AU631200B2 - Production of modified pe40 - Google Patents

Production of modified pe40 Download PDF

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AU631200B2
AU631200B2 AU52115/90A AU5211590A AU631200B2 AU 631200 B2 AU631200 B2 AU 631200B2 AU 52115/90 A AU52115/90 A AU 52115/90A AU 5211590 A AU5211590 A AU 5211590A AU 631200 B2 AU631200 B2 AU 631200B2
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Mark W. Riemen
Steven M. Stirdivant
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Merck and Co Inc
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

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  • Proteomics, Peptides & Aminoacids (AREA)
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Description

7437P/5478A o* C a S ae
I*
lA- 17879 TITLE OF THE INVENTION PRODUCTION OF MODIFIED PE 40 BACKGROUND OF THE INVENTION Traditional cancer chemotherapy relies on the ability of drugs to kill tumor cells in cancer patients. Unfortunately, these same drugs frequently kill normal cells as well as the tumor cells. The extent to which a cancer drug kills tumor cells rather than normal cells is an indication of the compound's degree of selectivity for tumor cells.
One method of increasing the tumor cell selectivity of cancer drugs is to deliver drugs preferentially to the tumor cells while avoiding normal cell populations. Another term for the selective delivery ii WF^WsP I 7437P/5478A 2 17879 of chemotherapeutic agents to specific cell populations is "targeting". Drug targeting to tumor cells can be accomplished in several ways. One method relies on the presence of specific receptor molecules found on the surface of tumor cells. Other molecules, referred to as "targeting agents", can recognize and bind to these cell surface receptors.
These "targeting agents" include, antibodies, growth factors, or hormones. "Targeting agents" i" which recognize and bind to specific cell surface i0 receptors are said to target the cells which possess 1 those receptors. For example, many tumor cells I possess a protein on their surfaces called ie epidermal growth factor receptor. Several growth factors including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) recognize and bind to the EGF receptor on tumor cells. EGF and TGF-alpha are therefore "targeting agents' for these tumor cells.
i "Targeting agents" by themselves do not kill 20 tumor cells. Other molecules including cellilar Si poisons or toxins can be linked to "targeting agents" to create hybrid molecules that possess both tumor cell targeting and cellular toxin domains. These hybrid molecules function as tutor cell selective 25 poisons by virtue of their abilities to target tumor cells and then kill those cells via their toxin compcnent. Some of the most potent cellular poisons used in constructing these hybrid molecules are bacterial toxins that inhibit protein synthesis in mammalian cells. Pseudomonas exotoxin A (PE-A) is one of these bacterial toxins, and has been used to 7437P/5478A 3 17879 construct hybrid "targeting toxin" molecules (U.S.
Patent 4,545,985).
PE-A is a 66 kD bacterial protein which is extremely toxic to mammalian cells. The PE-A molecule contains three functional domains: The amino-terminal binding domain, responsible for binding to a susceptible cell; The internally located "translocating" domain, responsible for delivery of the toxin to the cytosol; The carboxy-terminal enzymatic domain, responsible for S 10 cellular intoxication. PE-A has been used in the construction of "targeting-toxin" molecules, anti-cancer agents in which the 66 kD molecule is combined with the tumor-specific "targeting agent" .(monoclonal antibody or growth factor). The "targeting-toxin" molecules produced in this manner have enhanced toxicity for cells possessi;g receptors for the "targeting agent".
A problem with this approach is +hat the PE-A antibody or growth factor hybrid still has a reasonably high toxicity for normal cells. This S* toxicity is largely due to the binding of the hybrid protein to cells through the binding domain of the PE-A. In order to overcome this problem, a protein was recombinantly produced which contains only the 25 enzymatic and "translocating" domains of Pseudomonas exotoxin A (Hwang e-t al., Cell, 48:129-137 1987).
This protein was named PE 4 0 since it has a molecular weight of 40 kD. PE 4 0 lacks the binding domain of PE-A, and is unable to bind to mammalian cells. Thus, PE 4 0 is considerably less toxic than the intact 66 kD protein. As a result, hybrid "targeting-toxin" molecules produced with PE 4 0 were 7437P/5478A 4 17879 much more specific in their cellular toxicity (Chaudhary et al., Proc. Nat. Acad. Sci. USA, 84: 4583-4542 1987).
While working with PE 4 0 it was found that the cysteine residues at positions 265, 287, 372 and 379 (numbering from the native 66 kD PE-A molecules: Gray et al., Proc. Natl. Acad. Sci., USA, 81, 2645-2649 (1984)) interfered with the construction of "targeting-toxin" molecules using chemical conjugation methods. The reactive nature of the S 10 disulfide bonds that these residues form leads to ambiguity with regard to the chemical integrity of the product "targeting toxin".
S
DISCLOSURE STATEMENT 1. U.S. patent 4,545,985 teaches that pseudomonas exotoxin A can be conjugated to antibodies or to epidermal growth factor. Patent 4,545,985 further teaches that these conjugates can be used to kill human tumor cells.
2. U.S. patent 4,664,911 teaches that antibodies can be conjugated to the A chain or the B chain of ricin which is a toxin obtained from plants. Patent 25 4,664,911 further teaches that these conjugates can be used to kill human tumor cells.
7437P/5478A 5 17879 3. U.S. patent 4,675,382 teaches that hormones such as n-elanocyte stimulating hormone (MSH) can be linked to a portion of the diphtheria toxin protein via peptide bonds. Patent 4,675,382 further teaches that the genes which encode these proteins can be joined together to direct the synthesis of a hybrid fusion protein using recombinant DNA techniques. This fusion protein has the ability to bind to cells that possess MSH receptors.
4. Murphy Lt al., PNAS USA 83:8258-8262 1986, "Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria :toxin-related alpha-melanocyte-stimulating hormone fusion protein. This article teaches that a hybrid fusion protein produced in bacteria using recombinant DNA technology and consisting of a portion of the diphtheria toxin protein joined to alpha-melanocytestimulating hormone will bind to and kill human .melanoma cells.
5. Kelley et al., PNAS USA 85: 3980-3984 1988, Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo. This article teaches that a hybrid fusion protein produced in 25 bacteria using recombinant DNA technology and consisting of a portion of the diphtheria toxin protein joined to interleukin 2 functions in nude 9 mice to suppress cell mediated immunity.
6. Allured tej al., PNAS USA 83:1320-1324 1986, Structure of exotoxin A of Pseudomonas aeruginosa at Angstrom. This article teaches the three dimensional structure of the pseudomonas exotoxin A protein.
3~EI~P- 7437P/5,. :A 6 17879 a e, *0 7. Hwang t ail., Cell 48:129-136 1987, Functional Domains of Pseudomonas Exotoxin Identified by Deletion Analysis of the Gene Expressed in E. Coli.
This article teaches that the pseudomonas exotoxin A protein can be divided into three distinct functional domains responsible for: binding to mammalian cells, translocating the toxin protein across lysosomal membranes, and ADP ribosylating elongation factor 2 inside mammalian cells. This article further teaches that these functional domains correspond to distinct regions of the pseudomonas exotoxin A protein.
8. European patent application 0 261 671 published 30 March 1988 teaches that a portion of the pseudomonas exotoxin A protein can be produced which lacks the cellular binding function of the whole pseudomonas exotoxin A protein but possesses the translocating and ADP ribosylating functions of the whole pseudomonas exotoxin A protein. The portion of the pseudomonas exotoxin A protein that retains the 20 translocating and ADP ribosylating functions of the whole pseudoionas exotoxin A protein is called pseudomonas exotoxin 40 or PE-40. PE-40 consists of amino acid residues 252-613 of the whole pseudomonas exotoxin A protein as defined in Gray 25 aL., PNAS USA 81:2645-2649 1984. This patent application further teaches that PE-40 can be linked to transforming growth factor-alpha to form a hybrid fusion protein produced in bacteria using recombinant DNA techniques.
