JPH05502032A - Target-specific cytotoxic recombinant Pseudomonas exotoxin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Target-specific cytotoxic recombinant Pseudomonas exotoxin Technical field The present invention generally relates to the production of improved recombinant immunotoxins. Than In particular, the present invention provides at least a portion of Pseudomonas exotoxin (PE). A specific method for inserting recognition molecules into the carboxyl terminus to achieve targeted cytotoxicity. Recombinant Pseudomonas exotoxin (rPE) with cloning site Regarding creation.

Background technique The mechanism by which protein toxins kill cells is extremely complex. lots of toxins The protein binds to receptors on the surface of mammalian cells and is internalized by endocytosis. cytosyl, where it exhibits enzymatic activity and kills target cells. Therefore These toxins inactivate cell binding, migration and basic cellular functions. It has a separate domain for enzymatic activity. Pseudomonas exotoxin A (PE) is a single polypeptide chain consisting of 613 amino acids. PE minute X-ray crystallographic studies and mutational analysis of the offspring show that PE has amino-terminal cellular receptor binding. domain (domain I), central migration domain (domain ■), and carboxyl It was found that it is composed of three domains: a terminal active region (domain ■) did.

Domain ■ inhibits ADP ribosylation and protein synthesis, resulting in cell death. It catalyzes the inactivation of factor 2 (EF”-2). Mutational analysis of domain I revealed that It has been revealed that gin-57 plays a major role in receptor binding.

Similarly, glutamic acid 553, tyrosine 481 and histidium 426 are ADP - found to be important for ribosylation activity. Recently, mutation analysis of domain ■ This suggests that a portion of this domain is essential for the cytotoxic effects of PE. It turned out that.

Various growth factors fused to a form of PE lacking domain I (PE40) While constructing the chimera toxin, TGFα, TGFα, and Produced by attaching interleukin-2 or interleukin-4 The recombinant fusion protein was found to have poor cytotoxic activity. From this, We have started investigating the role of the carboxyl terminus of the PE molecule (domain ■).

Disclosure of invention Therefore, the object of the present invention is to improve the carboxyl terminus of PE molecules in their cytotoxic effects. The key is to determine the role of the edges.

Another object of the present invention is to provide a method for inserting recognition molecules to selectively kill target cells. The aim is to identify a specific region at the carboxyl terminus of the PE molecule.

A further object of the invention is to provide improved target-specific cytotoxic recombinant PE molecules. The target-specific recognition molecule is attached to at least the carboxyl-terminal domain of the PE molecule. A patent characterized in that it is inserted into The purpose of the present invention is to provide a modified PE molecule.

A further object of the invention is to modify the carboxyl terminus of PE to create chimeric toxins. is to increase the effectiveness of

A further object of the present invention is to use the same recognition molecule near the amino and carboxyl ends, etc. at two different ends to enhance cell binding, or at two different endpoints to enhance cell binding. The recognition elements are inserted into two different regions of the PE molecule, respectively, and the resulting PE molecule is Two or more different entities such as antigens, receptors etc. to which the recognition element can bind (en tities), allowing it to bind more effectively to cell surfaces with The aim is to produce cytotoxic PEs with recognition molecules (targeting ligands).

A further object of the invention is to provide repeating carboxyl-terminal cytotoxic sequences. The object of the present invention is to provide recombinant PE with enhanced cytotoxic activity.

Various other objects and advantages will become apparent from the following detailed description of the invention. Will.

Brief description of the drawing These and other objects, features and attendant advantages of the present invention will be apparent from the accompanying drawings. It will be better understood in the context from the detailed description below.

Figure 1 shows the cytotoxic effects of PE and PE mutants on Swiss cells. It is. Various dilutions of PE protein were added to a solution containing 0.2% human serum albumin. 1×10 = Swiss 37 cells prepared in PBS and placed in a 24-well plate. 3 cells. After 16 hours, cells were pulse-labeled with 3H-leucine and then TCA precipitable cell-associated radioactivity was then measured as a measure of protein synthesis. result is expressed as a percentage of control.・-・PE;0-OPE△613;Roro PE△61 2.613; and △-△P　E　611. −613° All assays were performed in duplicate. and repeated twice.

