AU766692B2 - Novel vectors and expression methods for producing mutant proteins - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2 r A

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventor: Address for service is: The Government of the United States of America, as represented by The Secretary, Department of Health and Human Services David M NEVILLE WRAY ASSOCIATES 239 Adelaide Terrace Perth, WA 6000 Attorney code: WR Invention Title: "Novel Vectors and Expression Methods for Producing Mutant Proteins" This application is a divisional application by virtue of Section 39 of Australian Patent Application 64459/98 (736501) filed on 5 March 1998.

The following statement is a full description of this invention, including the best method of performing it known to me:- 1/2 NOVEL VECTORS AND EXPRESSION METHODS FOR PRODUCING MUTANT PROTEINS BACKGROUND OF THE INVENTION Field of the Invention The invention relates to novel expression systems and vectors for engineered immunotoxins. More specifically the invention relates to a shuttle vector for E. coli and Corynebacteria. The invention also relates to a method of expressing engineered toxin mutants and toxin fusion proteins in a mutant form of Pichia pastoris, a mutant form Chinese hamster ovary cells, and mutant insect cells and a methods for producing these mutants.

Background Art U.S. Patent No. 5,167,956 describes in vivo T cell killing of 3 logs by immunotoxin anti-CD3-CRM9 or derivatives. This patent also describes the treatment of graft versus host disease, autoimmune disease and T cell leukemia.

A shuttle vector constructed for use in Corynebacterium and E. coli must contain a replication region (oriR) and a selectable marker that function in both host bacteria, or contain an oriR functional in E.

coli and an chromosomal integrative mechanism functional in Corynebacterium. A naturally occurring plasmid, pNG2, isolated from an erythromycin-resistant Corynebacterium strain fulfills the former criteria However this vector is large (14.4 kb), which reduces its 2 transformation frequency, which is additionally severely compromised by restriction incompatibilities between Corynebacterium and E. coli DNA In addition multiple cloning sites are not present in pNG2 which are required to facilitate splicing inserts of toxin and fusion protein toxin genes into the vector.

Thus, the invention meets an important need for an E.

coli and Corynebacteria shuttle vector with multiple cloning sites.

SUMMARY OF THE INVENTION The present invention describes a new shuttle vector 15 permitting the expression of engineered toxin mutants and toxin fusion proteins in Corynebacterium. In addition the invention provides a mutant Pichia pastoris, a method for producing this mutant and a method of expressing ~engineered toxin mutants and toxin fusion proteins in the mutant form of Pichia pastoris. The invention further provides a mutant Chinese hamster ovary (CHO) cell, a method for producing this mutant and a method of expressing engineered toxin mutants and toxin fusion proteins in the mutant CHO cells. These three systems have distinct advantages over E. coli expression systems because of their higher yields of secretion into the media compared to E. coli thus eliminating the need for refolding procedures from insoluble aggregates Refolding procedures employing denaturing agents although somewhat successful for single chain fusion proteins will not correctly refold divalent single chain fusion proteins due to their greater complexity. Because divalent immunotoxins are necessary for successful in vivo clinical application, the present expression system for engineered immunotoxins is the key to producing a successful immunotoxin.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 depicts the method for producing an E.

coli/Corynebacterium shuttle vector from the plasmid pNG2.

Fig. 2 depicts the new E. coli/ Corynebacterium shuttle vector, yCE96, cotaining nucleotides 1-3476 as shown in SEQ ID NO:1. Residues from positions 1 to 373 15 and 2153 to 3476 are from the vector LITMUS 29 and contain the polycloning linker sites and the ampicillin resistance marker respectively. Residues from positions 374 to 2152 were the origin sequences from the plasmid pNG2.

Fig. 3a depicts a single chain divalent antibody-mutant-toxin fusion protein produced in Corynebacterium. The toxin is CRM9 and is preceded by the CRM9 promoter and signal sequence. VL and VH linked by a spacer described in U.S. Serial No. 08/739,703, herein incorporated by reference, are from UCHT1. MCH3 and ACH2 are from human IgM. The fusion protein forms the disulfide dimer from the cysteine between the CH2 and CH3 domains during or shortly after secretion. The gene for this fusion protein is constructed by PCR overlap extension to avoid cloning CRM9 until its toxicity is further reduced by a second genetic event, in this case an additional carboxy terminal protein domain (NIH guidelines, see reference Fig. 3b shows a double mutant of DT containing the S525F mutation of CRM9 plus an additional replacement within the 514-525 exposed binding site loop to introduce a cysteine coupling site for example T521C can be produced in Corynebacterium ulcerans preceded by the CRM9 promoter and signal sequence. Residue numbering is based on the sequence provided by Shen et al. The double mutant is made in Corynebacterium ulcerans by a recombination event between the plasmid producing CRM9-antibody fusion protein and PCR generated mutant DNA with a stop codon at 526. This CRM9-C's can be used to form specific thioether 15 mutant toxin divalent antibody constructs by adding excess bismaleimidohexane to CRM9-C's and coupling to single chain divalent antibody containing a free cysteine at either the end of the ACH4 domain or the tCH3 domain.

DETAILED DESCRIPTION OF THE INVENTION Provided is an E. coli/Corynebacterium shuttle vector comprising the origin of replication of shuttle vector pNG2, polycloning linker sites, and an antibiotic resistance marker with a size less than 4 kb. There are multiple possibilities for the polylinker cloning site and antibiotic resistance marker, which can be selected from those known or later developed. An example of the vector described above is yCE96. The sequence of yCE96 is given below.

