NZ234674A

NZ234674A - Mutant interleukin-2,dna encoding it, host cells, cytotoxic and toxin-mutant il-2 conjugates

Info

Publication number: NZ234674A
Application number: NZ234674A
Authority: NZ
Inventors: Francis S Genbauffe; Donna E Ayiyoshi
Original assignee: Seragen Inc
Priority date: 1989-08-02
Filing date: 1990-07-27
Publication date: 1992-02-25
Also published as: EP0485497A1; EP0485497A4; CA2064696A1; JPH04507250A; WO1991002000A1; AU6179990A

Description

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P.O. Journal, No; NEW ZEALAND patents act. itsj NEW ZEALAND PATENT OFFICE 27JULI990 RECEIVED ~ COMPLETE SPECIFICATION "IL-2 DELETION MUTANTS" JP?Wc, SERAGEN, INC., a Delaware Corporation, United States of America, of 97 South Street, Hopkington, Massachusetts 01748, United States of America. h'freby declare the. invention for which'"XXK we pray that a patent may be granted to XXX#us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 1 - (followed by page la) I r> o 0369g - \a- IL-2 DELETION MUTANTS Background o£ the Invent ion 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 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 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 2 3 4 6 7 4 < 23 4 6 7 4 sequence that differs from the natural IL-2 encoding DNA in that it contains more prokaryotic preferred y 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 i acid residues 100-104 decreases the biological activity ; by two oders of magnitude; and deletion of amino acid j residues 124-126 renders the molecule inactive (Collins et al . , 1988, Proc. Nat. Aca. Sci. 85: 7709; Cohen et * j 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, 23 4 6 7 4 can 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 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 23 4 6 7 4 4 - 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 enzymatica1ly active fragment A into the cytosol. Once delivered to the cytosol, fragment A catalyzes the ADP-cibosylation 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/Notl 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 V r* y..

S 234 6 74 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 mxitants 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/Hindl 11 fragment of the IL-2 deletion mutant derived from pDWlS. This DNA fragment is ligated to Sphl/Hindl11 digested plasmid pABM6508 (Bishai et al. , 1987, J. Bacterid, 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 analysis, 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 *v. -V v- 234674 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 by comparison of the size of the expressed protein to known lL-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, Denmark) at a concentration of 105 per well in 100 pi complete medium. Il-2-toxin was added at varying — 12 —6 concentrations (10 M to 10 M) 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 pi leucine-free medium (DMEM Selectamine, Gibco) containing 2.5 yCi/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. IC50 refers to the concentration of IL2 required to inhibit J# protein synthesis to 50% of the untreated control.

IL-2 coding sequence 60 61 62 63 6* 65 66 67 68 69 70 71 72 73 74 73 76 77 79 79 80 81 82 83 (5' 3' ) T CTk GAA GAA GAA CTC iJU CCB CTO GA& GAA GTT CTG AAC CTG GCT CA& TCT AAA AAC TTC CAC CTG C&& CCG OS amino ac id —» Lev GL«. GCLa Glv Lev Ljrs Pro Lev GQ.u Glu Y*1 Lev A*n Lev AX* GXft Scr Lys A*tv fh* Rts Leu Arg Pro psl 133 (A74) T CTA GAA 6AA GAA^CTG AAA OC& CTG GAG 3AA GTT CTG AAC CTO GCA TCT AAA AAC TTC CAC CTG COG CC& C& lev Glu Glu Gl* Lev tys Pro Lev Gl* Glu Y%1 Lru A*n Lro Al* Ser Ly« A*» M» Ri« Leu Arg Pro psl 134 (A75) T CTA 6AA GAA GAA CTG AAA CCG CTS GA& GAA GTT CTO AAC CTG GCA CA& AAA AAC TTC CAC CTG COG CC8 OS Leu Glu Glv Glv Lev Ly* Pro Lru Glu 61* T*1 Leu Asa Leu Al* 81a Lfs Aa Pbe Hu Lru Arg Pro psl 136 (A78) T CTA GAA GAA 6AA CTG AAA CC& CTG 6A0 GAA GTT CT& AAC CTG GCA CAd TCT AAA AAC CAC CTG COG CC& C& Lru Glu Gin Glu Lru Ly* Pro Lru Glu SLu Y*1 Leu Asa Leu Al* 60a Ser Ly» Asa His Leu Arg Pro psl 137 (A79) t CTA GAA GAA GAA CTO AAA OCO CT& GA0 GAA GTT CT© AAC CT& GCA CAft TCT AAA. AAC TTC CT© OGG CC& C& Lev Glu Giv Glv Leu Lys Pro. Lev Glu Glv Y*1 Leu Asn Lev Al» Gl_a Ser Ly* A*n Tt» Lru. Arg Pro psl 14 3 (A75-77) T CTA 6AA GAA GAA CTG AAA CCG CTG 6A0 GAA STT CTG AAC CTG GCA CAG TTC CTO OGG CCG CO Lev Gl*. Gin Gl* Leu Ljv Pro Lru GLu Glu Yul Leu Asa Leu Al* 61a Pbe Leu Arg Pro * psl 141(A74-78) T CTA GAA GAA GAA CTO AAA COG CTG GAG GAA GTT CT& AAC CTG GCA CkC CTG CSG OCG C& Lev Giln Sin Glu Leu Lys Pro Leu Glu Glu Y*1 Leu Asa Leu Al* KLs Leu Arg Pro psl 150 (A76-79) T ^ GAA. GAA GAA CTG AAA CCG CTG GA& GAA GTT CTG AAC CT© GCA CAG TCT CAC CTG CGG CCG C& Leu Glo. Gau Glv Lev Lys Pro Leu Glu Glu Y*1 Leu Asa Leu Al* Gla Ser His Lev Arg Pro psl 145 ( A76-78) T CTA GAA GAA 0AA CTO AAA CCO CTO OA0 GAA GTT CTO AAC CTG GCA CAG TCT CTO C88 CCG CO L»u Glv Glu Glu Lev Lys Pro Leu Glu Glu TO. Lev Asa Leu Al* Sla Ser Lru Arg Pro TABLE I Table II 234 674 Plasmid amino acid(s} deleted C91/PL IC50 ^ psl133 $74 6xlO""1M psl 134 $7 5 1X10_10M Ps 1136 $78 . 5xlO_11M psl 137 $79 2xlO~10M psl 143 $75-77 2xl0~10M ps 1141 $74-78 1X10~10M ps114 5 $76-78 2xl0"10M psl150 $76-79 7xl0nM (psl129 control no deletion typically 5x10_11M) 4 234674 - 8 -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

234674 - 9 - >»"N y* 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. o
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. i > '£ | - 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. 23467 1
15. The molecule of claim 14 wherein said S | toxin molecule is diptheria toxin, and said portion of I diptheria toxin is linked to said mutant IL-2 molecule f. by a peptide bond. -j
| 16. A mutant IL-2 molecule as defined in claim 1 or ] claim 14 substantially as herein described with reference to any ! example thereof or to the accompanying drawing.
17. A DNA sequence as defined in claim 9 substantially as herein described with reference to any example thereof or to the accompanying drawing.
18. A cell as defined in claim 11 substantially as herein described with reference to any example thereof or to the accanpanying drawing.
19. A method as defined in claim 13 of producing mutant IL-2 ; substantially as herein described with reference to any example thereof. /.VL - ^if/Their authorised Agent £AJ. PARK & SON /// X