WO1991009949A1 - Exotoxine de pseudomonase, cytotoxique, recombinee specifique a une cible - Google Patents

Exotoxine de pseudomonase, cytotoxique, recombinee specifique a une cible Download PDF

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Publication number
WO1991009949A1
WO1991009949A1 PCT/US1990/007421 US9007421W WO9109949A1 WO 1991009949 A1 WO1991009949 A1 WO 1991009949A1 US 9007421 W US9007421 W US 9007421W WO 9109949 A1 WO9109949 A1 WO 9109949A1
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Prior art keywords
molecule
cytotoxic
recognition
sequence
recognition molecule
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PCT/US1990/007421
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English (en)
Inventor
Ira Pastan
Vijay Chaudhary
David Fitzgerald
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The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
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Priority to CA002072891A priority Critical patent/CA2072891C/fr
Priority to JP3504333A priority patent/JP2610740B2/ja
Publication of WO1991009949A1 publication Critical patent/WO1991009949A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/495—Transforming growth factor [TGF]
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
    • 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

Definitions

  • the present invention is related generally to the making of improved recombinant immunotoxins. More particularly, the present invention is related to the construction of a recombinant Pseudomonas exotoxin
  • Pseudomonas exotoxin A is a single polypeptide chain of 613 amino acids.
  • PE X-ray crystallographic studies and mutational analysis of the PE molecule have shown that PE consists of three domains: an amino terminal cell receptor binding domain (Domain I); a middle translocation domain (Domain II); and a carboxyl terminal activity domain (Domain III).
  • Domain III catalyzes the ADP ribosylation and inactivation of elongation Factor 2(EF-2) which inhibits protein synthesis and leads to cell death.
  • EF-2 elongation Factor 2
  • an object of the present invention to determine the role of the carboxyl terminus of the PE molecule in its cytotoxic action. It is another object of the present invention to identify specific regions at the carboxyl terminus of the PE molecule for the insertion of recognition molecules for selective killing of target cells.
  • a still further object of the present invention is to modify the carboxyl end of the PE to increase the potency of the chimeric toxin.
  • recognition molecules target ligands
  • Figure 1 demonstrates cytotoxicity of PE and PE mutants on Swiss cells.
  • Various dilutions of PE proteins were made in PBS containing 0.2% human serum albumin and added to 1x10 5 Swiss 3T3 cells in 24-well plates. Sixteen hours later the cells were pulse labeled with 3H-leucine arid TCA precipitable cell associated radioactivity was determined as a measure of protein synthesis. The results are expressed as percent of control where no toxin was added. •------•
  • Figure 2 shows the results of competition for the cellular uptake of recombinant PE.
  • Swiss 3T3 mouse cells were incubated with 400 ng 3 H-PE (specific activity 3.5x10 5 DPM/ ⁇ g) and increasing concentrations of purified mutant proteins for one hour at 37°C. Cell monolayers were washed and cell-associated radio- activity was determined. •-------• PE; ⁇ -------- ⁇ PEglu57;
  • Figure 3 shows immunofluorescence detection of binding and internalization of Pseudomonas exotoxin and its recombinant variants in Swiss 3T3 cells.
  • Swiss 3T3 cells were incubated at 37°C for 30 minutes in the absence of toxin (A) or in the presence of 10 ⁇ g/ml of native Pseudomonas exotoxin (PE) (B), recombinant PE gly 57 (C) or recombinant PE 612,613 (D). Following this incubation, the cells were fixed in formaldehyde and further incubated in the continuous presence of saponin.
  • PE native Pseudomonas exotoxin
  • mice monoclonal anti PE (M40-1) (10 ⁇ g/ml), followed by affinity-purified rhodamine-labeled goat anti-mouse IgG (25 ⁇ g/ml).
  • M40-1 mouse monoclonal anti PE
  • affinity-purified rhodamine-labeled goat anti-mouse IgG 25 ⁇ g/ml.
  • a cytotoxic recombinant Pseudomonas exotoxin having a recognition molecule inserted at least in domain III at the carboxyl terminus of the PE for selective killing of target cells recognized by said recognition molecule without substantial cytotoxicity to other cells not recognized by said recognition molecule, and by a rPE with modified "cytotoxic sequence" with increased cell killing activity.
  • rPE cytotoxic recombinant Pseudomonas exotoxin
  • recognition molecule means those molecules or ligands which recognize only target cells desired to be killed. Examples of such recognition molecules are antibodies or portions thereof that can recognize the target cells, growth factors, lymphokines, cytokine ⁇ , hormones and the like which specifically bind to molecules on the surface of the target cells.