The vector can further comprise an insert consisting of a protein-encoding nucleic acid. The encoded protein can contain a disulfide bond. For example, the proteinencoding nucleic acid can encode the binding site mutant DT toxin, CRM9. Alternatively, the protein-encoding nucleic acid can encode a CRM9 further comprising a second attenuating mutation. The second attenuating mutation can be the insertion of a COOH terminal protein domain. The second attenuating mutation can be a COOH terminal mutation that reduces binding activity, but not translocating activity. The second attenuating mutation can be selected from the group consisting of S508F, Y514A/C K516A/C, V523A/C, N524A/C, K526A/C and F530A/C.

15 A protein encoding nucleic acid construct of the invention can include a mutation that introduces a cysteine residue into CRM9 or its derivatives. For e* example, the mutation can be selected from the group consisting of K530C, K516C, D519C and S535C.

A protein encoding nucleic acid construct of the invention can include a mutation that introduces residues or the replacement of residues in CRM9 or its derivatives, to attenuate the blocking effects of anti-diphtheria toxin antibodies. Examples of these mutations are described in the related applications.

A vector of the invention can further comprise the CRM9 iron-independent promoter, wherein the proteinencoding nucleic acid encodes a binding site mutant of diphtheria toxin, and the protein-encoding nucleic acid is under the control of the CRM9 iron-independent promoter and is preceded by the CRM9 signal sequence.

A method of expressing a diphtheria toxin moiety or other protein is provided. The expression method comprises transfecting a Corynebacterium ulcerans or a Corynebacterium diphtheriae cell with a vector of the invention under conditions that permit expression of the protein-encoding nucleic acid. The conditions required for expression are the same as those previously used or described herein, including limited iron in the medium.

A method of making a vector of the invention is provided. For example the method comprises a) deleting 15 COOH terminal base pairs of the attenuated CRM9 toxinencoding nucleic acid using the restriction site Sph I at the toxin nucleotide position 1523 and a restriction site used to clone the COOH terminal part of the toxin into the polylinker cloning sites of yCE96, to produce a gapped, linear, plasmid deleted in the COOH terminal coding region; b) amplifying a product that corresponds to the COOH terminal region of CRM9 deleted in step with a PCR primer that includes the desired mutation and 30-40 base pairs homologous to the down stream and upstream regions adjacent to the deletion; c) purifying the amplified product of step b) on an electrophoretic gel; and d) electroporating the product of step c) into a Corynebacterium along with the gapped plasmid of step a), under conditions which permit homologous recombination to occur intracellularly.

A method of mutating a protein-encoding nucleic acid in a vector of the invention -is provided. The method can comprise a) deleting a region of the COOH terminusencoding nucleotides of the protein-encoding nucleic acid using a unique restriction site and a restriction site used to clone the COOH terminal part of the toxin into the polylinker cloning sites of the shuttle vector, to produce a gapped, linear plasmid deleted in the COOH terminal coding region is produced; b) amplifying a product that corresponds to the COOH terminus-encoding region deleted in step using a PCR primer that includes the desired mutation and 30-40 base pairs homologous to the downstream and upstream regions adjacent to the deletion; c) purifying the amplified product of step b) on an 15 electrophoretic gel; and d) electroporating the purified product of step c) into a Corynebacterium along with the gapped plasmid of step under conditions which permit "homologous recombination to occur intracellularly.

The Corynebacterium used in the methods of making the present vectors or mutating proteins can be Corynebacterium ulcerans. Alternatively, the Corynebacterium can be a Corynebacterium diphtheriae, which has been mutated by chemical mutagenesis to exhibit less DNA restriction. Any number of mutations can accomplish this. The presence of the desired mutation is measured by a reduction in restriction in the organism.

For example, this can be determined by measuring the efficiency of transformation, if number of transformants per jg of vector DNA increases over wild type or other mutants.

8 A mutant strain of Pichia pastoris is provided. The mutant strain comprises a mutation in at least one gene encoding elongation factor 2 (EF2). This the mutation comprises a Gly>Arg replacement at a position two residues to the carboxyl side of the modified histidine residue diphthamide. In this manner, the strain is made resistant to the toxic ADP-ribosylating activity of diphtheria and pseudomonas toxins.

A method of expressing a diphtheria toxin protein moiety or a pseudomonas exotoxin A toxin protein moiety is provided. Such a method of the invention comprises transfecting a mutated Pichia cell of the invention with a vector comprising a toxin protein-encoding nucleic acid 15 under conditions that permit expression of the proteinencoding nucleic acid in the cell. The conditions are those used for Pichia cells and can be optimized for the •particular system.

In an expression method using a mutant Pichia strain, the encoded protein is glycosylated in the cell to produce of immunotoxins that are resistant to the blocking effects of anti-diphtheria toxin antibodies on the T cell depleting function of CRM9-containing immunotoxins in human patients in vivo. There is a consensus sequence for glycosylation (NXS/T), which may be removed or inserted to control glycosylation which occurs in all eukaryotes, Pichia.

A method of expressing a diphtheria toxin or a pseudomonas exotoxin A toxin in Chinese hamster ovary (CHO) cells, insect cells or other eukaryote cells is provided. The method can comprise first making a mutant strain of cells, comprising a mutation in at least one gene encoding elongation factor 2 (EF2), wherein the mutation comprises a Gly>Arg replacement at a position two residues to the carboxyl side of the modified histidine residue diphthamide, and the strain is resistant to the toxic ADP-ribosylating activity of diphtheria and pseudomonas toxins. This homologous recombination method can be used in any eukaryote, due to the high conservation of diphthamide in ekaryotes. Then the mutant cells are transfected with a vector of a type appropriate for the particular cell used, under conditions that permit expression of protein-encoding nucleic acid in the cells.