9. Chaudhary at ail., PNAS USA 84:4538-4542 1987, Activity of a recombinant fusion protein between i i j i i i 0* S 5* as
S
o ~l 7437P/5478A 7 17879 transforming growth factor type alpha and Pseudomonas exotoxin. This article teaches that hybrid fusion proteins formed between PE-40 and transforming growth factor-alpha and produced in bacteria using recombinant DNA technique.' will bind to and kill human tumor cells possessing epidermal growth factor receptors.
Bailon et al., Biotechnology, pp. 1326-1329 Nov.
1988. Purification and Partial Characterization of S* 10 an Interleukin 2-Pseudomonas Exotoxin Fusion Protein. This article teaches that hybrid fusion proteins fformed between PE-40 and interleukin 2 and produced in bacteria using recombinant DNA techniques will bind to and kill human cell lines possessing S 15 interleukin 2 receptors.
OBJECTS OF THE INVENTION It is an object of the present invention to too provide modifications of PE 4 0 which provide 20 improved chemical integrity and defined structure of conjugate molecules formed between "targeting agents" and modified PE 4 0 It is another object of this invention to provide a method for preparing and recovering the modified PE 4 0 domain from fusion 25 proteins formed between "targeting agents" and S* modified PE 4 0 These and other objects of the present invention will be apparent fro., the following description.
SUMMARY OF THE INVENTION The present invention provides modifications of the PE 40 domain which eliminate the chemical ambiguities caused by the cysteines in PE 1 -8- Substitution of other amino acids such as, ala;nne for the cysteine residues in PE 40 or deletion of two or more of the cysteine residues improves the biological and chemical properties of the conjugates formed between modified PE 40 and a targeting agent.
According to a broad format therefore, this invention provides a polypeptide selected from the group consisting of PE 40 aB, PE 40 Ab, or PE 40 ab, as herein defined.
DETAILED DESCRIPTION OF THE INVENTION Hybrid molecules produced by conjugation of EGF and PE40 are characterized in three primary assay systems. These assays include: ADP ribosylation of elongation factor 2 which measures the enzymatic activity of EGF-PE 40 which inhibits mammalian protein synthesis; inhibition of radiolabled EGF binding to the EGF receptor on membrane vesicles from A431 cells which measures the EGF receptor binding activity of EGF-PE 40 and cell viability as assessed by conversion of 3-C4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan which Is used to measure the survival of tumor cells following exposure to EGF-PE 40 These assays are performed as .previously described (Chung et al., Infection and Immunity, 16:832-841 20 1977, Cohen et al., J. Biol. Chem., 257:1523-1531 1982, Riemen et al., SPeptldes 8:877-885 1987, Mossman, J. Immunol. Methods, 65:55-63 1983).
To create EGF-PE 40 protein conjugates with defined chemical structure and superior biologic characteristics, we used recombinantly *J expressed TGF-alpha PE 40 molecules as the source material for modified PL 40 We first produced a series of recomblnant DNA molecules that encoded either TGF-alpha PE 40 or specifically modified versions e*
IN
ST
tSA0 7437P/ .478A 9 17879 of TGF-alpha PE 4 0 The original or parental TGF-alpha PE 4 0 gene was molecularly cloned in a bacterial TAC expression plasmid vector (pTAC TGF57-PE40) using distinct segments of cloned DNA as described in Example 1. The pTAC TGF57-PE40 DNA clone was used as the starting reagent for constructing specifically modified versions of TGF-alpha PE 4 0 DNA. The specific modifications of the pTAC TGF57-PE40 DNA involve site specific mutations in the DNA coding sequence required to 10 replace two or four of the cysteine codons within the
PE
4 0 domain of the pTAC TGF57-PE40 ENA with codons •for other amino acids. Alternatively, the site r** specific mutations can be engineered to delete two or four of the cysteine codons within the PE40 domain of a.
15 pTAC TGF57-PE40. The site specific mutations in the pTAC TGF57-PE40 DNA were constructed using the methods of Winter £t al., Nature 211:756-758 1982.
Specific examples of the mutated pTAC TGF57-PE40 DNAs are presented in Example 2.
20 The amino acid sequence of the parent TGF-alpha PE 40 is presented in Table 3. The four cysteine residues in the PE 40 domain of the parental TGF-alpha PE hybrid fusion protein are designated residues Cys Cys 287 Cys 372 and r ,y 379 25 Cys Amino acid residues are numbered as defined for the native 66 kD PE-A molecule (Gray t al, Proc. Natl. Acad. Sci., USA, 81, 2645-2649 1984). The modified TGF-alpha PE 40 fusion proteins used to generate the modified PE 40 molecules contain substitutions or deletions of residues [Cys 265 and Cys 287 or [Cys 372 and 379 265 Cy 287 372 and Cys 3 or [Cys Cys Cys and c 7437P/5478A 10 17879 Cys 37 To simplify the nomenclature for the modified PE 40 molecules generated from the modified fusion proteins, we have designated the amino acid residues at positions 265 and 287 as the locus, and the residues at positions 372 and 379 the "B" locus. When cysteines are present at amino acid residues 265 and 287 as in the parental TGF-alpha
PE
40 fusion protein, the locus is capitalized (i.e.
When the cysteines are substituted with other I amino acids or deleted from residues 265 and 287, the 10 locus is represented by a lower case Similarly, when the amino acid residues at positions 372 and 379 Sare cysteines, the locus is represented by an upper case while a lower case represents this locus when the amino acid residues at positions 372 or 379 15 are substituted with other amino acids or deleted.
Thus when all four cysteine residues in the PE 4 0 domain are substituted with alanines or deleted the modified PE 40 is designated PE 40 ab. In a similar fashion the parental PE 40 derived from the 20 parental TGF-alpha PE 40 fusion protein with j cysteines at amino acid residue positions 265, 287, 372, and 379 can be designated PE 40
AB.
The source materials the TGF-alpha
PE
40 AB hybrid protein, and the modified TGF-alpha 25 PE 40 Ab, aB and ab hybrid proteins), are produced i in E. coli using the TAC expression vector system I "described by Linemeyer at al., Biotechnology 5:960-965 1987. The source proteins produced in these bacteria are harvested and purified by lysing the bacteria in guanidine hydrochloride followed by the addition of sodium sulfite and sodium tetrathionate. This reaction mixture is subsequently C JI~II~BB~ l~ V~ ili~i*-..
7437P/5478A 11 17879
S
S
a.
9 4
S@
a.
9 9*S* dialzyed and urea is added to solubilize proteins which have precipitated from solution. The mixture is centrifuged to remove insoluble material and the recombinant hybrid TGF-alpha PE 4 0 source proteins are separated using ion exchange chromatography, followed by size exclusion chromatography, followed once again by ion exchange chromatography.
Since the single methionine residue in the hybrid source proteins is located between the TGF-alpha and PE 4 0 domains, treatment with CNBr 10 would cleave the source proteins, yielding the modified PE 4 0 proteins and TGF-alpha. The purified S-sulfonate derivatives of TGF-alpha PE 4 0 are thus subjected to CNBr treatment to remove the TGF portion of the molecule. The desired modified PE 4 0 portion is purified by ion-exchange chromatography followed by size exclusion chromatography. The purified modified PE 4 0 is then derivatized with a suitable heterobifunctional reagent, e.g. SPDP, to allow conjugation of the desired targeting agent.
