Note: Descriptions are shown in the official language in which they were submitted.
WO~t/02000 2 ~ PCT/US90/04258
IL-2 DELETION MUTANTS
Backqround of the Invention
This invention relates to the use of
recombinant DNA techniques to make mutant interleukin-2
(IL-2) molecules and chimeric IL-2/toxin molecules.
Il-2 is a protein secreted by human
T-lymphocytes which is capable of binding to IL-2
receptors on activated T-lymphocytes and effecting
T-lymphocyte proliferation. IL-~ has been shown to be a
therapeutic immunostimulant in humans (Rosenberg, 1988,
Immunology Today 9 2: 5a-62), and IL-2 or a specific
binding portion thereof can be coupled to the
enzymatically active portion of diphtheria toxin to form
a hybrid molecule with a number of therapeutic
applications ~Murphy U.S. Patent No. 4,675,382, hereby
incorporated by reference). IL-2/diphtheria toxin
hybrid proteins of Murphy '382, which were made using
recombinant DNA techniques, have been shown to inhibit
rejection of transplanted organs (Pankewycz et al.,
~ransplantation 47:318-322 (1989)), and are also
potential therapeutic agents in the treatment of certain
cancers and autoimmune diseases in which the IL-2
receptor plays a role.
IL-2 encoding DNA sequences are reported in a
2~ number of publications, and in addition, a modified
IL-2-encoding gene, in which a cysteine codon is changed
to enhance stability, is described in U.S. Pat. No.
4,518,584, hereby incorporated by reference. U.S.S.N.
834,900, filed Feb. 28, 1986, hereby incorporated by
reference, describes a synthetic IL-2-encoding DNA
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sequence that dlffers ~rom the natural IL-2 encoding DNA
in that it contains more prokaryotic preferred
translation codons than the naturally occurring
sequence.
Amino acid deletions or substitutions have been
made in the IL-2 amino acid sequence (European Pat.
Appln. Nos. 86114468.1 and 87101839.6, U.S. Pat. No.
4,604,377). Although the DNA and amino acid sequences
of IL-2 and its crystal structure are known (Brandhuber
lo et al., 1987, Science 238, 1707), there is little data
available that allows accurate prediction of the regions
of IL-2 that are responsible for biological activity or
are sensitive to proteolytic breakdown; e.g., a single
substitution of the cysteine residue at position 125 of
the IL-2 amino acid sequence with a serine results in
increased stability of the molecule (U.S. Patent No.
4,604,377); a substitution of the tryptophan residue at
position 121 inactivates the molecule; deletion of amino
acid residues 100-104 decreases the biological activity
by two oders of magnitude; and deletion of amino acid
residues 124-126 renders the molecule inactive (Collins
et al., 198~, Proc. Nat. Aca. Sci. 85: 7709; Cohen et
al., 1986, Science 234:349).
SummarY of the Invention
The present invention provides IL-2 mutant
polypeptides that bear a deletion of one to five amino
acids, yet retain the ability to bind to IL-2
receptor-bearing cells. It is known that lysine 76 is a
proteolytic sîte in the IL-2 molecule (Cohen et al.,
1986, Science 234:349). These mutants either delete
this proteolytic site completely, or alter the s.ructure
of that area in an effort to reduce proteolysis. The
IL-2 mutants can be used as immunostimulants or, when
coupled to a toxin to form a hybrid IL2-toxin molecule,
WO91/02000 3 2 ~ ~ 4 ~ ~ ~ ` PcT/us9o/o4258
an be used to treat immune and other disorders
characterized by the presence of the IL-2 receptor.
The invention,thus generally features eight new
mutant IL-2 polypeptides capabl~ of binding to the IL-2
receptor; the IL-2 polypeptides have deletions of one or
more amino acid residues, as follows; 74; 74-78; 75-77;
76-78; 76-79; 15, 78; and 79 (according to the numbering
convention of the Figure, taken from Williams et al.,
Nucleic Acids Res., vol. 16, no. 22 (1988).
In some preferred embodiments, the mutant IL-2
polypeptide may be part of a fusion protein consisting
of a toxin portion (e.g., derived from diphtheria toxin)
covalently linked, preferably through a peptide bond at
its carboxy terminal end, to the mutant IL-2
polypeptide. The diphtheria toxin portion is large
enough to exhibit cytotoxic activity and small enouah to
fail to exhibit generalized eukaryotic cell binding
Preferably, the DNA sequence encoding the IL-2
polypeptide contains nucleotide substitutions designed
to maximize gene expression in the cells used for
expressior i.e., where prokaryotic cells such as E.
coli are used, preferred prokaryotic codons are
substituted for some of the natural codons (this has
been done in the sequence shown in the Figure).
The hybrid molecules of the invention are
useful for treating diseases in which the IL-2 receptor
plays a role, e.g., IL-2 receptor positive malignancies,
allergic reactions, ~nd systemic lupus erythmatosis
(SLE), or to prevent an immune response by IL-2 receptor
bearing T cells that occurs in graft rejec~ion. This
targeted toxin functions by the following mechanism: the
IL-2/toxin, by virtue of the IL-2 domain, binds to high
affinity IL-2 receptor-bearing cells. The IL-2-toxin is
internalized into endocytic vesicles by IL-2
receptor-mediated endocytosis. Acidification of t~e
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endosome causes a conformational change in the toxin,
allowing its me~orane-associating domains to interact
with the endocytic vesicle's membrane and facilitate
translocation of the enzymatically active fragment A
into the cytosol. Once delivered to the cytosol,
fragment A catalyzes the ADP-ribosylation of elongation
factor 2, resulting in inhibition of protein synthesis
and subseguent death of the IL-2-receptor bearing cell.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
Descri~ticn _ the Preferred Embodiments
The drawing is first described.