The conditions can be those used for CHO cells and can be routinely optimized for the particular system and cells used.

As with the Pichia-expressed nucleic acids, the CHO method produces a glycosylated protein. This can produce immunotoxins that are resistant the 15 blocking effects of anti-diphtheria toxin antibodies on the T cell depleting function of CRM9-containing immunotoxins in human patients in vivo.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

EXAMPLES

Example 1 A Corynebacterium/Escherichia coli Shuttle Vector for Gene Expression In brief, a new shuttle vector is constructed using the oriR of pNG2 and the antibiotic resistance marker and multiple cloning sites of the vector Litmus p29 (New England Bio Labs, Inc. Figure 1 depicts a method of making of a vector according to the invention. The new vector, yCE96, is only 3.4 kb in size and can transform both E. coli and Corynebacterium ulcerans and, thus, can be used to produce toxins and mutant toxins S Cloning OriR from pNG2 plasmid: pNG2 plasmid DNA was purified from E. coli JM109 ooo*** strain. The 2.6 kb fragment containing oriR was released from pNG2 plasmid by restriction digestion with EcoRI and 20 Clal endonuclease. After separation and isolation, the 2.6 kb fragment was cloned into pBR322 vector (4361 bp) by the same two restriction sites EcoRI and Clal, to form a construct of pBRNG with a size of 6.96 kb. This new vector is of limited use, because of its relatively large size and lack of multiple cloning sites.

Sub cloning pNG2 plasmid OriR to Generate a New Vector: To generate a functional expression vector in Corynebacterium with a small size and a multiple cloning sites, the oriR DNA fragment, released from pBRNG constructs, is combined with a DNA fragment borrowed from vector litmus p29. The DNA fragment containing the pNG2 oriR was released from pBRNG vector by endonucleases EcoRI and SnaBI, and decreased in size from 2.6 kb to 1.77 kb by cleavage with these enzymes. It is noted that the sequence of the oriR is published, such that it could be constructed without the pNG2 plasmid. Its nt numbers are 374-2152 in the sequence disclosed for yCE96. The DNA fragment borrowed from litmus 29 vector was released by restriction enzyme SnaBI and DraIII, and contains an ampicillin resistant gene and a multiple cloning sites.

The two DNA pieces could not be ligated together, because the sticky end of the EcoRI site was not compatible with the sticky end of DraIII. To turn the sticky end into a blunted end, these DNA fragments were treated with T4 polymerase in the presence of 0.1-mM dNTP.

After digestion with restriction enzymes SnaBI and DraIII, and treatment with T4 polymerase in the presence of 0.1 mM dNTP, the litmus 29 vector DNA was dephosphorylated with alkaline phosphatase to remove the phosphate group. The recombined vector was selected by transformation of the ligation mixture into Novablue E.

coli cells. Five colonies were picked up. All of them contained a plasmid vector. The restriction digestion patterns of purified DNA from these five colonies demonstrated that four colonies contained the correct vector size of 3.4 kb.

The selection of endonuclease to release the oriR from pBRNG was based on the nucleotide sequence of plasmid pNG2 oriR identified by Messerotti et The 12 replication region of pNG2 is 1854 bp, consists of a single oriR ans one major open reading frame. When digested with SnaBI, 75 nucleotides were deleted from end of the identified 1.85 kb oriR sequence. Therefore, the oriR cloned into the new vector, yCE96, was nucleotides smaller than the previously identified 1.85 kb oriR sequence of plasmid pNG2, that is 1779 bp.

Transformation of Corynebacterium and E. coli by yCE96 E. coli cells were rendered competent by overnight growth followed by resuspension in LB medium containing 10% PEG 8000, 5% DMSO and 50 mM MgCL,, pH 6.5. The cells were heat shocked in the presence of 20 ng of vector DNA per 106 cells. C. ulcerans were converted to protoplasts 15 as described and transformed by electroporation of 404g of vector DNA S"Unless they have undergone transformation by yCE96, no colonies of Corynebacterium ulcerans or E. coli grow up 20 under the selection pressure of the presence of carbenicillin. Many colonies are formed after transformation of E. coli cells and C. ulcerans with the yCE96 vector on the LB plate with 0.1 mg/ml of carbenicillin. The results demonstrate that yCE96 can stably transform E. coli and C. ulcerans; thus it is an effective shuttle vector between E. coli and C. ulcerans.

yCE96 is used to introduce foreign proteins into C.

ulcerans, for example, diphtheria toxin mutants made by PCR site directed mutagenesis. To this end the CRM9 promoter carrying the iron-insensitive mutation (see U.S.

Serial No. 08/739,703, hereby incorporated by reference), as well as the toxin signal sequence have been cloned from CRM9 chromosomal DNA and precede mutant toxin constructs and mutant toxin single chain antibody fusion proteins.

Thus, high level synthesis of these engineered proteins, and their secretion into the medium, is expected.

Insertion of tandem repeats of these protein-encoding constructs into yCE96 can also be made to achieve even higher production levels. Several constructs and their uses are detailed in Fig. 3 and Fig. 3 legend.

Example 2 Corynebacteriophage-based Vector An alternate type of shuttle vector containing the 15 integrative mechanism of Corynebacteriophages can be constructed by cloning the corynephage attachment site (attP) and the integrase gene (int) sites from beta Corynebacteriophages into small E. coli vectors such as pUC19) not capable of replicating in Corynebacteriae.