23 Following conjugation, size exclusion chromatography is used to isolate the conjugate from non-conjugated materials. Once the purified conjugate is isolated, it ii tested for biologic activity using the ADP ribosylation assay and the relevant receptor binding and cell viability assays.
The following examples illustrate the present invention without, however, limiting the same thereto. All of the enzymatic reactions required for molecular biology manipulations, unless ccherwise 30 specified, are carried out as described in Maniatis it i 1 (1982) In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press.
1
F~-
7437P/5478A 12 17879 Example 1 Construction of recombinant DNA clones containing TGF-alpha PE0 DNA The TGF-alpha DNA segment was constructed using three sets of synthetic oligonucleotides as described by Defeo-Jones et al., Molecular and Cellular Biology 8:2999-3007 1988. This synthetic TGF-alpha gene was cloned into pUC-19. DNA from the pUC-19 clone containing recombinant human TGF-alpha was digested with Sph I and Eco RI. The digestion generated a 2.8 kb DNA fragment containing all of pUC-19 and the 5' portion of TGF-alpha. The 2 8 kb fragment was purified and isolated by gel electrophoresis. An Eco RI to Sph I oligonucleotide S 15 cassette was synthesized. This synthetic cassette oe had the sequence indicated below: 5'-CCGACCTCCTGGCTGCGCATCTAGG-3' For convenience, this oligonucleotide S cassette was named 57. Cassette 57 was annealed and I ligated to the TGF-alpha containing 2.8 kb fragment j forming a circularized plasmid. Clones which S 2F contained the cassette were identified by hybridization to radiolabeled cassette 57 DNA. The *I presence of human TGF-alpha was confirmed by DNA Ssiquencing. Sequencing also confirmed the presence of a newly introduced Fsp I site at the 3' end of th1 TGF-alpha sequence. This plasmid, named TGF-alpha-57/pUC-19, was digested with HinD 111 and Fsp I which generated a 168 bp fragment containing r c 7437P/5478A 13 17879 the TGF-alpha gene (TGF-alpha-57). A separate preparation of pUC-19 was digested with HinD III and Eco RI which generated a 2.68 kb pUC-19 vector DNA.
The PE 40 DNA was isolated from plasmid pVC 8 (Chaudhary at al., PNAS USA 84:4538-4542 1987). pVC 8 was digested using Nde I. A flush end was then generated on this DNA by using the standard conditions of the Klenow reaction (Maniatis et al., pupra, p.113). The flush-,nded DNA was then subjected to a second digestion with Eco RI to 10 generate a 1.3 kb Eco RI to Nde I (flush ended) fragment containing PE 40 The TGF-alpha-57 HinD III to Fsp I fragment (168 bp) was ligated to the 2.68 kb pUC-19 vector. Following overnight incubation, the 1.3 kb EcoRI to Nde I (flush ended) 15 PE 40 DNA fragment was added to the ligation mixture. Th.i second ligatior was allowed to proceed i overnight. The ligation reaction product was then iused to transform J' 109 cells. Clones containing i TGF-alpha-57 PE 40 in pUC-19 were identified by S 20 hybridization to radiolabeled TGF-alpha-57 PE 0 DNA and the uNA from this clone was isolated. T''e STGF-alpha-57 PE 40 4as removed from the pUC-19 vector and transferred to a TAC vector system described by Linemeyer et al., Bio-Technology .:960-965 1987). The TGF-a.pha-57 PE 4 0 in pUC-19 *i was digested with HinD III aad Eco RI to generate a kb fragment containing TGF-alpha-57 PE 40
A
flush end was generated on this DNA fragment using stai.dard Klenow reaction conditions (Maniatia et Z1., op. cIt.). The TAC vector was digested with HinD II and Eco RI. A flush end was generated on the digested TAC vector DNA using standard Klenow nw~raga~la~* 9~B~Bl s~~lBB~igFA fiawyM/ar 7437P/5478A 14 17879 reaction conditions (Maniatis at al., gE. Lit. The 2.7 kb flush ended vector was isolated using gel electrophoresis. The flush ended TGF-alpha-57 PE 40 fragment was then ligated to the flush ended TAC vector. The plasmid generated by this ligation was used to transform JM 109 cells. Candidate clones containing TGF-alpha-57 PE 4 0 were identified by hybridization as indicated above and sequenced. The clone containing the desired construction was named pTAC TGF57-PE40. The plasmid generated by these manipulations is depicted in Table 1. The nucleotide sequence of the amino acid codons of the TGF-alpha
PE
40 fusion protein encoded in the pTAC TGF-57-PE40 DNA are depicted in Table 2. The amino acid sequence encoded by the TGF-5/-PE40 gene is shown in Table 3.
1 S..Example 2 Constru tion of modified veisiors of recombinant TGF-alpha PE 40 containing DNA clones: Substitution of alanines for cysteines.
TGF-alpha PE 40 aB: The clone pTAC TGF57-PE40 was digested with SphI and BamHI and the 750 bp SphI-BamHI fragment (specifying the C-terminal 5 amino acids of TGF-alpha •i and the N-terminal 243 amino acids of PE40) was isolated. M13 mpl9 vector DNA was cut with SphI and BamHI and the vector DNA was isolated. The 750 bp SphI-BamHI TGF-alpha PE 40 fragment was ligated into the M13 vector DNA overnight at 15*C. Bacterial host cells were transformed with this ligation mixture, candidate clones were isolated and their WAN WON-
'IMMOOM
r I:_17- n? "-C13)li l: 7437P/5478A 15 17879 plasmid DNA was sequenced to insure that these clones contained the proper recombinant DNAP. Single stranded DNA was prepared for mutagenesis.
An oligonucleotide (oligo #132) was synthesized and used in site directed mutagenesis to introduce a Hpal site into the TGF-alpha PE/0 DNA at amino acid position 272 of CTGGAGACGTTAACCCGTC 3' (oligo #132) S. 10 One consequence of this site directed mutagenesis was the conversion of residue number 272 S' in PE 40 from phenylalanine to leucine. The mutagenesis was performed as described by Winter et aL Nature, 29_:756-758 1982.
15 A candidate clone containing the newly i created Hpal site was isolated and sequenced to validate the presence of the mutated genetic sequence. This clone was then cut with SphI and SalI. A 210 bp fragment specifying the C-terminal 20 amino acids of TGF-alpha and the N-terminal 70 amino acids of PE 40 and containing the newly introduced Hpal site was isolated and subcloned back into the parent pTAC TGF57-PE40 plasmid at the SphI-SalI sites. Bacterial host cells were transformed, a 25 candidate clone was isolated and its plasmid DNA was .i sequenced to insure that this clone contained the proper recombinant DNA. For convenience this clone was named pTAC TGF57-PE40-132. pTAC TGF57-PE40-132 was digested with SphI and HpaI and a 3.96 Kb DNA fragment was isolated. A synthetic oligonucleotide cassette (oligo #153) spanning the C-terminal 5 amino acids of TGF-alpha and the N-terminal 32 6mino acids 7437P/5478A 16 17879 of PE 40 and containing SphI and HpaI compatible ends was synthesized and ligated to the digested pTAC TGF57-PE40-132:
CGGACCTCCTGGCCATCGCCGMAGAGGGC(GCAGCCTGGCCGCGCTGACCGCGOA
3' GTACGCCTGGAGC.'cGCcTACCGGCTTcTCC~cCCGTCGGACCGGCGCGACTGGCGCGT CCAGCT(nCACACCTGCCGCTGGAGACGTT 31 GGTCGAc.,TGTGGACGGcGACCTCTGCAA 5' (oligo #153) 2* 10 This oligonucleotide cassette incorporated a change in the TGF-alpha PE 40 DNA so that the codon specifying cysteine at residue 265 now specified alanine. For convenience this plasmid DNA *.:was called pTAC TGF57-PE4O--132,153. Bacterial host K 15 cells were transformed with pTAC TGF57-PE40-*132,153 DNA. Candidate clones were identified by hybridization, isolated and their plasmid DNA was sequenced to insure that it contained the proper recoalbir ,t DNA.