Figure 2 shows the results of competition for cellular uptake of recombinant PE. Switzerland 37 3 mouse cells with 400ng3H-PE (specific activity: 3.5X10” DPM /μg) and increasing concentrations of purified mutein at 37°C for 1 h. It was cultured for a period of time. Monolayers of cells were washed and cell-associated radioactivity was measured.・-・P E.

Mumu PEglu57;△-△PE612.613;0-OP　E△613;■ −■PEg1y276・Roro PE609-613;+−−+PE△609 -613+598-613° Figure 3 shows Pseudomonas exotoxin and its combination in Swiss 373 cells. Figure 3 shows immunofluorescence detection of binding and internalization of recombinant mutants. Switzerland 37 3 cells in the absence of toxin (A) or with 1.0 μg/m1 of natural Pseudomonas exotoxin (P E) (B), mb modified P E g 1 y57 (C) Also in the presence of i; t, m-transformed PE612.613 (D) The cells were incubated at 37°C for 30 minutes. After this incubation, the cells were fixed in formaldehyde and Further cultivation was carried out in the continuous presence of saponin. cells, mouse monoclonal After incubation with anti-PE (M2O-1) (10 μg/ml), affinity purification loader Cultured with Min-labeled goat anti-mouse IgG (25 μg/ml) (Mags- X400; bar -10 μm).

Detailed description of the invention The above and various other objects and advantages of the invention are not recognized by recognition molecules. Recognized by the recognition molecule without substantially exhibiting cytotoxic effects on other cells A recognition molecule is attached to the carboxyl terminus of PH to selectively kill target cells. Cytotoxic recombinant Pseudomonas aeruginosa having inserted at least the domain ■ xotoxin (rPE) and modified “cytotoxicity” with increased cell-killing activity. Achieved by rPE containing cytotoxic sequence J be done.

All technical and scientific terms used herein, unless otherwise defined. has the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Ru.

Any methods and materials similar or equivalent to those described herein may be used by the present invention. Preferred methods and materials for the present invention are described here, although they can be used to carry out and test the present invention. I will clarify.

The disclosures in all publications mentioned below are incorporated herein by reference. be done. Unless otherwise specified, techniques used or contemplated herein are: Standard methods well known to those skilled in the art. Materials, methods and examples described herein are only for illustrating the present invention, and the present invention is not limited thereto. stomach.

The term “recognition molecule” used herein. le) J is a molecule or ligand that recognizes only the target cells that are intended to be killed. It means "do". Such recognition molecules include, for example, molecules on the surface of target cells. Target cells, growth factors, lymphokines, cytokines, and hormones that specifically bind to Examples include antibodies and parts thereof that can recognize molecules such as cancer.

The term "Cytotoxic Sequence" as used herein nce) J refers to various cytotoxic sequences at the carboxyl terminus of PE. means column. The presence of the cytotoxic sequence negates the cytolethal activity of the toxin. and its repetitive sequences may determine the level of cytotoxicity.

Such sequences include, for example, various of the sequences described herein below. Examples include KDEL, RDELK, etc., which will become clear from the embodiments of .

Materials and methods material Unless otherwise specified, materials and reagents used herein are commercially available. Polymerization chain reaction PCR kit, Gene Amp Kit , Perkin Elmer Φ Cetus (P The erkin Elmer-Cetu mutant strain was developed by Jinno et al. l), 1988, J. Biol, Cheml, 263.13203-13207 and Zink et al., 1989, J. Biol.