Addition of another protein coding sequence such as modified CRM sequences (see below) will permit the integration of these sequences into tox-Corynebacteriae.

These sequences can be further modified by excision followed by a gapped plasmid methodology described below using a PCR product to achieve any desired mutation or combination of mutations. An advantage of the integrative vector is that it recombines at high efficiency.

14 Example 3 Using Shuttle Vector yCE96 to Perform Site-Specific Mutagenesis On Diphtheria Toxin Binding Site Mutants in Corynebacteria A binding site mutant of full length diphtheria toxin residues 1-535 (16) S525F (17) is further modified for chemical coupling by changing a residue in the binding domain (residues 379-535) to cysteine. Preferred residues are those with exposed solvent areas greater than 38%.

These residues are K516, V518, D519, H520, T521, V523, K526, F530, E532, K534 and S535 Of these, K516 and F530 are presently preferred, since they are likely to block any residual binding activity However, maximal coupling of the new cysteine residue will be enhanced by the highest exposed solvent surface and proximity to a positively charged residue (which has the effect of lowering cysteine -SH pKa). These residues are at D519 and S535, so that these are also preferred from the above list of possibilities.

These mutations are accomplished by gapped plasmid PCR mutagenesis (18) using the newly designed E. coli/C.

uicerans shuttle vector yCE96 containing either the double mutant DT S508F S525F or a CRM9 COOH terminus fusion protein construct having reduced toxicity due to the COOH terminal added protein domain Both of these constructs follow current NIH guidelines for cloning DT derivatives into E. coli (Federal Register, Notices, May 7, 1986), Appendix F-II-B, p. 16971) in that they contain two mutations which both individually diminish toxicity and, therefore, greatly reduce the chance of introducing a wild type toxin into E. coli by a single base pair reversion. This mutation is made by deleting the COOH terminal 52 base pairs of the toxin construct using the restriction site Sph I at the toxin nucleotide position 1523 (16) and the restriction site used to clone the COOH terminal part of the toxin into the polylinker cloning sites of yCE96 (Xba or BamHI for example). Since Sph I, Xba, and BamHI only occur singly within vector yCE96 containing the inserted toxin construct, a gapped, linear plasmid, deleted in the COOH terminal coding region is the result. Using PCR the COOH terminal region of CRM9 is rebuilt introducing the desired mutation and including 30-40 base pairs homologous to the down stream and 15 upstream regions adjacent to the gap. The amplified product is gel purified and electroporated into C.

ulcerans along with the gapped plasmid (18).

Recombination at the homologous regions occurs 20 intracellularly, accomplishing site specific mutagenesis of DT products within Corynebacteriae which are not specifically subject to NIH toxin cloning restrictions Using methods analogous to those described above, but with different restriction enzymes, mutagenesis can be performed anywhere within the toxin molecule. This would be highly useful for the construction of toxin B chain mutations having full translocating activity that are relatively free from the antitoxin blocking activity variably present in the sera of patient populations resulting from prior immunizations with diphtheria toxoid.

Because of the unique features of the present vector, a virtually unlimited number of mutated proteins can be expressed.

Example 4 Expression of mutant ADP-ribosylating toxins and toxin fusion proteins in an EF2 mutant of Pichia pastoris The invention provides a system for expressing mutant ADP-ribosylating toxins and toxin fusion proteins in a Pichia pastoris mutant. The presently preferred mutant is one that has been rendered insensitive to these toxins by *0 15 substituting arginine for glycine at a position two amino acids carboxyl to the modified histidine residue diphthamide (position 701 in the EF2 gene based on the S* numbering system in S. cerevisiae) S. 20 This mutation has been performed in S. cerevisiae and prevents toxin induced ADP-ribosylation rendering the toxin inactive However, the literature has not described or proposed the use of this mutation to generate mutant cells for toxin production. To date, all DT based mutant toxins generated by site directed mutagenesis have been expressed in E coli. To date, toxin mutants generated by the application of mutating reagents which are not site specific have only been produced in C.

diphtheriae and E. coli.

Among the advantages of using this mutant Pichia are that for antibody toxin fusion proteins, which must have appropriate folding of critical antibody disulfide bonds, the endoplasmic reticulum compartment of the eukaryote yeast offers a similar oxidizing environment to natural eukaryote antibody producing cells such as hybridomas.

Pichia pastoris has numerous advantages over S. cerevisiae including the observation that, for therapeutic proteins, the glycosylation pattern of Pichia is much more similar to that of humans compared to S. cerevisiae.

The Pichia mutant can be constructed by direct transformation of yeast spheroplasts with a mutating oligonucleotide complimentary to the sense strand of S.

S: 15 cerevisiae EF2 in the region of the desired mutation but not at the desired mutation Since homology in this area is very high across phyla this sequence will very likely undergo homologous recombination in P.

*oe pastoris. One example of such a mutating oligonucleotide ~20 has the following sequence: o ACT TTA CAT GCC GAT GCT ATC CAC AGA AGA GGT GGT CAA ATC ATC CCA-3 (SEQ ID NO:2) .0 The arginine substitution has been underlined. This nucleic acid can be modified, for example, by shortening on both ends, but this may result in reduced recombination frequency.

The transformation is done in the presence of 10 iM wild type diphtheria toxin or a binding site mutant DT toxin, such as CRM9, as a selecting agent Several repeat rounds of selection can be utilized. The final selection is performed by transforming Pichia with a toxin-containing construct, which will inhibit growth in any cells which lack toxin-resistance mutation in EF2.

