IL-2 DELETION MUTANTS

Abstract: A mutant IL-2 molecule capable of binding an IL-2 receptor-bearing cell, having a deletion of one to five amino acid residues of IL-2, the deletion resulting in active IL-2 molecules that have increased resistance to proteolysis.

Description: IL-2 DELETION MUTANTS Background " 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. 11-2 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-2 has been shown to be a therapeutic immunostimulant in humans (Rosenberg, 1988, Immunology Today 9:2: 58-62), 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. , Transplantation 4.7:318-322 (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 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 sequence that differs from 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 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., 1988, 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 site in the IL-2 molecule (Cohen et al., 1986, Science 234:349). These mutants either delete this proteolytic site completely, or alter the structure 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, can be used to treat immune and other disorders characterized by the presence of the IL-2 receptor. The invention thus 'jenerally features eight new mutant IL-2 polypeptides capable 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; 75, 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 enough 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 expression; 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, and systemic lupus erythmatosis (SLE), or to prevent an immune response by IL-2 receptor bearing T cells that occurs in graft rejection. 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 the endosome causes a conformational change in the toxin, allowing its membrane-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 subsequent 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. Description of the Preferred Embodiments The drawing is first described. Drawing The Figure is a DNA sequence, encoding IL-2, in which preferred prokaryotic translation codons are employed; the numbers correspond to the numbering referred to in this specification. Construction of the Genes Encoding IL-2 Deletion Mutants/Toxin Amino acids 74 through 79 are contained within the Xbal/Not fragment of the synthetic IL-2 gene (see Figure). For each of the eight deletion mutants, an Xbal Notl fragment with a deletion 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 1/2 Xbal site at the 5' end and a 1/2 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 into the expression plasmid, pDWl5 (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 of the IL-2 deletion mutants are confirmed by the dideoxy method of Sanger et al . (1977, Proc. Nat. Acad. Sci., 74:5463). The genes encoding the IL-2/diphtheria toxin fusion proteins are constructed by standard recombinant DNA techniques, as follows. The IL-2 portion of the fusion gene is contained within the Sphl/Hindlll fragment of the IL-2 deletion mutant derived from pDW15. This DNA fragment is ligated to Sphl/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 into a suitable E. coli host and plated onto Luria broth plates plus an appropriate antibiotic for selection, according to conventional techniques. Transformants are screened by Ddel restriction digest analysis of minilysate DNA and by Western blot analy is, as follows. Western Blot Analysis Total bacterial cell lysates are analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970, Nature 227:680) for the production of IL-2/toxin protein. Proteins are electroblotted onto nylon membrane and iiranunoblot 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 by comparison of the size of 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. Cytotoxicity assay Referring to Table II, C91/P1 cells (a high-affinity IL2 receptor-bearing cell line) were seeded in 96-well V-bottom plates (Nunc, Roskilde, 5 Denmark) at a concentration of 10 per well in 100 μl complete medium. Il-2-toxin was added at varying concentrations (10 —12M to 10—6M) in complete medium. Cells cultured with medium alone were included as the control. Following 18 hours incubation at 37°C in a 5% Co, atmosphere, the plates were centrifuged for 5 minutes at 170 x g, the medium was removed and replaced with " 100 μl leucine-free medium (DMEM Selectamine, Gibco) containing 2.5 μCi/ml 14 [ C]-leucine (New England Nuclear, Boston, MA). 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. IC 50 refers to the concentration of IL2 required to inhibit protein synthesis to 50% of the untreated control. IL-2 coding sequence ""* • 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 8 (5' - 3') - CTA GAA GAA. GAA CTG AAA CCG CTG GAG GAA GTT CTG .AAC CTG GCT CAG TCT AAA AAC TTC CAC CTG CGG CCG C amino acid -* leu Glu Gin Glu Leu Lys Fro LeuG n Glu Tal Leu .Asa Leu Al* Gin Ser Lys Asn Phe His Leu krg fro psl 133 (Δ74) T CTA GAA SAΛ GAA.CTG AAACCGCT&GAβGAAGtT α*AAC <TOGCATCT .AAAΛAC TTCCAC CTGCΘ&CCGC& Leu Glu GLu Glu Leu Lys Pro Leu Glu Glu Y»l Leu .Asa Leu .Al* Ser Lys Asn Pl»e His Leu .AΓQ- Pro psl 134 (Δ75) TCTλGAAGAAGAACreAAAC<»CTGGAfrGΛAΘTT CT8AAC CT&ΘCA<aG CTG C8G CCG CG Leu Glu Glu Glm Leu Lys Pro Leu β_u Gin Tnl Leu Asn Leu Al* Gin Lys .Asa Phe His Leu krg Pro psl 136 (Δ78) T CTlGAAGAAGAACreAAACCGCreeA&GAAGTTCT&AAC CTO Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Tnl Leu Asa Leu Al* Gin Ser Lys Asa His Leu Ar-g Pro psl 137 (Δ 79 ) τ CTJ^ QAA GAA -GAA CTG AAA CCG CTG GAG GAA OTT CTG AAC CTG GCA CAG TCT -AAA AAC TTC CTG CGG CCG CG Leu Glu Glu Gin Leu Lys PrαLeu Glu G n Y*l Leu Asa Leu Al* Gin Ser Ly* Asn Phe Leu Arg Pro psl 143 (Δ75-77) T CTA. GAA GAA GAA CTG JAA<X8<^GAGGAAGTT <«AAC CT8GCACAGTrCCT&CGGCCG<» Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Tel Leu .Asa Leu All 61a Phe Leu Ar_r Pro psl 141(Δ74-78) CTAGAAGAAGAACTOAAACCGCTG6AGGAAGTTCTGAAe <HO6*<^ Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Tn Leu .Asa Leu .Al*. HLs Leu _τq Pro psl 150 (Δ76-79) τ CTA GAA GAA GAACTGAAACCG<π«GAGGAAGrr CTGAA CTGCGGCCG C& Leu Glu Glu Glu _%_ Lys Pro Leu G n Glu T*l Leu Asa Leu λl_ Gn Ser His Leu _τq Pro psl 145(Δ76-78) T CTA GAA GAA GAA CTG A1Λ CCG CTG GAG GAA GTT CTG AAC CTG GCA CAG TCT CTG CG8 CCG CG Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Tal Leu Asa Leu Al*S»la Ser Leu Λrg Pro TABLE I Table I I Plasmid amino acid(s) deleted C91/PL IC50 psI133 74 6xlO _1 M psI134 Φ75 lxlO ~10 M PSI136 Φ78 - 5xlO -11 M psI137 Φ79 2X10~ 10 psI143 Φ75-77 2X10 ~10 M psI141 Φ74-78 lχlθ "10 M psI145 Φ76-78 2X10 ~10 M psI150 Φ76-79 7X10 1:L (psI129 no deletion typically control 5xlO -11 M) 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 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.