  • cytotoxic sequence means those variety of amino acid sequences at the carboxyl end of the PE, the presence of which is a prerequisite for the cytocidal activity of the toxin and the repeat sequences of which may determine the level of cytotoxicity.
  • the examples of such sequences are KDEL, RDELK and the like as will become apparent from the various embodiments of the sequences discussed herein below.
  • Mutants were created by oligonucleotide directed mutagenesis using plasmid pVC45f+T as described by
  • pVC45f+T carries a PE gene under a T7 promoter and also contains a T7 transcriptional terminator and a fl phage origin.
  • the PE gene also contains an OmpA signal sequence which is cleaved upon secretion of PE into the periplasm leaving a 3 amino acid (ala asn leu) extension at the amino terminus (Chaudhary et al, 1988, Proc. Natl. Acad. Sci. USA 85, 2939-2943).
  • oligonucleotides and a 1.0 Kb Sall- EcoRI fragment of pVC45f+T were employed.
  • One oligonucleotide was the same as nucleotides 2216-2236 of the PE gene (Gray et al, 1984, Proc. Natl. Acad. Sci. USA 81, 2645-2649).
  • Other oligonucleotides were complementary to the 3' end of the coding sequence PE gene, contained desired mutations and created an EcoRI site after the stop codon. Other unique restriction sites were also created without changing amino acids to identify the mutants.
  • a 30 cycle PCR was performed with denaturation at 94°C for 2 minutes, annealing at 55C for 1 min and polymerization at 72C for 3 min with 10 seconds extension per cycle using a gene amplification thermal cycler (Perkin Elmer Cetus). After the PCR, amplified fragment was cut with EcoRI and BamHI, it was purified using low melting point agarose. PCR fragments were ligated with a 4.5 Kb dephosphoylated EcoRI-BamHI fragment of pVC45f+T. Mutants were identified by unique restriction sites which were created during mutagenesis and finally confirmed by sequencing by Snager's dideoxy-chain termination procedure using Sequenase (US Biochemical Corp.). pVC4915f+T
  • This plasmid contains two mutations; Codon 608, CCG and 609, CGC were changed to CCC and GGG, respectively. This mutation results in glycine at 609 in place of arginine and creates a Smal site between codons 608 and 609. This plasmid was used to clone various carboxyl terminal fragments of PE.
  • pVC4975f+T A 1 Kb BamHI- Pstl fragment of pVC8 (Wozniak et al, 1988, Proc. Natl. Acad. Sci.
  • pVC4995f+T A synthetic oligonucleotide duplex VK192/193 (not shown), containing codons 598-613 of PE with a stop codon and an EcoRI compatible 3' end, was ligated to the 4.9 Kb Smal-EcoRI fragment of pVC4915f+T.
  • pVC4715f+T This plasmid was created by PCR mutagenesis and contains restriction sites Stul, Ndel, Smal, EcoRV and EcoRI within the 3' end of the PE gene and encodes amino acids RPHMPGDILK in place of PREDLK at 608 to 613. These unique sites were later used to make insertions and to attach various DNA segments encoding carboxyl terminal portions of PE.
  • PVC47195f+T A synthetic oligonucleotide duplex VK192/193 (not shown), containing codons 598-613 of PE with a stop codon and an EcoRI compatible 3' end, was ligated to the 4.9 Kb S
  • Plasmids for the insertion of receptor binding domains in the carboxyl end of PE Plasmid pVC4715f+T consists of DNA sequences encoding PE from amino acids l to 607 followed by a polylinker that contains Stul, Ndel, Smal, EcoRV and Aflll sites as well as encodes amino acids RPHMPGDILK.
  • Plasmid pVC 4715/4E f+T is similar to pVC 4715 f+T, but also contains mutations in the receptor binding domain of PE (Domain I). These mutations are Lys 57 —> Glu, His 246,249 —> Glu and Arg 247 —> Glu.
  • Plasmid pVC 47195/f+T is similar to pVC4715 f+T but contains a polylinker with Stul, Ndel, Smal sites encoding amino acids RPHMPGI followed by the last 16 codons of PE that encode PDYASQPGKPPREDLK.
  • Plasmid pVC 47195/4# f+T contains insertions of a cDNA encoding transforming growth factor a in the Ndel site of plasmids pVC 4715f+T and 4715/4Ef+T respectively.
  • Plasmids pVC47395f+T and pVC 47195/4Ef+T are derived from 47195f+T and pVC 47195f+T by inserting TGF ⁇ sequences into the Ndel site.
  • Plasmid pVC 47355/4Ef+t was derived from pVC47395/4Ef+T by deleting 6 amino acids, inserting TGF ⁇ and following it with 10 amino acids of the PE carboxyl end.
  • Periplasmic fractions were prepared as described by Chaudhary et al, supra. By virtue of having an OmpA signal sequence, more than 90% of each of the expressed toxin proteins were secreted into the periplasm. These proteins have a residual ala asn leu sequence at the amino end left behind after the processing of the OmpA signal sequence. Periplasmic fractions were assayed for ADP ribosylation activity and cytotoxicity.