The invention also provides a Pichia EF2 toxinresistant clone, which is free of toxin-encoding constructs, to be used for to express alternative constructs. This cell can be generated by popping out the construct via homologous recombination (12) Many Pichia pastoris strains and E. coli/Pichia shuttle vectors are available (Invitrogen Corporation), 15 and can be used in an expression system as described *above. This technology will be useful for diphtheria toxin and pseudomonas exotoxin A constructs and fusion immunotoxins based on these toxins. The same mutant toxins and toxin-antibody fusion proteins produced in Corynebacterium shown in Fig 2 can also be produced in P.

pastoris. The genetic constructs differ in that in P.

pastoris the promotor is AOXI and the secretion or signal sequence is either PHO1 or @-factor. In addition certain codons unique to Corynebacterium may require changing in 25 P. pastoris, as will some codons specifying potential glycosylation sites which would be active in Pichia but inactive in Corynebacterium. Alternatively, the introduction of glycosylation sites within the CRM9 gene could be used as a method to block antibodies directed at the toxin in patients with high antitoxin titers secondary to recent immunization with diphtheria toxoid.

19 Example Expression of mutant ADP-ribosylating toxins and toxin fusion proteins in an EF2 mutant of CHO cells.

CHO cells have certain advantages for the production of fusion proteins used as therapeutic reagents: a) Hamster cells are relatively free from retrovirus contamination, b) Protein glycosylation patterns are relatively similar to that seen in humans, and c) CHO cells generally, respond well to gene amplification systems that increase the yield of proteins introduced through DNA transfection. Although it has been reported that mammalian cells can secrete an ETA based fusion *e protein without succumbing to ETA induced cytotoxicity via 15 ADP-ribosylation this situation is likely due to the e resistance of ETA to proteolytic processing at pH feature not shared by DT. Therefore the production of DT based fusion immunotoxins must utilize a line of CHO cells S. that has been rendered DT insensitive by mutating EF2 by substituting arginine for glycine at a position two amino .acids carboxyl to the modified histidine residue diphthamide (position 717 in the EF2 gene based on the numbering system in CHO cell EF2). Two such cell lines have been reported. RE1.22c (14) and KEE1 (15) were 25 isolated by double rounds of chemical mutagenesis and selection with DT or ETA. These mutants were used to determine the physiologic role of dipthamide, the modified histidine residue whose formation is blocked by this mutation. No mention is made in the art of using these cells to make toxins or immunotoxins. The same procedure can be applied to other eukaryote cell lines expressing toxin sensitivity, such as insect cell lines. These lines have the advantage of secreting large amounts of foreign proteins introduced through baculovirus (21).

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Throughout this application various publications are referenced by numbers within parentheses. Full citations for these publications are as follows. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

REFERENCES

1. Greenfield, Targeted Diagnosis and Therapy, Chapter 16, p. 307; 1992.

2. vanderSpek, J. Mindell, J. et al. J. Biol.

Chem. 268:12077-82, 1993.

3. Serwold-Davis, T. Groman, N. et al. FEMS Microbiology Letters. 66:119-124, 1990.

4. Serwold-David, T. Groman, N. et al. Proc.

Natl. Acad. Sci. 84:4964-4968, 1987.

25 5. Groman, Schiller, et al. Infection and Immunity 45;511-517, 1984 6. Messerotti, L. Radford, A. J. et al. Gene 96:147-8, 1990.

21 7. Kimata, Harashima, et al. Biochemical and Biophysical Research Commnunications 191:1145-1151, 1993.

8. Buckholz, R. and Gleeson, M. A. G.

Bio/Technology. 9:1067, 1991.

9. Moerschell, R. Das, et al. Mechods in Enzymology. 194:362, 1991.

Perenetesis, J. Phan, L. et al. J. Biol.

Chem. 267:1190-1197, 1992.

0: 0.11. Murakami, Bodley, J. et al. Molecular and Cellular Biology 2:588-592, 1982.

12. Rothstein, R. Methods in Enzyrnology 194:281, 1991.

13. Chen, et a1. Nature 385 :78, January 2, 1997.

14. Foley, B.T. et al. J. Biolog. Chem. 370(39) :23218- *~**23225, September 29, 1995.

Kohno, K. and Uchida, T. J. Biolog. Chem.

262 (25) :12298-12305, 1987.

16,. Kaczorek M, Delpeyroux F, Chenciner N, Streeck R (1983) Science; 221:855.

17. Shen WE, Choe S, Eisenberg D, Collier RJ (1994) Biol. Chem.; 469(46) :29077-29084.

18. Muhirad D, Hunter R, Parker R (1992) Yeast; 8:79-82.

19. Madshus IH, Stenmark H, Snadvig K, Qisries S (1991) J.

Biol. Chem. 266(26) :17446-53.

Federal Register, Notices, May 7, 1986) Appendix F-IT-B, P. 106971.

21. Bei, R, Schioi, J arnd Kashmiri, SVS (1995) J.

Immunol. Meth.; 186:245-255.

SEQUENCE LISTING GENERAL INFORMATION APPLICANT: The Government of the United States of America, as represented by the Secretary, Department of Health and Human Services (ii) TITLE OF THE INVENTION: NOVEL VECTORS AND EXPRESSION METHODS FOR PRODUCING MUTANT PROTEINS (iii) NUMBER.OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: NEEDLE ROSENBERG, P.C.