Plasmid I as described in Chem., 264.15963-15959. ) using VC45f+T or by polymerase chain reaction (PCR) as described below. was generated by oligonucleotide-directed mutagenesis using pVC45f+T carries the PE gene under the T7 promoter and the T7 transcription terminus. It contains data and f1 phage origin. In addition, the PE gene is It is cleaved with secretion to become the periplasm, and the amino terminal contains three amino acids (ala asn 1eu) contains the extended ompA signal sequence (Chowdary et al. Chaudhary et al), 1988, Proc, Natl, Aca d, Sci., USA, 85.2939-2943). Place for PCR mutagenesis In this case, two oligonucleotides and 1.0 KbSa I I- of pVC45f+T An EcoRI fragment was used. One oligonucleotide is a nucleonucleotide of the PE gene. It was the same as Chido 2216-2236 [Gray et al. 1984, Proc, Nat 1. Acad, 5ci0, USA, 81.2645 ~2649]. The other oligonucleotide is the 3' end of the coding sequence PE gene. is complementary to , contains the desired mutation, and contains an EcoRI site after the stop codon. It was forming. Other unique restriction sites can also be used without mutating amino acids. The mutant strain was identified. Denature at 94°C for 2 minutes and anneal at 55°C for 1 minute. 30 cycles of PCR were performed, including ringing and polymerization for 3 minutes at 72°C. On this occasion , gene amplification thermocycler (Perkin-Elmer Cetus) A 10 second extension was performed per cycle using a 10 sec. P After CR, the amplified fragment was cut with EcoRI and BamHI and placed in low melting point agarose. It was purified using I) VC45f+T 4.5Kb dephosphorylated E The coRI-BamHI fragment was ligated. Mutant strains are unique strains formed during mutagenesis. Finally, Sequanase [ Ninis-Biochemical Fist Corporation (US Biochemical Snager's dideoxy chain termination method (Snager's Corp.) dideoxy-chaintermfnation procedure) This was confirmed by further sequencing.

pVC4915f +T This plasmid contains two mutations: codon 608, CCG and codon 609, CGC. These were mutated into CCC and GGG, respectively. This mutation is Glycine occurs at 609 instead of arginine, and codon 608 and codon 609 During this time, Sma 1 site is generated. Using this plasmid, various cultures of PE The boxyl terminal fragment was cloned. pVC4975f +T: pVc8 IKbBamHI-PstI fragment [Wozniak et al. al), 1988, Proc.

Natl. Acad. Sci., USA, 85.8880-8884). After restriction with arI and treatment with T4 DNA polymerase to create blunt ends, EcoR I and the 286 bp fragment was ligated with 4.9 Kb of pVc4915f+T. ligated to the original SmaI-EcoRI fragment. pVC4985f+T: pVC8 The IKbBamHI-PstI fragment was restricted with Hinfl and added to the T4 DNA polymerizer. I) VC45f+T was ligated to a 4,9 Kb SmallEcoRI fragment. pVC4995f +T: Contains codons 598-613 of PE with a stop codon and an EcoRI compatible 3' end. Synthetic oligonucleotide duplex VK192/193 (not shown) containing It was ligated to the 4.9 Kb Small-EcoRI fragment of pVC4915f+T. p Vc4715f+T: This plasmid was generated by sudden PCR induction Then, within the 3' end of the PE gene, there are restriction sites S tul, Nde 1% Sma l5Ec.

Contains RV and EcoRI, and replaces PREDLK in 608-613. It encodes the amino acid RPHMPGD ILK. These unique parts are , later create insertions and inserts of various Used to attach DNA segments This is an oligonucleotide duplex VK 191/192, pVC4715f-+-r 4,9KbEcoRV-Eco It was generated by linking to Rr. The carboxyl terminus of this PE mutant is Amino acids RP H instead of amino acids 608-613 (PREDLK) of PE Contains MPGDPDYASQPGKPPREDLK.

Plasmid pVc4715f+T encodes PE from amino acids 1-607. DNA sequence followed by < S t u I, Nde I, Sma 1. , Contains EcoRV and AfIII sites and the amino acids RPHMPGD ILK It consists of a polylinker that codes. These sequences are T7 promoter Shine-Darga derived from E. coli OmpA. It also contains a region and a signal sequence. Plasmid pVc4715/4Ef+T is similar to pVc4715f+T, but the receptor binding of PE (domain I) It also contains mutations in the domain. These mutations include Lys57-Glu, H1s 246°249-bGlu and Arg-bGlu.