20 pTAC GF7I0-132,153 DNA was digested with ilpaI and SalI and a 3.95 Kb vector DNA was isolated. A synthetic oligonucleotide cassette (oligo #142) spanning amino acid residues 272 to 309 of PE 40 and containing HpaI and Sail compatible 25 ends was synthesized and ligated to the 3.95 Kb pTAG TGFIPE4O 132,153 DNA.
5i MCCCGTCATCGCCAGCCGCGCGGCTGOGMACAACTGGAGCAGGCTGGCTATCCGGTGC V, "CGGCAGTAGCGOTCGGCGCGCCOACCCTTGTTGACCTCGTCCGACCGATAOGCCACG AGCGGCTGGTCGCCCTCTACCTGGCCCGCGCCTGTCGTGMACCAGG 3' TCGCCACCAGCGGGAGATGGACCGCCGCCOACAGCACCTTGOTcCAGCT 5' (oligo #142)
IXI~
7437P/5478A 17 17879 This oligonucleotide cassette changes the codon specifying cysteine at residue 287 so that this codon now specifies alanine. For convenience this mutated plasmid DNA was called pTAC TGF57-PE40- 132,153,142. Bacterial host cells were transformed with this plasmid and candidate clones were identified by hybridization. These clones were isolated and their plasmid DNA was sequenced to insure that it contained the proper recombinant DNA. The pTAC TGF57-PE40-132,153,142 plasmid encodes the TGF-alpha 10 PE 40 variant with both cysteines at locus "A" replaced by alanines. Therefore, following the nomenclature described previously this modified version of TGF-alpha PE 4 0 is called TGF-alpha
PE
40 aB. The amino acid sequence encoded by the 15 TGF-alpha-PE 40 aB gene is shown in Table 4.
TGF-alpha PE 4 0 Ab: The clone pTAC TGF57-PE40 was digested with 20 SphI and BamHI and the 750 bp SphI-BamHI fragment (specifying the C-terminal 5 amino acids of TGF-alpha and the N-terminal 252 amino acids of PE 4) was 40 isolated. M13 mpl9 vector DNA was cut with SphI and BamHI and the vector DNA was isolated. The 750 bp 25 SphI-BamHI TGF-alpha PE40 fragment was ligated into the M13 vector DNA overnight at 15 0 C. Bacterial Shost cells were transformed with this ligation mixture, candidate clones were isolated and their plasmid DNA was sequenced to insure that these clones contained the proper recombinant DNAs. Single stranded DNA was prepared for mutagenesis.
7437P/5478A 18 17879 An oligonucleotide (oligo #133) was synthesized and used in site directed mutagenesis to introduce a BsteII site into the TGF-alpha PE 40 DNA at amino acid position 369 of 5' GACGTGGTGACCCTGAC 3' (oligo #133) One consequence of this mutagenesis was the conversion of the serine residue at position 369 of
PE
40 to a threonine.
A DNA clone containing the newly created S* BsteII site was identified, isolated and sequenced to ensure the presence of the proper recombinant DNA.
This clone was next digested with Apal and SalI restriction enzymes. A 120 bp insert DNA fragment 15 containing the newly created BsteII site was isolated and ligated into pTAC TGF57-PE40 that had also been digested with Apal and SalI. Bacterial host cells were transformed, and a candidate clone was isolated *i and sequenced to insure that the proper recombinant 20 DNA was present. This newly created plasmid DNA was called pTAC TGF57-PE40-133. It was digested with BsteII and Apal and 2.65 Kb vector DNA fragment was Sisolated.
A BsteII to Apal oligonucleotide cassette 25 (oligo #155) was synthesized which spanned the region of TGF-alpha PE 4 0 deleted from the pTAC TGF57-PE40-133 clone digested with BsteII and Apal restriction enzymes. This cassette also specified the nucleotide sequence for BsteII and Apal compatible ends.
GTGACCCTGACCGCGCCGGTCGCCGCCGGTGAAGCTGCGGGCC 3' 3' GGACTGGCGCGGCCAGCGGCGGCCACTTCGACGC 5' (oligo #155) 7437P/5478A 19 17879 This oligonucleotide cassette changed the codons for cysteines at residues 372 and 379 of
PE
40 to codons specifying alanines. 01igonucleotide cassette #155 was ligated to the 2.65 Kb vector DNA fragment. Bacterial host cells were transformed and candidate clones were isolated and sequenced to insure that the proper recombinant DNA was present.
This newly created DNA clone was called pTAC TGF57-PE40-133,155. It encodes the TGF-alpha
PE
40 variant with both cysteines at locus "B" f. 10 replaced by alanines. Therefore, following the a nomenclature described previously this modified :i .version of TGF-alpha PE40 is called TGF-alpha
PE
40 Ab. The amino acid sequence encoded by the TGF-alpha-PE 40 Ab gene is shown in Table a. S 'TGF-alpha
PE
4 0 ab: The pTAC-TGF57-PE40-132,153,142 plasmid encoding TGF-alpha PE 40 aB was digested with SalI 20 and Apal and the resultant 3.8 Kb vector DNA fragment was isolated. The pTAC TGF57-PE40-133,155 plasmid ,j encoding TGF-alpha PE 40 Ab was also digested with SalI and Apal and the resultant 140 bp DNA fragment containing the cysteine to alanine changes at amino 5 acid residues 372 and 379 of PE 40 was isolated.
These two DNAs were ligated together and used to transform bacterial host cells. Candidate clones were identified by hybridization with a radiolabeled 140 bp DNA from pTAC TGF57-PE40-133,155. Plasmid DNA from the candidate clones was isolated and sequenced to insure the presence of the proper recombinant DNA. This newly created DNA clone was called pTAC 11 'Mill am I I in 11 -1 1111 7437P/5478A 20 17879 TGF57-PE40-132,153,142,133,155. Thiq plasmid encodes the TGF-alpha PE 40 variant with ali 'ur cysteines at loci and replaced by alanines.
Therefore, following the nomenclature described previously this modified version of TGF-alpha
PE
40 is called TGF-alpha PE 40 ab. The amino acid sequence encoded by the TGF-alpha-PE 40 ab gene is shown in Table 6.
Example 3 10 Production and isolation of recombinant TGF-alpha PE40 source proteins Transformed E. oli JM-109 cells were cultured in 1 L shake flasks in 500 mL LB-Broth in i the presence of 100 ug/mL ampicillin at 37 0 C. After 4 I; 15 the A 600 spectrophotometric absorbance value reached 0.6, isopropyl B-D-thiogalactopyranoside was added to a final concentration of 1 mM. After 2 hours the cells were harvested by centrifugation.
