeted receptor
by mèans of assays such as those described below.
~ 092/203~ 2 1 u 3 2 5 8 PCT/US92/04093
-- 19 --
Toxicity towards IL-2R bearing cells can be tested
as follows. Cultured HUT 102/6TG (Tsudo et al., Proc.
Natl . Acad . Sci . USA 83:9694, 1986) or YT2C2 (Teshigiwari
et al., J . Exp. Med . 165:223, 1987) cells are maintained
5 in RPMI 1640 me~ium (Gibco, Grand Island, NY~
supplemented with 25 mM HEPES (pH 7.4), 2mM l-glutamine,
100 U/ml penicillin, 100 ~g/ml streptomycin, and 10%
fetal calf serum (Hazelton, Lenexa, KS). Cells are
seeded in 96-well V-bottomed plates (Linbro-FIow
10 Laboratories, McLean, VA) at a concentration of 1 x 105
per well in complete medium. Putative toxins are added
to varying c~ncentrations (10 12M to 10 6M) and the
cultures are incubated for 18 hrs. at 37C in a 5% C02
atmosphere. Following incubation, the plates are
15 centrifuged for 5 min. at 170 x g, and the medium removed
and replaced with 100 ~1 leucine-free medium (MEM, Gibco)
containing 8 ~Ci/ml (3H-leucine; New England Nuclear,
Boston, MA). After an additional 90 min. at 37C, the
plates are centrifuged for 5 min. at 170 x g, the medium
20 is removed, and the cells are collected on glass fiber
filters using a cell harvester (Skatron, Sterling, VA).
Filters are washed, dried, and counted according to
standard methods. Cells cultured with medium alone serve
as the control.
Toxicity towards cells bearing IL-4R may be tested
by an assay similar to that described above for IL-2R
bearing cells, but utilizing MLA144 cells (Rabin et al.
J. Immunol . 127 :1852,-1981) or HUT 102/6TG cells, seeded
at 1 x 105 cells per well and incubated for 40 hours.
30 Therapv
Generally, the molecules of the invention will be
administered by intravenous infusion. They may also be
administered subcutaneously. Dosages of molecules useful
in the methods of the invention will vary, depending on
35 factors such as whether the substance is a cytotoxin, a
W092/20~ PCT/USg2/~3
2 i ~ 8 - 20 -
lytic antibody, or an IL-2R blocking molecule. In the
case of toxic molecules that act intracellularly, the
extent of cell uptake is an important factor; less
permeable molecules must be administered at a higher
S dose.
More than 60 patients have received DAB~86IL-2 in
Phase I/II clinical protocols. The maximum tolerated
dose (MTD) established by transient asymptomatic hepatic
transaminase elevations in about 30% of patients treated
10 at the NTD. Anti-tumor effects have been seen in
approximately 40% of patients; responses were noted in B-
cell leukemias and lymphomas, cutaneous T-cell lymphoma
and Hodgkin's disease (LeMaistre et al., Blood
360à:abstract 1429, 1990; Woodworth et al., Fourth
15 International Conference on Human Retrovirology, 1991).
Serum concentrations of 10 8M DAB~86IL-2 have been
achieved in patients with IL-2 receptor expressing
malignancies. Anti-tumor effects have been observed in
highly refractory leukemia/lymphoma patients and these
20 effects have occurred despite the presence o~f elevated
soluble IL-2R levels in all patients. This observation
is consistent with data which suggest that soluble IL-2R ,
does not interfere with binding of IL-2 to the high
affinity interleukin-2 receptor. Animal and human
25 studies have demonstrated that DAB486IL-2 has no general
immunosuppressive effect (LeMaistre et al., supra ;
Woodworth et al., supra ) .
- Experiments indicate that binding and
internalization of DAB~86IL-2 by cells bearing the high
30 affinity IL-2 receptor occurs within 30 minutes of
exposure, resulting in maximal inhibition of protein
synthesis within several hours. Therefore, the molecule
should be effective even if the serum half-life is rather
short.