STREET: 127 Peachtree Street, Suite 1200 CITY: Atlanta STATE: GA COUNTRY: USA ZIP: 30303-1811 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: o. APPLICATION NUMBER: FILING DATE: 05-MAR-1998

CLASSIFICATION:

:(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 60/037,196 FILING DATE: 0S-MAR-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: Spratt, Gwendolyn D.

REGISTRATION NUMBER: 36,016 REFERENCE/DOCKET NUMBER: 14014.0286/P (ix) TELECOMMUNICATION INFORMATION: ot TELEPHONE: 404 688 0770 TELEFAX: 404 688 9880

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3476 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: *t

AAAAAAAAGC

TGCGCAACTG

AAGGGGGATG

GTTGTAAAAC

TCTGGATGCA

AAGCTTCACC

TGAGTCGTAT

TAACACCGGT

GGCAACCCGA

GTTTACAGAC

GTGGTGGACT

TAGGCCGCGA

ACGCTGACGG

GCGAGTACGC

AAGTAGGTAC

GCtCACTGAT

AAGCCCAGTT

AGATGAAGCT

ACTTTTCCCA

GT TGGTATAG

ATATGGCAGG

AACTTATCAA

AGGACGTAGA

GTGTGCGTGT

ATGCGCTTA

TCATCGACGC

ACAACGAGAT

TCGCCCAATC

GCGAGCGGPA

GGAAAACAGA

ACCGTAAGAA

ATCAGTATTT

TCTCTCGCGC

ACGTTTAAGG

TAAAACCTCA

CTTCCTCTGT

TCGCCCTGAT

CTC7TGTTCC

GGGATTTTGC

GCGAATTTTA

TTTTTGGGGC

CTTGGTCTGA

TTCGTTCATC

TACCATCTGG

TATCAGCAAT

CCGCCTCCAT

ATAGT=GCG

GTATGGCTTC

TGTGCAAAAA

CAGTGTTATC

CCGCCGAAGC

TTGGGAAGGG

TGCTGCAAGG

GACGGCCAGT

TTCGCGCGCA

ATGGGAGACG

TACGTAACCG

GTCATTTAGA

TGACTTTCGC

TCATGCGCAG

GGACAACACC

TGTACTCCAC

CACGAACTCG

CATGCTCACC

CGCAGGCGGT

TACTCATAGC

CATATGGCTT

TCTTGCAGCA

CCGCTTTAGC

GCAGCACAAC

ACACGACCAG

CGCGGTCAAG

CGCGGAAATC

GCTCTGGATT

GACTGGCCAC

CTATGAGCAC

GCCACCCATG

CAAGAGCGAG

AGCCTTGGCC

CCCCGAGGGC

AAAGGcTCAA

CCAGACAGGG

CACGG'rTGCT

GGTCTCATAC

CTTGAAGAAA

TCTCCTAGAC

AGACGGTT?