  • PE mutants were purified ssing a MonoQ anion exchange column (HR5/5) attached to a Pharmacia FPLC system. PE and mutant proteins eluted at NaCl concentrations of 0.22-0.26M. Upon SDS-PAGE the toxins were more than 90% pure. Protein concentration was measured by the Bradford assay Reagent (BioRad.Richmond, CA) using bovine serum albumin as a standard. ADP Ribosylation and Cytotoxicity Assays
  • ADP ribosylation activity was assayed after the activation of PE and mutant proteins with 4M urea and 50 mM DTT unless otherwise stated (Collier et al, 1971, J. Biol. Chem. 246, 1496-1503). Cytotoxicity of PE mutants was determined by adding various dilutions of periplasmic proteins or purified proteins to 1 x 10 5 Swiss 3T3 cells in 24-2311 plates as described by Jinno et al, (1988) supra, and Jinno et al, (1989) supra. ADP-ribosylation and cytotoxic activities of recombinant PE and native PE (from Swiss Serum and Vaccine Institute, Berne, Switzerland) were indistinguishable.
  • PE expression vectors with the cloning sites in the carboxyl end of PE to produce selective cytotoxic molecules are illustrated here with TGF ⁇ , a recognition molecule which recognizes only EGF receptor bearing cells. These cloning sites were used to insert TGF ⁇ near the carboxyl end of PE which produced a very active molecule that killed EGF receptor bearing cells when the insertion was followed by the last 10 amino acids of PE (Table A). The details of the procedure are now described.
  • lysine 613 was converted to glutamine, asparagine or aspartate. All these mutations produced a less cytotoxic molecule (Table 5). Addition of 6 or 11 amino acids to the carboxyl terminus of PE also produced a less cytotoxic molecule (data not shown). However, replacement of lys 613 with the basic amino acid, arginine, did not decrease cytotoxicity.
  • positions 609 and 613 both require a basic amino acid for full cytotoxic activity.
  • PE molecule with the carboxyl terminus of RPHMPGDILK in place of PREDLK.
  • This molecule in which arg 609 and asp 611 were altered, was not cytotoxic. But attaching the last 16 amino acids of an intact PE molecule to give a carboxyl terminus of RPHMPGDPDYASQPGKPPREDLK restored cytotoxicity to this molecule.
  • constructs were made in which a cDNA TGF ⁇ was inserted at the carboxyl end of PE with an inactive carboxyl terminus (Table A pVC 47315/4Ef+T) and an active carboxyl terminus (Table A, pVC 47355f+T and pVC 47395f+T).
  • the constructs with good carboxyl termini were more than 50 times as cytotoxic to cells with EGF receptors (TGF ⁇ binds to the EGF receptor) as the ones with the bad carboxyl ends. This clearly indicates that for the highest cytotoxic activity, a suitable carboxyl end is an essential requirement.
  • the results presented herein clearly show that mutations at the carboxyl end of PE and particularly in the last five amino acids of PE result in a molecule with full ADP ribosylation activity, but greatly reduced cytotoxicity.
  • the data show that the amino acid sequence at the carboxyl end of PE is Arg, Glu, Asp, Leu, Lys (REDLK, Table 2).
  • the arginine at 609 can be replaced by lysine but non basic amino acids cannot be tolerated (Table 3).
  • Lysine at 613 is not essential and can be deleted without loss of cytotoxic activity (Table 1), but it cannot be replaced with a non-basic amino acid (Table 5).
  • the cell targeting ligands can be inserted at two cloning regions in the PE molecule (at the amino terminus as previously described or near the carboxyl end as described herein), the same or different targeting ligands can be inserted at these two regions thereby increasing either cell binding, cytotoxicity or both.
  • Different targeting molecules at each of the two cloning regions would enable the chimeric toxin to bind to two different types of receptors present on the same cell. This is important because some antigens on target cells do not internalize well and are, therefore, poor targets for immunotoxins. But, if the toxin also binds to another antigen that is well internalized, specific cell killing is increased greatly.
  • the present invention for the first time shows that: 1. An appropriate carboxyl end sequence is absolutely required for cytotoxicity of the PE; 2. Deletion of as few as two amino acids from the carboxyl end of PE yields a molecule that contains full ADP ribosylation and receptor binding activity, but is nontoxic to target cells (Table 1); 3. Mutational analyses indicate that PE should possess a positively charged amino acid at 609, negatively charged amino acids at 610 and 611 and a leucine at 612;
  • Lysine at 613 can be deleted but cannot be substituted with several other amino acid residues; 5. Addition of random amino acid residues at the carboxyl end of PE produce relatively inactive molecules (data not shown).
  • target-specific immunotoxins are made similar to the method described herein supra by using appropriate recognition molecules, toxins and cytotoxic sequences including such modified recombinants as TGF ⁇ -PE40 and the like.