~.~VO 92/20364 PC~r/US92/04093
t3 U
- 21 -
Generally, drugs targeted to IL-2 receptor will be
administered immediately after (e.g., within several
minutes or less) vascular injury. Preferably, treatment
begins before the accumulation of platelets and
5 leukocytes. Animal models of denuding balloon catheter
injury have been used to show that platelet aggregation
and thrombus formation occurs immediately after injury
and that leukocyte adhesion begins within several hours.
Autopsy of a patient who died S days after angioplasty
10 revealed that proliferating smooth muscle cells were
invading the dilated region (Austin et al., J. Amer.
Coll . Cardiol . 6:369, 1985).
Once initiated, endothelial regeneration is
complete within one to two weeks. Since re-
15 endothelialization of the vessel wall appears to inhibitsmooth muscle cell proliferation (Ip et al., J. Amer.
Coll . Cardiol . 15:1667, 1990), treatment may need to last
for only a few weeks. Accordingly, it is desirable to
administer the compounds of the invention periodically
20 over a period adequate to allow regeneration of the
endothelium.
The hybrid molecule can be administered as an
unmodified molecule or in the form of a pharmaceutically
acceptable salt, admixed with a therapeutically -
25 acceptable carrier, e.g., saline. Examples of preferredsalts are therapeutically acceptable organic acids, e.g.,
acetic, lactic, maleic, citric, or salicylic. For
example, the hybrid molecule may be purified and sterile
filtered using 2 micron filters and suspended in sterile
30 phosphated buffer saline (0.15M NaCl; 0.02M phosphate
buffer, pH 7.2).
Other Embodiments
Derivatives of IL-2 which block utilization of
endogenous IL-2 are useful for preventing proliferation
W092/20~ PCT/US92/04~93
~ i ù ~, 2 ~ 8
- 22 -
of IL-2R bearing cells. Activated cells deprived of IL-
2 fail to proliferate and, in the absence of the
essential anabolic stimulus provided by IL-2, will
eventually die. The ability of a given IL-2 derivative
5 to interfere with IL-2 function can be tested in an IL-2
bioactivity assay such as the one described by Ju et al.
(J. Biol . Chem. 262: 5723, 1987). IL-2R/toxin hybrids in
which the toxin has been rendered inactive can be also
used to block IL-2 receptors. A non-toxic mutant
10 diphtheria toxin molecule has been described (Uchida et
al. J. Biol. Chem. 248:3838, 1973), and this molecule can
be used to produce a non-toxic IL-2/diphtheria toxin
hybrid. See Svrluga et al. U.S. Serial No. 590,113,
hereby incorporated by reference, for an example of such
15 a hybrid molecule.
~-~ Monoclonal antibodies which interfere with the
binding and/or uptake of IL-2 are useful in the method of
the invention because IL-2R bearing cells deprived of IL-
2 fail to proliferate. Blocking monoclonal antibodies
20 can be tested for their ability to interfere with IL-2
~ bioactivity using the method of Ju et al.,(supra) .
`~ Nonoclonal antibodies which induce complement can
be used to destroy IL-2R-bearing cells. Complement
inducing antibodies are generally those of the Ig~l,
25 IgG2, IgG3, and IgM isotypes. Monoclonal anti-IL-2R
antibodies can be screened for those able to induce
complement using a complement-dependent cytotoxicity
test, as follows.
Human T-lymphocytes and EBV transformed B-
30 lymphocytes are labeled with 51Cr sodium chromate and used
as target cells; these cells are incubated with hybridoma
culture supernatants and with complement, and then the
supernatants are collected and counted with a gamma
counter. Those supernatants exhibiting toxicity against
35 activated T-lymphocytes, but not resting T- or B-
~092/20~ 2 1 '~ 3 2 5 8 PCT/US92/04093
. - 23 -
lymphocytes, are selected, and then subjected to a
further screening step to select those supernatants
containing antibody which precipitates (i.e., is
specifically reactive with) the 50 kd glycoprotein IL-2
5 receptor (described in detail in Leonard et al. (Proc.
Natl . Ac~d . Sci . USA 80:6957, 1983). The desired anti-
IL-2 rèceptor antibody is purified from the supernatants
using conventional methods.