AAACTGGAAC

CGATTTCGGC

ACAAAATA'rr

TTTTCTGATT

CAGTTACCAA

CATAGTTGCC

CCCCAGTGCT

AAACCAGCCA

CCAGTCTATT

CAACGTTGT

ATTCAGCTCC

AGCGGTTAGC

ACTCATGGTT

GGGCTTTATT

CGATCGGTGC

CGATTAAGTT

CCGTAATACG

CGTACGGTCT

TCACCGGTTC

CAGGTAAAAG

GTCAGGGAAA

TTATCACCCA

AATGCGCACA

CCAGCATCTG

GGTTCAGTCA

CCGCGTATGT

ACCAAGCAGT

GACCCCGCAG

GTCGGGCCAG

ATTGACCCTG

ACCACGCGTG

CGCAACCCGT

CGGGTGATGC

TTCAACCCCA

ACCCGCCGTG

GCCGGTGGTC

GTCCAAGGAA

CGCTTGCGCC

GCCTACAACG

CGCGACCGCC

ACCTACAGCG

ACGATGGGAC

AAATATGCGC

GGACGATCTA

ACAGTTCCCA

AGGCATGTCG

CGTAAGCAAT

ACCTrTGAGGG CTrCGCAACCC

TCGCCCTTTG

AACACTCAAC

CTATTGGTTA

AACGTTTACA

ATCAACCGGG

TGCI'AATCA

TGACTCCCCG

GCAATGATAC

GCCGGAAGGG

AATTGTTGCC

GCCATTGCTA

GGTTCCCAAC

TCCT1'CGGTC

ATGGCAGCAC

ACCAAGCGAA

GGGCCTCTTC

GGGTAACGCC

ACTCACTTAA

CGAGGAATTC

TAGAACCTAG

GCATATTTTT

GACAATGAAA

GCACACACCT

CTAAAACACC

CCAGTGACCG

CACGAGACTT

ATCGCTTCGA

ACGCCGCCGT

ACTTAAACCC

CCTGGGTGGG

TCTACGCTGA

TGCTGGGTGA

TCTACACAGG

GCCTTGGAGA

CCCCACGCCA

AAGAAGCCCA

TCGACCAGTA

CCGCAGCAC!G

AGCAAGGCCA

TCGCACACAC

AAACCATGGC

GCTCTAACGC

GCAGAGGCGG

AAGCACAAAG

CGAAGTCCCG

CGTGGGCTGA

CGGAGCTAAA

ATACGGTTC!C

GCAGGGCAGC

TCCGCCATAA

ACGTTGGAGT

CCTATCTCGG

AAAAATGAGC

ATTTAAATAT

GTAAATCAAT

GTGAGGCAcc

TCGTGTAGAT

CGCGAGACcc

CCGAGCGCAG

GGGAAGCTAG

CAGGCATCGT

GATCAAGGCG

CTCCGATCGT

TGCATAATTC

GCGCCATTCG

GCTATTACGC

AGGGTTTTCC

GGCCTTGACT

CTGCAGGATA

GGAGCTCTGG

CGCGTGTCAT

AACGAAGAA-A

GGGAGAAATC

TACCCGCGTC

CGACCTTT TA

TAAAAAGGCC

GACTGATGCT

CCTGGTCGTA

GTACGTCCGC

TATTAACCCA

CCGTAACGGT

GCTTTTAGAC

CAAAGCCCCT

CTTGATAAAG

GCAATTCAGC

AGCATTCAAA

TGACCCGGAA

CGACGAAACA

ACGCCTGACA

CCACGGCGGT

AAGGCGCGTG

ACCAGGTAAA

ACAAAAAGCC

GTCGAAGCTT

CCATTCAGGC

CAGCTGGCGA

CAGTCACGAC

AGAGGGAAGA

TCGTGGATCC

TACCCACTAG

GGCTAGTAAA

GCCACCGGGC

ACGGTCATGA

GAGCGCGACC

CGCGATCATC

TATCG.ACGCA

TTAGGACGGT

GACGTTGACC

GACGTGGTGC

ACTAACGGCA

AAATCTGCGC

CATGACCCGC

ACCGCTTATC

CAGGTAAGGG

TCTGGCCGCG

GCACTCGCCC

CTTATCGACG

GCCTTTAGAC

GACGCAGCAA

GCAGGCCGCG

CGCGGGTATG

GCCACCAGCA

GCACAACGCT

GAAAAGACGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 TATTAGCCAA ATGGTGAACG AATAGGGGCA GAGGTAGGAG GAAGAGCGGT GACTATCCGG CCTGCCGTTA GGCAGTTAGA TTATATGCTT CAAAGCATGA CC'TCACCGAA TTGTGGGCCA CCACGTTCTT TAATAGTGGA GCTATTCTrI' TGATTTATAA TGATTTAACA AAAATTTAAC TTGCTTATAC AATCTTCCTG CTA.AAGTATA TATGAGTAAA TATCTCAGCG ATCTGTCTAT AACTACGATA CGGGAGGGCT ACGCTCACCG GCTCCAGATT AAGTGGTCCT GCAACTTTAT AGTAAGTAGT TCGCCAGTTA GGTGTCACGC TCGTCGTTTG AGTTACATGA TCCCCCATGT TGTCAGAAGT AAGTTGGCCG TCTTACTGTC ATGCCATCCG

TAAGATGCTT

GGCGACCGAG

CTTTAAAAGT

CGCTGTFTGAG

TTACTTTCAC

GAATAAGGGC

GCATTTATCA

AACAAATAGG

TTCTGTGACT

TTGCTCT~TGC

GCTCATCATT

ATCCAGTTCG

CAGCGTTTCT

GACACGGAAA

GGGTTATTGT

GGTTCCGCGC

GGTGAGTACT

CCGGCGTCA.A

GGAGAACGTT

ATGTAACCCA

GGGTGAGCAA

TGTTGAATAC

CTCATGAGCG

ACATTTCCCC

CAACCAAGTC

CACGGGATAA

CTTCGGGGCG

CTCGTGCACC

AAACAGGAAG

TCATACTCTT

GATACATATT

GAAAAGTGCC

ATTCTGAGAA

TACCGCGCCA

AAAACTCTCA

CAACTGATCT

GCAAAATGCC

CCTTTTTCAA

TGAATGTATT

ACCTGACGTA

TAGTGTATGC

CATAGCAGAA

AGGATCTTAC

TCAGCATCTT

GCAAAAAAGG

TATTATTGAA

TAGAAAAATA

GTTAAC

3060 3120 3180o 3240 3300 3360 3420 3476 INFORMATION~ FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GrITTTAC ATGCCGATGC TATCCACAGA AGAGGTGGTC AAATCATCCC A

U

Claims (28)

1. An E. coli/Corynebacterium shuttle vector comprising the origin of replication of shuttle vector pNG2, polycloning linker sites, and an antibiotic resistance marker with a size less than 4 kb, wherein the vector is yCE96.

2. An E. coli/Corynebacterium shuttle vector comprising the origin of replication of shuttle vector pNG2, polycloning linker sites, an antibiotic resistance marker with a size less than 4 kb, an insert consisting of a protein-encoding nucleic acid encoding a binding site mutant of diphtheria toxin, the CRM9 iron-independent promoter and the CRM9 signal sequence, wherein the protein-encoding nucleic acid is under the control of the CRM9 iron- independent promoter and is preceded by the CRM9 signal sequence.

3. The vector of claim 2, wherein the protein-encoding nucleic acid encodes CRM9. The vector of claim 3, wherein the CRM9 further comprises a second 15 attenuating mutation.

5. The vector of claim 4, wherein the second attenuating mutation is the insertion of a COOH terminal protein domain.

6. The vector of claim 4, wherein the second attenuating mutation is a COOH terminal mutation that reduces binding activity, but not translocating activity.

7. The vector of claim 4, wherein the second attenuating mutation is selected from the group consisting of S508F, Y514A/C, K516A/C, V523A/C, N524A/C, K526A/C and F530A/C.