The cells were lysed in 8 M guanidine 20 hydrochloride, 50 mM Tris, 1 mM EDTA, pH 8.0 by stirring at room temperature for 2 hours. The lysis mixture was brought to 0.4 M sodium sulfite and 0.1 M I sodium tetrathionate by adding solid reagents and the j pH was adjusted to 9.0 with 1 M NaOH. The reaction 25 was allowed to proceed at room temperature for 16 hours.
i The protein solution was dialysed against a S10,000 fold excess volume of 1 mM EDTA at 4 0 C. The mixture was then brought to 6 M urea, 50 mM NaCI, mM Tris, pH 8.0, at room temperature and stirred for 2 hours. Any undissolved material was removee by centrifugation at 32,000 x g for 30 minutes.
I L I I I wki I I am 11, 11 7437P/5478A 21 17879 *9
S~
tt. The cleared supernatant from the previous step was applied to a 26 x 40 cm DEAE Sepharose Fast-Flow column (Pharmacia LKB Biotechnology, Inc.) equilibrated with 6 M urea, 50 mM Tris, 50 mN NaC1, pH 8.0, at a flow rate of 1 mL/minute. The column was washed with the equilibration buffer until all unadsorbed materials were removed as evidenced by a T
A
280 spectrophotometric absorbance below 0.1 in the equilibration buffer as it exits th" column. The adsorbed fusion protein was eluted fom the .olumn with a 1000 mL 50-350 mM NaCI gradient and then concentrated in a stirred cell Amicon concentrator fitted with a YM-30 membrane.
The concentrated fusion protein (8 mL) was applied to 2.6 x 100 cm Sephacryl S-300 column 15 (Pharmacia LKB Biotechnology, Inc.) equilibrated with 6 M urea, 50 mM 50 mM NaC1, pH 8.0, at a flow rate of 0.25 mL/minute. The column was eluted with additional equilibration buffer and 3 mL fractions collected. Fractions containing TGF-alpha Ph 40 20 activity were pooled.
The pooled fractions from the S-300 column were applied to a 1.6 x 40 cm Q Sepharose Fast-Flow column (Pharmacia LKB Biotechnology, Inc.) equilibrated with 6 M urea, 50 mM Tris, 50 mM NaC1, 25 pH 8.0 at a flow rate of 0.7 mL/minute. The column was washed with the equilibration buffer and then eluted with a 600 mL 50-450 mM NaC1 gradient. The fractions containing the TGF-alpha PE40 activity were pooled and then dialyzed against 50 mM glycine pH 9.0 and stored at
*I,
'p~g 4 '0
I
S. S 7437P/5478A 22 17879 Example 4 CNBR cleavage of TGF-alpha PE 40 source proteins and isolation of modified PE40s (PE 4 0 AB, PE Ab. PE4 aB, PE ab). The desired fusion protein, still in the S-sulfonated form, is dialysed versus 10% (v/v) acetic acid in water, then lyophilized. The lyophilized protein is dissolved in a sufficient amount of deaerated 0.1 M HC1 to give a protein concentration of 1 mg/mL. The protein/HCl solution 10 contains 5 moles tryptophan/mole fusion protein.
CNBr (500 equivalents per equivalent of methionine) is added, and the reaction allowed to proceed vur 18 hours, at room temperature in the dark. Large digestion fragments, including the desired modified 15 PE 40 are then separated from the reaction mixture by gel filtration Sephadex G-25) in 25% acetic acid Fractions containing the modified PE 40 are pooled and lyophilized.
In the case of the modified proteins 20 containing cysteine (i.e PE 40 AB, PE 40 aB, and
PE
40 Ab) it is necessary to form the requisite disulfide bonds before proceeding with purification.
The lyophilized protein is therefore dissolved in a sufficient amount of 50 mM glycine, pH 10.5 to give a 25 UV A 28 0 0.1. Beta-mercaptoethanol is added to give a 4:1 molar ratio over the theoretical number of SS-sulfonate groups present in the protein sample.
The reaction is allowed to proceed for 16 hours at 4°C, after whic' time the solution is dialysed against a 10,000 fold excess of a buffer containing mM Tris, 1 mM EDTA, 100 mM NaCl, pH ___111 7437P/5478A 23 17879 Fractions from the anion exchange column containing the desired PE 40 are pooled based on ADP-ribosylation activity and protein content as determined by SDS-PAGE. The pooled fractions are concentrated using a 30,000 molecular weight cutoff membrane (YM-30, Amicon).
The pooled fractions are applied to a 2.6 x 100 cm Sephacryl '-200 gel filtration column H (Pharmacia LKB Biotechnology, Inc.), equilibrated in, and eluted with 20 mM Tris, 50 mM NaC1, 1 mM EDTA, pH i: 10 8.0 at a flow rate of 0.75 mL/minute. Fractions from the gel filtration chromatography are pooled based on ADP-ribosylation and SDS-PAGE.
Ii Though this procedure yields material sufficiently pure for most purposes, another 15 chromatographic step is included in order to produce highly homogeneous material. This final chromatographic step is high resolution gel filtration, using a 0.75 x 60 cm Bio-Sil TSK-250 I column (Bio-Rad). In preparation for chromatography 20 on the TSK-250 column, samples are concentrated on devices (Amicon) and protein concentration adjusted to 5 mg/mL. The sample is dissolved in 6 M urea, 100 mM sodium phosphate, 100 mM .,aCl, pH 7.1. The column is eluted with 6 M urea, 25 100 mM sodium jhosphate, 100 mM NaC1, pH 7.1, at a flow rate of 0 5 mL/minute. Fractions from the high A resolution gel filtration step are pooled based on ADP-ribosylation and SDS-PAGE.
7437P/5478A 24 17879 Example Conjugation of EGF to modified PE 40 s and isolation of conjugates In order to conjugate EGF to modified it is necessary to derivatize both the EGF and PE40 with heterobifunctional agents, so that a covalent connection between the two molecules can be achieved. In preparation for the derivatization, samples of modified PE 40 are dialyzed against 0.1 M NaC1, 0.1 M sodium phosphate, pH 7.0. Following 10 dialysis, the solution of modified PE 4 0 is adjusted to 4 mg/mL PE 40 using the dialysis buffer, giving a concentration of 100 uM. A sufficient amount of a s: mM solution of N-succinimidyl 3-(3-pyridyldithio)propionate (SPDP, Pierce) in ethanol is added to the 15 protein solution to give a final concentration of 300 uM SPDP. This concentration represents a 3:1 ratio of SPDP to PE 40 The derivatization reaction is allowed to proceed at room temperature for :minutes, with occasiontl agitation of the mixture.
20 The reaction is terminated by adding a large excess of glycine (approximately a 50-fold molar excess over the initial amount of SPDP). The resulting 3-(2-pyridyldithio)propionyl-derivative is called
PDP-PE
4 0 The non-protein reagents are removed 25 from the product by extensive dialysis versus 6 M urea, 0.1 h NaCl, 0.1 M sodium phosphate, pH The number of PDP-groups introduced into the modified
PE
40 is determined an described by Carlsson _t al., Biochem. 173:723-737 in78.
The PDP-EGF derivative is prepared by dissolving lyophilized EGF (Receptor grade, Collaborative Research) in a sufficient amount of r, L~"lllll~s ll~r''Iwo NNIM!"01 1 WHOM 11 7437P/5478A 25 17879 0.1 M NaC1, 0.1 M sodium phosphate, pH 7.0 to give a final concentration of 150 uM EGF. A sufficient amount of a 20 mM solution of SPDF in ethanol is added to the EGF solution give a final concentration of 450 uM SPDP, representing a 3:1 ratio of SPDP to EGF. The derivatization reaction is allowed to proceed at room temperature for minutes, with occasional agitation of the mixture.