Abstract

Exotoxine de Pseudomonas, cytotoxique, recombinée spécifique à une cible. Lesdites toxines sont obtenues par insertion de molécules de reconnaissance spécifiques sur des sites de clonage spécifiques, dans au moins le domaine III au niveau de la terminaison carboxyle de la molécule PE. Diverses modifications de la terminaison carboxyle de la molécule PE augmentant la cytotoxicité, sont présentées.
PCT/US1990/007421 1990-01-02 1990-12-27 Exotoxine de pseudomonase, cytotoxique, recombinee specifique a une cible WO1991009949A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002072891A CA2072891C (fr) 1990-01-02 1990-12-27 Proteines de fusion d'exotoxine de pseudomonas, avec modifications carboxyliques pour une cytotoxicite plus grande
JP3504333A JP2610740B2 (ja) 1990-01-02 1990-12-27 標的特異的細胞障害性組み換えシュードモナスエキソトキシン

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US45963590A 1990-01-02 1990-01-02
US459,635 1990-01-02

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AU (1) AU644139B2 (fr)
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WO (1) WO1991009949A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0531434A1 (fr) * 1990-05-11 1993-03-17 THE UNITED STATES OF AMERICA as represented by the Secretary UNITED STATES DEPARTMENT OF COMMERCE Exotoxines de pseudomonas ameliorees ayant une toxicite animale faible et une forte activite cytocide
EP0597844A1 (fr) * 1990-05-14 1994-05-25 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Exotoxine recombinee de pseudomonas cytotoxique, specifique de cibles
EP2382990A1 (fr) 2003-04-30 2011-11-02 Universität Zürich Procédés permettant de traiter le cancer à l'aide d'une immunotoxine
US8932586B2 (en) 2011-09-06 2015-01-13 Intrexon Corporation Modified forms of Pseudomonas exotoxin A
WO2016146833A1 (fr) 2015-03-19 2016-09-22 F. Hoffmann-La Roche Ag Biomarqueurs de résistance à la nad(+)-diphtamide adp-ribosyltransférase
EP3184547A1 (fr) 2015-10-29 2017-06-28 F. Hoffmann-La Roche AG Anticorps anti-tpbg et procédés d'utilisation

Families Citing this family (2)

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AU631200B2 (en) * 1989-02-17 1992-11-19 Merck & Co., Inc. Production of modified pe40
NZ237758A (en) * 1990-04-17 1992-12-23 Shell Int Research (phenylalkyl) triphenylphosphonium salt derivatives and fungicidal compositions