8. A method of making the vector of claim 4, comprising: deleting COOH terminal base pairs of the attenuated CRM9 toxin- encoding nucleic acid using the restriction site Sph I at the toxin nucleotide position 1523 and a restriction site used to clone the COOH -27- terminal part of the toxin into the polylinker cloning sites of yCE96, to produce a gapped, linear, plasmid deleted in the COOH terminal coding region; amplifying a product that corresponds to the COOH terminal region of CRM9 deleted in step with a PCR primer that includes the desired mutation and 30-40 base pairs homologous to the down stream and upstream regions adjacent to the deletion; purifying the amplified product of step b) on an electrophoretic gel; and electroporating the product of step c) into a Corynebacterium along with the gapped plasmid of step under conditions which permit homologous recombination to occur intracellularly.

9. The method of claim 8, wherein the Corynebacterium is Corynebacterium ulcerans. method of claim 8, wherein the Corynebacterium is Corynebacterium diphtheriae mutated by chemical mutagenesis to exhibit less DNA restriction. 11 .A method of making the vector of claim 2 comprising: deleting a region of the COOH terminus-encoding nucleotides of the protein-encoding nucleic acid using a unique restriction site and a o°* restriction site used to clone the COOH terminal part of the toxin into the polylinker cloning sites of the shuttle vector, to produce a gapped, linear plasmid deleted in the COOH terminal coding region; amplifying a product that corresponds to the COOH terminus-encoding region deleted in step using a PCR primer that includes the desired mutation and 30-40 base pairs homologous to the downstream and upstream regions adjacent to the deletion; purifying the amplified product of step b) on an electrophoretic gel; and electroporating the purified product of step c) into a Corynebacterium along with the gapped plasmid of step under conditions which permit homologous recombination to occur intracellularly.

12. The method of claim 11, wherein the Corynebacterium is Corynebacterium ulcerans. -28-

13.The method of claim 11, wherein the Corynebacterium is Corynebacterium diphtheriae mutated to exhibit less DNA restriction.

14.The method of claim 11, wherein the protein encoding nucleic acid encodes a protein containing a disulfide bond.

15.The method of claim 11, wherein the encoded protein is a binding site mutant of diphtheria toxin, further comprising the CRM9 iron-independent promoter, wherein the protein- encoding nucleic acid is under the control of the CRM9 iron-independent promoter.

16.The method of claim 15, wherein the mutation is the introduction of a cysteine residue into CRM9 or its derivatives.

17.The method of claim 15, wherein the mutation is selected from the group consisting of K530C, K516C, D519C and S535C.

18.The method of claim 15, further comprising a second attenuating mutation, 15 which is the insertion of a COOH terminal protein domain.

19.The method of claim 15, further comprising a second attenuating mutation, which is a COOH terminal mutation that reduces binding activity, but not translocating activity. method of claims 15, further comprising a second attenuating mutation 20 selected from the group consisting of S508E, Y514A/C, K516A/C, V523A/C, N524A/C, K526A/C and F530A/C.

21.The method of claim 15, wherein the mutation is the introduction of residues or the replacement of residues in CRM9 or its derivatives, to attenuate the blocking effects of anti-diphtheria toxin antibodies.

22.A mutant strain of Pichia pastoris, comprising a mutation in at least one gene encoding elongation factor 2 (EF2), -29- wherein the mutation comprises a Gly to Arg replacement at a position two residues to the carboxyl side of the modified histidine residue diphthamide, whereby the strain is resistant to toxic ADP-ribosylating activity of a diphtheria toxin.

23.A method of producing a diphtheria toxin moiety, comprising transfecting the cell of claim 22 with a vector comprising a nucleic acid that encodes the toxin moiety, under conditions that permit expression of the nucleic acid to produce the diphtheria toxin moiety in the cell.

24.The method of claim 23, wherein the toxin moiety is glycosylated in the cell.

25.A method of expressing a diphtheria toxin or a pseudomonas exotoxin A toxin, comprising: transfecting, with a vector of claim 2, under conditions that permit expression of protein-encoding nucleic acid, a mutant strain of Chinese hamster ovary cells, comprising a mutation in at least one gene encoding elongation factor 2 (EF2), wherein the mutation comprises a Gly>Arg replacement at a position two residues to the carboxyl side of the modified histidine residue diphthamide, whereby the strain is resistant to the toxic ADP-ribosylating activity of diphtheria and pseudomonas toxins.

26.The method of claim 25, wherein the encoded protein is glycosylated in the cell.

27.The method of claim 23, wherein the diphtheria toxin moiety comprises a mutant diphtheria toxin moiety. 9* S* 28.The method of claim 27, wherein glycosylation sites are removed from the 25 toxin moiety.

29.A method of producing a toxin-antibody fusion protein, comprising transfecting the cell of claim 22 with a vector comprising a toxin protein- encoding nucleic acid and an antibody-encoding nucleic acid under conditions that permit expression of the toxin protein-encoding nucleic acid and the antibody-encoding nucleic acid to produce the toxin-antibody fusion protein acid in the cell. method of claim 29, wherein the toxin moiety is glycosylated in the cell.

31.The method of claim 29, wherein the toxin is a diphtheria toxin.

32.The method of claim 31, wherein the diphtheria toxin is a mutant diphtheria toxin.

33. The method of claim 32, wherein the toxin moiety of the toxin-antibody fusion protein lacks one or more glycosylation sites.

34. A vector according to claim 1 or 2 substantially as herein before described with reference to the examples. A method according to claims 8 or 11 substantially as herein before described with reference to the examples. Dated this TWENTY-NINTH day of AUGUST 2003. The Government of the United States of America, as represented by The Secretary, Department of health and Human Services Applicant Wray Associates Perth, Western Australia o; Patent Attorneys for the Applicant e**