Claims: Claims 1, A mutant IL-2 molecule in which only amino acid residue 74 has been deleted. 2. A mutant IL-2 molecule in which only amino acid residues 74-78 have been deleted. 3. A mutant IL-2 molecule in which only amino acid residues 76-78 have been deleted. 4. A mutant IL-2 molecule in which only amino acid residues 76-79 have been deleted. 5. A mutant IL-2 molecule in which only amino acid residue 75 has been deleted. 6. A mutant IL-2 molecule in which only amino acid residue 78 has been deleted. 7. A mutant IL-2 molecule in which only amino acid residues 75-77 have been deleted. 8. A mutant IL-2 molecule in which only amino acid residue 79 has been deleted. 9. A DNA sequence encoding the mutant IL-2 molecule of any of claims 1-8. 10. The DNA sequence of claim 9, contained in an expression vector. 11. A cell containing the expression vector of claim 10. 12. The DNA sequence of claim 9 wherein said DNA sequence is a synthetic sequence containing more prokaryotic preferred translation codons than naturally occurring IL-2 encoding DNA. 13. A method of producing mutant IL-2 comprising culturing the cell of claim 12 and recovering mutant IL-2 therefrom. 14. The mutant IL-2 molecule of any of claims 1-8, covalently linked to a portion of a toxin molecule which is large enough to exhibit cytotoxic activity and small enough to fail to exhibit generalized eukaryotic cell binding. 15. The molecule of claim 14 wherein said toxin molecule is diptheria toxin, and said portion of diptheria toxin is linked to said mutant IL-2 molecule by a peptide bond.

Inventors: AKIYOSHI DONNA E, GENBAUFFE FRANCIS S JR, AKIYOSHI, DONNA, E., GENBAUFFE, FRANCIS, S., JR.
Applicants: SERAGEN INC, SERAGEN, INC.
Application year: 1990
Main classification: C07K13
IPC codes: A61K38, A61K47, A61P37, C07K14, C07K19, C12N1, C12N15, C12P21, C12R1