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US4545985A (en) * 1984-01-26 1985-10-08 The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services Pseudomonas exotoxin conjugate immunotoxins
US4892827A (en) * 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects

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US5458878A (en) * 1990-01-02 1995-10-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services P. exotoxin fusio proteins have COOHG220101al alterations which increase cytotoxicity

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US4545985A (en) * 1984-01-26 1985-10-08 The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services Pseudomonas exotoxin conjugate immunotoxins
US4892827A (en) * 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects

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Title
Biological Abstracts, Volume 88, No. 9, issued 01 November 1989, C.B. SIEGALL et al. "Functional Analysis of Domains II, IB, and III of Pseudomonas Exotoxin", See Page 1181 column 1, Abstract No. 103,070, J. Biol. Chem., 264(24), 14256-14261. *
Cell, Volume 48, issued 13 March 1987, S. MUNRO et al., "A C-terminal Signal Prevents Secretion of Luminal ER Proteins", pages 899-907, see Figure 1 and the Abstract. *
Proceedings of the National Academy of Science, USA, Volume 87, issued January 1990, V.K. CHAUDHARY et al., "Pseudomonas Exotoxin Contains a Specific Sequence at the Carboxyl Terminus that is Required for Cytotoxicity", pages 308-312, see entire document. *
Proceedings of the National Academy of Sciences, USA, Volume 84, issued July 1987, V.K. CHAUDHARY et al., "Activity of a Recombinant Fusion Protein Between Transforming Growth Factor type and Pseudomonas toxin", pages 4538-4542, see the Abstract. *
See also references of EP0509056A4 *
The Journal of Biological Chemistry, Volume 264, No. 31, issued 05 November 1989, J.T. CHOW et al., "Identification of the Carboxy-terminal Amino Acids Important for the ADP- Ribosylation Activity of Pseudomonas exotoxin A", pages 18818-18823, see the Abstract and Discussion. *
The Journal of Biological Chemistry, Volume 265, No. 27, issued 25 September 1990, V.K. CHAUDHARY et al., "Mutagenesis of Pseudomonas Exotoxin in Identification of Sequences Responsible for the Animal Toxicity", pages 16306-16310, see the Abstract and Discussion. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0531434A1 (fr) * 1990-05-11 1993-03-17 THE UNITED STATES OF AMERICA as represented by the Secretary UNITED STATES DEPARTMENT OF COMMERCE Exotoxines de pseudomonas ameliorees ayant une toxicite animale faible et une forte activite cytocide
EP0531434A4 (en) * 1990-05-11 1993-10-13 Us Health Improved pseudomonas exotoxins of low animal toxicity and high cytocidal activity
EP0597844A1 (fr) * 1990-05-14 1994-05-25 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Exotoxine recombinee de pseudomonas cytotoxique, specifique de cibles
EP2382990A1 (fr) 2003-04-30 2011-11-02 Universität Zürich Procédés permettant de traiter le cancer à l'aide d'une immunotoxine
US8932586B2 (en) 2011-09-06 2015-01-13 Intrexon Corporation Modified forms of Pseudomonas exotoxin A
US9371517B2 (en) 2011-09-06 2016-06-21 Intrexon Corporation Modified forms of Pseudomonas exotoxin A
US9447387B2 (en) 2011-09-06 2016-09-20 Intrexon Corporation Modified forms of pseudomonas exotoxin A
US9677055B2 (en) 2011-09-06 2017-06-13 Interxon Corporation Modified forms of Pseudomonas exotoxin A
WO2016146833A1 (fr) 2015-03-19 2016-09-22 F. Hoffmann-La Roche Ag Biomarqueurs de résistance à la nad(+)-diphtamide adp-ribosyltransférase
EP3184547A1 (fr) 2015-10-29 2017-06-28 F. Hoffmann-La Roche AG Anticorps anti-tpbg et procédés d'utilisation

Also Published As

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JPH05502032A (ja) 1993-04-15
AU644139B2 (en) 1993-12-02
CA2072891A1 (fr) 1991-07-03
JP2610740B2 (ja) 1997-05-14
AU7242491A (en) 1991-07-24
EP0509056A1 (fr) 1992-10-21
CA2072891C (fr) 1999-12-21
EP0509056A4 (en) 1993-02-17

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