The reaction is terminated by .ddiing a large excess of glycine (approximately a 50-fold mAlar excess over the initial amount of SPDF). The non-protein reagents are removed from the product by extensive dialysis versus 6 M urea, 0.1 M NaCI, 0.1 M sodium phosphate, pH 7.5. The number of PDP-groups introduced .nto EGF is determined as described by 15 Carlsson et i. Biochem. J 17:723-737 1978.
Using the derivatives described above, either PDP-PE 40 or PDP-EGF can be reduced at acidic pH, in order to generate the 3-thioproDionyl derivative, in the presence of the intact, native 20 disulfides (Carlsson It al. Eupra). However, the preferred strategy is the generation of a free thiol on the modified PE 40 0* 40
PDP-PE
4 0 (0.4 ml of a 100 uM solution of
PDP-PE
4 0 in 6 M urea, 0.1 M NaC1, 0.1 M sodium 25 phosphate, pH 7.5) is dialyzed against several 500 mL changes of a buffer containing 6 M urea, 25 mM sodium "acetate, pH 5.5, at 4°C. Following the dialysis, uL of 100 mM dithiothreitol (final concentration mM) is added to the PDP-PE 4 0 The reduction is al.owed to proceed for 10 minutes at room temperature, 7437P/5478A 26 17879 and is then terminated by dialysis of the reaction mixture against 6 M urea, 23 mM sodium acetate, i mM EDTA, pH 5.5, at 4°C. Dialysis against this buffer is repeated, and then the sample is dialyzed against 0.1 M NaCl, 0.1 M sodium phosphate, pH 7.5. The material generated by these manipulations is called thiopropionyl-PE 40 In preparation for conjugation, PDP-EGF (0.8 mL of a 150 uM solution in 6 M urea, 0.1 M NaCI, 0.1 M sodium phosphate, pH 7.5) is dialyzed against 10 several changes of 0.1 M NaCI, 0.1 M sodium phosphate, pH 7.5, at 4 0 C, to free the sample of urea. Following this dialysis, the PDP-EGF solution S"and the thiopropionyl-PE 40 solution are combined and the reaction mixture is incubated at room 9* 15 temperature foi 1 hour. The progress of the reaction can be monitored by measuring the release of pyridine-2-thione as described (Carlsson et al., supra). The reaction is terminated by dialysis against ueveral changes of 6 M urea, 0.1 M NaCI, 0.1 20 M sodium phosphate, pH 7.5, at 4 0
C.
The conjugates are purified by size a exclusion chromatography, using a high resolution 0.75 x 60 cm Bio-Sil TSK-250 column (Bio-Rad). The column is eluted with 6 M urea, 0.1 M sodium phosphate, 0.1 M NaCI, pH 7.1, at a flow rate uf mL/minute. Fractions from the high resolution gel filtration step are pooled based on ADP-ribosylation and SD' '\GE.
7437P/5478A -27 17879 Examvle 6 Conjugation of TGF-alpha to modified PE 4 0 s and isolation of conjugates Conjugation of TGF-alpha to modified PE 4 0 s and isolation of conjugates. Conjugates of TGF-alpha and the modified FE 4 0 are prepared in a fashion analogous to the *rif-conjugates describe in Example ,,xample 7 Biologic activites of TGF-alpha modified
PE
4 0 10 conjugates The relevant biologi activities of the 0* 690 0 TGF-alpha modified FE conjugates are similar to theresectvebiolsic 4activities of the hybrid .TGF-alpha PE E 40 de sc r ib ed in S a 9...u 52115/90 7437P/5478A 28 TABLE 1 (EC RD 17879
I.
I
I
4*4*1*
I
I. Ii
I
4 -II 4 Hi)
I
a I S .4 S. II 4
S
S S SOS a 55 a I I
II
acids of TGF-alpha and the N-terminal 32 amino acids 74~37P/5478A 29 17879 TABLE-2 9.
V
4.
4.
C
4* 4 4 *4 0
S
ATGGCTGCAGCAGTGGTGTCCCATI TTAATGACTGCCCAGATTCCCACACTCAGTTCTGCTTCCATGGAACATGCAGG TTTTTGGTGCAGGAGGACAAGCCGt3CATGT' TC TGCCATTCTGGGTAC GTTGGTGCGCGCTGTGAGCA TGCGGACCTC CTGGCT GCTATGGCCGAAGAGGGCGGCAGCCTGGCCGCGC TGACCGCGCACCAGGCTTGCCACCTGCCGCTGGAGACT TTCACCCGTCATCGCCAGCCGCGCGGCTGGGAACAAC TGGAGCAGTGCGGCTATC CGGTGCAGCGGCTGGTCGCCC TC Tk.CCTGGC GGCGCG3C TGTCGTGGAACCAGGTC UACCAGGTGATCCGCAACGCCCTGGCCAGCCCCGGCAGC GGCGGC GACC TGGGC GAAGCGA TCCGCGAGCAGC CG(GAGCAGGCCC TGGC CCTGACCCTGGCCGCCGCCGAGAGCGAGCGCTTC GTCCGGCAGGGCAC CGGCAACGACGAGGCC GGC GCGGCCAACGCCGACGT GGTGAGCC TGACC TGC CCGGTC GCCGCC GGTGAATGCGCGGGCCCGGCGGACAGCGGCGACGCCCT GCTGGAGCGCAACTATCC CA CTGGCGCGGAGTTCCTCGGC 10 GA CGGCGGCGAC GTCAGCTTCAGCACCC GCGGCACGCAGAAC T -AC GGTGGAGC GGC TGC TCCAGGCGCACCGCCAA C TGGAGGAGCGCGGC TAT GTGTT CGTCGGC TACCACGGCACC Ii CTCGAAGCGGCGCAAAGCATCGTCTTCGGCGGG GTGCGCGCq;CGCAGCCAGGACCTCGACGCGATCTGGCGCGGTTTCTATATCGCCGGCGATCCGGCGCTGGCCTACGGC
TACGCCCAGGACCAGGAACCCGACGCACGCGGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGCTCG
AGCCTGCCGGGC TTCTACCGCACCAGCCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCGAACGGCTGATCGGC 1 5 CATCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCCGAGGAGGAAGGCGGGCGCCTGGAGACCATTCTCGGCTGG CCGC TGGCCGAGC GCAC CGT GGT GAT TCCC TC GGCGATCCCCACCGAC CCGC GCMACCT CGGCGGCGACC TC GACC CG
TCCAGCATCCCCGACAAGGAACAGGCGATCAGCGCCCTGCCGGACTACGCCAGCCAGCCCGGCAAACCGCCGCGCGAG
GACCTGAAGTAA
4* *4 4 4 0~
S
Si @4 4 4 4
S
@0494 5 eq U S I 7437P/5478A 30 17879 TABLE 3 TGF-aipha-PE 40AMINO ACID SEQUENCE -4 Met Phe Gi y Gly -1 ITGF(X Ala 61a Val Thr Cys Arg Gly Ala Arg Ala Ala Leu S
S
S
*5
S
0* S S
S
*SSS
S
S
S. 55 S
S
555555
S
*5 S ft *5 Arg His Arg Gin Pro Arg Leu Val Ala Leu Tyr Leu Asn Ala Glu Gin Gly Thr Val Ala Tyr Pro 25 Thr Gin Tyr Val Ser Pro Leu Ala Asp Glu Giu Cys Ala Glu Thr Val Gly Tyr His Phe Asn Asp Cys 26 Leu Val Gi n Glu Asp 46 Glu His Ala Asp Leu 263 Ala His Gin Ala Cys 283 Trp Glu Gin Leu Glu 303 Ala Arg Leu Ser Trp 323 Ser Gly Gly Asp Leu 343 Thr Leu Ala Ala Ala 363 Gly Ala Ala Asn Ala 383 Gly Pro Ala Asp Ser 403 Leu Gly Asp Gly Gly 423 Arg Leu Leu Gin Ala 443 Gly Thr Phe Leu Glu Pro Asp Ser Lys Pro Alp.