Drawinq
The Figure is a DNA sequence, encoding IL-2, in
which preferred prokaryotic translation codons are
employed; the numbers correspond to the nu~oering
referred to in this specification.
Construction of the Genes Encodinq IL-2 Deletion
Mutants/Toxin
Amino acids 74 through 79 are contained within
the Xbal/Notl fragment of ~he synthetic IL-2 gene (see
Figure). For each of the eight deletion mutants, an
Xbal/Notl fragment with a dele~ion of DNA encoding
between one and five amino acids is synthesized using an
automated DNA synthesizer according to conventional
techniques. The DNA sequences of the oligonucleotides
are shown in Table I.,
Each Xbal/Notl fragment is synthesized as two
complementary strands with a l/2 Xb 1 site at the 5' end
and a l/2 Notl site at the 3' end. The synthetic DNA's
are gel purified on a denaturing polyacrylamide-urea gel
and complementary strands are annealed according to
conventional methods. The annealed DNA's are ligated
WOgl/02000 - 5 - 2 ~ 9 ~ PCT/US90/04258
into the expression plasmid, pDW15 (Williams et al.,
1987, Prot. Engineering 1: 493 ), which contains the
synthetic IL-2 gene shown in the Figure. Ligation
reactions are transformed into a suitable E. coli host
according to conventional techniques.
Transformants are screened by restriction
digest analysis of minilysate DNA using the restriction
enzyme Ddel. The Ddel restriction digest profile of the
IL-2 mutants differs from that of non-deleted IL-2 due
to elimination of a Ddel site within the Xbal/Notl
fragment of the deletion mutants. The DNA sequence o~
the rL-2 deletion mutants are confirmed by the dideoxy
method of Sanger et al. (1977, Proc. Nat. Acad. Sci.,
74:S463).
The genes encoding the IL-2/diphtheria toxin
fusion proteins are constructed by standard recombinant
D~A techniques, as follows. The IL-2 portion of the
fusion gene is contained within the S~hl/Hindlll
fragment of the IL-2 deletion mutant derived from
pDW15. This DNA fragment is ligated to S~hl/Hindlll
digested plasmid pABM6508 (Bishai et al., 1987, J.
Bacteriol, 169:5140), which contains the diphtheria
toxin-related portion of the fusion up to and including
the amino acid residue Ala 486. The DNA is transformed
2S into a suitable E. coli host an~ plated onto Luria broth
plates plus an appropriate antibiotic for selection,
according to conventional techniques. Transformants are
screened by _del restriction digest analysis of
minilysate DNA and by Western blot analysis, as follows.
Western Blot Anal~sis
-
Total bacterial cell lysates are analyzed by
SDS-polyacrylamide gel electrophoresis (Laemmli, 1970,
Nature 227:680) for the production of IL-2ttoxin
protein. Proteins are electro~lotted onto nylon
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WO91/02000 2 ~ 9 6 - 6 - PCT/US90/04258
membrane and immunoblot analysis is performed according
to conventional techniques. Confirmation of the
expected construct is made by positive cross-reactivity
to both anti-diphtheria toxin (Connaught Laboratories,
Toronto, Ontario, Canada) and to a monoclonal anti-IL-2
antibody, as well as ~y comparison of the size cf the
expressed protein to known IL-2/toxin standard. Final
confirmation of the construct is made by DNA sequence
analysis of the IL-2//toxin gene.
CvtotoxicitY assaY
Referring to Table II, C9l/Pl cells (a
high-affinity IL2 receptor-bearing cell line) were
seeded in 96-well V-bottom plates (Nunc, Roskilde,
Denmark) at a concentration of l05 per well in l00
~1 complete medium. Il-2-toxin was added at varying
concentrations (l0-l2M to l0-6M) in complete
medium. Cells cultured with medium alone were included
as the control. Following 18 hours incubation at 37C
in a 5~ Co2 atmosphere, the plates were centrifuged
for 5 minutes at 170 x g, the medium was removed and
replaced with l00 ~l leucine-free medium (DMEM
Selectamine, Gibco) containing 2.5 ~Ci/ml
~14Cl-leucine ~New England Nuclear, Boston, ~A).
Cells were then incubated at 37 for 90 minutes and
collected on glass fiber filters using a cell harvester
(Skatron, Sterling, VA). Filters were washed, dried,
and counted according to standard methods. All
determinations were performed in pentuplicate. IC50
refers to the concentration of IL2 required to inhibit
protein synthesis to 50~ of the untreated control.
WO 91/02000 2 ~ P~/US90/04258
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Plasmid amino acid(s)
deleted C9l/PL IC50 ~;
psI133 ~74 6xlO lM
psI134 ~75 lxlO lOM
PsI136 ~78 5xlO llM
psIl37 ~79 2xlO lOM
psI143 ~75-77 2xlO lOM
psI141 ~74-78 lxlO 1OM
psIl45 ~76-78 2xlO 1OM
psI150 ~76-79 7xlO1lM
(psI129 no deletion typically
control 5 1o~ll
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WO91/02000 - 8 ~ 2 ~ q ~ PCT/US90/04258
Other Embodiments
Other embodiments are within the following
claims. For example, the deletion mutant IL-2 molecules
can be used alone, in addition to their use in toxic
S hybrids, the deletions can advantagously provide
resistance to proteolysis in both contexts. In
addition, toxins other than diphtheria toxin can be
coupled to the mutants, e.g., the enzymatically active
portion of Pseudomonas exotoxin can be used.
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