TGRX 50 1 Pro Leu Gly Tyr Val Asp Ala Ilie Glu Arg Val Ser Ala Leu Ser Plie Gin Leu Gin Ser Thr Phe Val Gin Val Ilie Glu Gin Val Arg Thr Cys Glu Arg Thr Arg Glu Arg Val Phe His Thr Cys Val Gin Phe Cys 36 Cys His Ser
F
252 Leu Ala kl Met Ala 'Ulu 1 31u Gly 463 Gly Val Arg Ala Arg Ser Gin Asp Leu Asp Ala Ilie Trp Arg Gly Phe Tyr Ile Ala Gly 7437P/5478A -31 -17879 TABLE 3 CONT'D TGF-aipha-PE 40AMINO ACID SEQUENCE 483 493 Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gin Asp Gin Glu Pro Asp Ala Arg Giy Arg Ilie 503 513 Arg Asn Gly Ala Leu Leu Arg Val Tyr Vai Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg 523 533 Thr Ser Leu Thr Leu Ala Ala Pro Giu Ala Ala Gly Glu Val Giu Arg Leu Ile Giy His 543 553 Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Giu Glu Giu Gly Giy Arg Leu Glu 563 573 Thr Ile Leu Giy Trp Pro Leu Ala Giu Arg Thr Val Val Ile Pro Ser Ala Ile ?ro Thr 9. 9583 593 Asp Pro Arg Asn Val Gly Gly Asr., Leu Asp Pro Ser Ser Ile Pro Asp Lys Giu Gin Ala 603 613 Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gin Pro Gly Lys Pro Pro Arg Giu Asp Leu Lys ~GTGACGCTGACCGCGCCGGTCGCCGCCGGTGMAGCTGCGGGCC 3' 3t GGACTGGCGCGGCCAGCGGCGGCCACTTCGACGC 5' (Oligo #155) 7437 P/5478A 32 17879 TABLE 4 TGF-alpha-PE 40-aB AMINO ACID SEQUENCE 0 0 *0000*
S
*00000 0 0 *0 0* S S S SO
S
5055
*SS*
5 0 *5 S S. 50 S S
S
S
S. 0 *0 -4 Met Phe 61 y 10 Gly Arg Leu Asn Glu 20 Gly Val Tyr 25 Thr Tyr Gly GhFa I/al Cys Ala Ala Pro Tyr Leu Ala Ser Ala Arg Leu Gly Asn Asp Ala Gly Glu Thr Glu Ala Asn Trp Thr Phe Val Gly Arg Ala Arg Val Ser His Arg Phe Leu Arg Cys Giu Leu Thr Ala Arg Gly Trp Leu Ala Ala Pro Gly Ser Ala Leu Thr Glu Ala Gly Cys Ala Gly Glu Phe Leu Val Glu Arg Tyr His Gly Ser Gin Asp Asn Asp Cys 26 Gin Glu Asp 46 Ala Asp Leu 263 Gin Ala Ala 283 Gin Leu Glu 303 Leu Ser Trp 323 Giy Asp Leu 343 Ala Ala Ala 3 63 Ala Asn Ala 383 Ala Asp Ser 403 Asp Gly Gly 423 Leu Gin Ala 443 Phe Leu Glu 463 Asp Ala Ilie TGFa 50 1 Leu Ala l a His Leu Pro Gin Ala Gly Asn Gin Val Gly Glu Ala Giu Ser Giu Asp Val Val Gly Asp Ala Asp Val Ser His Arg Gin Ala Ala Gin Trp Arg Gly Pro Acp Ser His Thr Gln Phe Cys 36 Lys Pro Ala Cys Val Cys His Ser Glu blu Gly 273 Thr Leu Thr 293 Val Gin Arg 313 Val Ilie Arg 333 Giu Gin Pro 353 Val Arg Gin 373 Thr Cys Pro 393 Giu Arg Asn 413 Thr Arg Gly 433 Giu Arg Gly 453 Val Phe Gly 473 Ile Ala Gly f.
7437 P/5478A 33 17879 TABLE 4 CONT'D TGF-aiphs.-PE 4 aB AMINO ACID SEQUENCE 9 9 999999 9 9 9. 9 9 9 o .9 99 9 9 9.9 9 9999 9 99*9 9 9 *9 99 *9 9 9 999999 9 .9 9 9 9 99 Asp Pro Ala Arg Asn Gly Thr Ser Leu 10 Pro Leu Pro Thr Ile Leu Asp Pro Arg Ilie Ser Ala Leu Ala Tyr Gly Ala Leu Leu Arg Thr Leu Ala Ala Leu Arg Leu Asp Gly Trp Pro Leu Asn Val Gly Gly Leu Pro Asp Tyr Ala Gin 503 Tyr Val 523 Giu Ala 543 Ilie Thr 563 Giu Arg 583 Leu Asp 603 Ser Gin Asp Gin Pro Arg Ala Gly Gly Pro Thr Val Pro Ser Pro Gly Giu Pro Ser Ser Giu Val Giu Giu Val Ilie Ser Ilie Lys Pro Ala Arg Pro Gly Arg Leu Gly Giy Ser Ala Asp Lys Arg Giu 493 Arg Ilie 513 Tyr Arg 533 Gly His 553 Leu Giu 573 Pro Thr 593 Gin Ala 613 Leu Lys 7437P/5478A 34 17879 TABLE TGF-.alpha-PE 40Ab AMINO ACID SEQUENCE -4 -3 -2 -1 FrGFC~l 6 16 Met Ala Ala Ala 1 Jal Val Ser His Phe Asn Asp Cys Pro Asp Ser His Thr Gin Phe Cys 26 36 Phe His Gly Thr Cys Arg Phe Leu Val Gin Giu Asp Lys Pro Ala Cys Val Cys His Ser 46 TGFCC 50 1 E 252 Gly Tyr Val Gly Ala Arg Cys Glu His Ala Asp Leu Leu Ala 1 la Met Ala Giu 1 31u Gly ::263 273 10 Gly Ser Leu Ala Ala Leu Thr Ala His Gin Ala Cys His Leu Pro Leu Glu Thr Phe Thr *283 293 *Arg His Arg Gin Pro Arg Gly Trp Giu Gin Leu Giu Gin Cys Gly Tyr Pro Val Gin Arg 303 313 Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gin Val Asp Gin Val Ile Arg 323 333 Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Giu Ala Ile Arg Glu Gin Pro 343 353 Giu Gin Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gin ~363 373 Gly Thr Gly Asn Asp Giu Ala Gly Ala Ala Asn Ala Asp Val Val Thr Leu Thr Ala Pro 393 Val Ala Ala Gly Glu Ala Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn 403 413 Tyr Pro Thr Gly Ala Giu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly 25 423 433 Thr Gin Asn Trp Thr Val Giu Arg Leu Leu Gin Ala His Arg Gin Leu Giu Giu Arg Gly 443 453 Tyr Val Phe Val Gly Tyr His Gly Thr Phe Lcu Giu Ala Ala Gin Ser Ile Val Phe Gly 463 473 Gly Val Arg Ala Arg Ser Gin Asp Leu Asp Ala Ile Trp Arg Gly Plie Tyr Ile Ala Gl y 7437 P/5478A 35 17879 TABLE 5 CONTID TGF-alpha-PE 40Ab AMINO ACID SEQUENCE 9
S
*9 S *9 99
S
S
Asp A rg Thr Pro Thr Asp Ile Ala Leu Gly Ala Leu Thr Pro Leu Leu Gly Arg Asn Ala Leu Ala Tyr Gly Leu Leu Arg Leu Ala Ala Arg Leu Asp Trp Pro Leu Val Gly Gly Pro Asp Tyr Ala Gin Asp Gin Giu 503 Tyr Val Pro Arg Ser 523 Glu Ala Ala Glv Glu 543 Ilie Thr Giy Pro Giu 563 Giu Arg Thr Val Val 583 Leu Asp Pro Ser Sor 603 Ser Gin Pro Giy Lys Pro Asp Ala Arg Ser Leu Pro Gly Val Giu Arg Leu Giu Giu Giy Gly Ile Pro Ser Ala Ile Pro Asp Lys Pro Pro Arg Glu 493 Gly Arg Ilie 513 Phe Tyr Arg 533 Ilie Gly His 553 Arg Leu Giu 573 Ilie Pro Thr 593 Glu Gin Ala 613 Asp Leu Lys L.J as" .L 0L .1 11111 AL.J.Lrl, .LVV HI~ 1'I1\L%.L JJI, Ui 7437 P/5478A 36 17879 TABLE 6 TGF-.alpha-PE 40ab AMINO ACID SEQUENCE 4 -3 -2 -1 IGFC Met Ala Ala Ala Phe His Gly Thr Gly Tyr Val Gly Gly Ser Leu Ala Arg His Arg Gin .Leu Val Ala Lev Asn Ala Leu Ala Glu Gln Ala Arg Gly Thr Gly Asn Val Ala Ala Gly Tyr Pro Thr Gly 25 Thr Gin Asn Trp Tyr Val Phe Val 1 Val Ser Cys Arg Phe Ala Arg Cys Ala Leu Thr Pro Arg Gly Tyr Leu Ala Ser Pro Gly Leu Ala Leu Asp Giu Ala Glu Ala Ala Ala Glu Phe Thr Val Glu Gly Tyr His Phe Asn 26 Val Gin His Al a 263 His Gin 283 Giu Gin 303 Arg Leu 323 C'iy Gly 343 Leu Ala 363 Ala Ala 383 Pro Ala 403 Gly Asp 423 Leu Leu 443 Thr Phe Asp Cys Pro Asp Ser Glu Asp Lys Proj Ala TGF(X 50 1 Asp Lev~ Leu Ala kia Ala Ala His Leu Pro Leu Glu Gin Ala Giy Ser Trp Asn Gin Val Asp Leu Gly Glu Ala Ala Ala Glu Ser Glu Asn Ala Asp Val Val Asp Ser Gly Asp Ala Gly Gly Asp Val Ser Gin Ala His Arg Gin Leu Glu Ala Ala Gin Gin Phe Cys 36 Cys His Ser Glu 1 31u Gly 273 Thr Lev Thr 293 Val Gin Arg 313 Val Ilie Arg 333 Glu Gin Pro 353 Val Arg Gin 373 Thr Ala Pro 393 Glu Arg Asn 413 Thr Arg Gly 433 Giu Arg Gly 453 Val Phe Gly 463 473 Giy Val Arg Ala Arg Ser Gin Asp Leu Asp Ala Ilie Trp Arg Gly Phe Tyr Ile Ala Gly lb- 7437P/5478A 37 -17879 TABLE 6 CONT'D TGF-aipha-PE 40ab AMINO ACID SEQUENCE 483 493 Asp Pro Ala Leu Ala Tyr Giy Tyr Ala Gin Asp Gin Giu Pro Asp Ala Arg Gly Arg Ile 503 513 Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg 523 533 Thr Ser Leu Thr Leu Ala Ala Pro Giu Ala Ala Gly Glu Val Giu Arg Le Ile Gly His 0*543 553 Pro Leu Pro Leu Arg Leu Asp Ala Ilie Thr Gly Pro Giu Giu Giu Gly Gly Arg Leu Glu Thr Ilie Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ilie Pro Ser Ala Ilie Pro Thr :583 593 *Asp Pro Arg Asn Vai Giy Gly Asp Leu Asp Pro Ser SE- Ile Pro Asp Lys Giu Gin Ala 603 613 Ilie Ser Aia Leu Pro Asp Tyr Ala Ser Gin Pro Gly L~s Pro Pro Arg Glu Asp Leu Lys

Claims (12)

1. A polypeptide selected from the group consisting of PE 40 Ab, or PE 40 ab, as hereinbefore defined. PE 40 aB, a repr
2. A polypeptide according
3. A polypeptide according
4. A polypeptide according A polypeptide according esents a peptide bond.
6. A polypeptide according -esents a peptide bond.
7. A polypeptide according claim 1 claim 1 claim 1 any one that is PE 40 aB. that is PE 40 Ab. that is PE 40 ab. of claims 1, 2 or 4 wherein b repr to any one of claims 1, 3 or 4 wherein to claim I or claim 4 wherein both a to any one of claims 1, 2 or 4 wherein and b represent peptide bonds.
8. A polypeptide according a is an amino acid other than Cys.
9. A polypeptlde according A polypeptide according b Is an amino .cid other than Cys.
11. A polypeptide according
12. A process for preparing to claim 8 to any one wherein a is Ala. of claims 1, 3 or 4 wherein to claim 10 wherein b is Ala. a modified PE 40 which process is S S 20 substantially as hereinbefore described with reference to Example 3 or Example 4.
13. Conjugates of EGF or TGF-alpha and -rdified PE 40 which conjugates are substantially as hereinbefore Ibed with reference to F ample 5 or Example 6.
14. k process for preparing conjugates of EGF or TGF-alpha and modified PE 40 which process Is substantially as hereinbefore described reference to Example 5 or Example 6. DATED this TNENTY-FIFTH day of AUGUST 1992 Merck Co., Inc. Patent Attorneys for the A[plicant SPRUSON FERGUSON
AU52115/90A 1989-02-17 1990-03-21 Production of modified pe40 Ceased AU631200B2 (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US31254089A 1989-02-17 1989-02-17
US312540 1989-02-17
US32721489A 1989-03-22 1989-03-22
US38909289A 1989-08-03 1989-08-03
US389092 1989-08-03
US44918789A 1989-12-21 1989-12-21
US449187 1989-12-21

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5562790A (en) * 1989-04-21 1990-11-16 Protein Design Laboratories, Inc. Recombinant antibody-toxin fusion protein
AU7242491A (en) * 1990-01-02 1991-07-24 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Target-specific, cytotoxic, recombinant pseudomonas exotoxin
AU617039B2 (en) * 1989-02-17 1991-11-14 Merck & Co., Inc. Protein anti-cancer agent

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU617039B2 (en) * 1989-02-17 1991-11-14 Merck & Co., Inc. Protein anti-cancer agent
AU5562790A (en) * 1989-04-21 1990-11-16 Protein Design Laboratories, Inc. Recombinant antibody-toxin fusion protein
AU7242491A (en) * 1990-01-02 1991-07-24 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Target-specific, cytotoxic, recombinant pseudomonas exotoxin

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