Plasmid pVc47195/f+T is similar to pVC4715f+T is 5tuI, which encodes the amino acid RPHMPGI, and NdeISSma1 part. PDYASQPGKPPREDLK, followed by a polylinker with a It contains the last 16 codons of the coded PE. Plasmid pVc4719 5/4#f10T C, plasmid pVc4715f+T and 4715/4Ef+T Contains insurgidine of transforming growth factor α at the NdeI site. Plasmi pVC47395f+T and pVc47195/4Ef+T are 47195f +T and pvC47195f+T, insert TGFa sequence into NdeI site This is what was induced by this. Plasmid p　V　C47355/　4　E　 f　10T is obtained by deleting 6 amino acids from pVC47395/4Ef+T. Insert GFα, followed by the PE carboxyl-terminal 10 amino acids was induced by inserting.

pVc49415f+T and hip VC47355/4 Ef+t are -Deposited with the ATCC in Rockville, R.D., on December 28, 1989. (Deposit numbers: 68198 and 68199). The Deposit shall survive and remain a patent. 5 years from the date of the last request for a sample of the deposit, either during the term of the deposit or for 30 years from the deposit. If it becomes non-viable during the longest period of the year, it will be replaced and the patent Upon publication, it will be made available to the public without restriction in accordance with the provisions of the law. . The Commissioner of Patents and Trademarks shall have access to the deposit upon request.

Protein expression and purification Cultures of E. coli BL21 (λDE3) containing various plasmids were Grow to 0D650 of 0.6-0.8 and add 1mM isopropyl-thiogalacto. The cells were induced with CID for 90 minutes at 37°C. Chaudhary et al. (Cha, udhar) The veliplasmic fraction was prepared by the method described by Y et al. Manufactured. Because it has OmpA signal sequence, more than 90% of each toxin is emitted. The present protein was secreted into the periplasm. These proteins are OmpA signal After processing of the null sequence, a residual ala asn leu sequence is left at the amino terminus. left behind. The veliplasmic fraction was analyzed for ADP ribosylation activity and cytotoxicity. was assayed for. Then, the PE mutant strain was analyzed using the Pharmacia FPLC system. Purification using a MonoQ anion exchange column (HR515) attached to did. PE and mutant proteins were eluted at 111 degrees of 0.22-0.26M NaCl. did. The purity of the toxin after 5DS-PAGE was over 90%.

Protein concentrations were measured using bovine serum albumin as a standard in Bradford (B radford) assay reagents (available from Schimmond, California, USA). 'aJ was determined using a method (manufactured by Orad).

ADP-ribosylation and cytotoxicity assay ADP-ribosylation activity was determined unless otherwise specified. After activating PE and mutant protein with 4M urea and 50mM DTT as long as possible. Assayed C. Collieret al., 1971, J.B. iol, Chem, 246.1496-1503]. Cytotoxicity of PE mutants Adverse effects are described in Zink et al., supra (1988) and Zink et al., supra (1989). The method described above is based on the production of veliplasmic proteins or purified proteins. ×105 Swiss 3T3 plates containing various dilutions in 24-2311 plates. It was measured by adding it to cells. Recombinant PE and natural PE [Bern, Switzerland] Swiss Serum and Vaccine Institute (SwissS) Erumand Vaccine (obtained from In5 titute)] Bosylation and cytotoxic activity could not be determined.

Toxin activity and internalization Regarding the binding of 3H-labeled PE to Swiss cells for various mutant PE proteins. on the competition and internalization of various mutant PE derivatives examined by immunofluorescence. Regarding competitive ability, see Jinno et al. (1989) above. is stated.

Preparation of target-specific immunotoxins Cloning moieties at the carboxyl terminus of PE to generate selective cytotoxic molecules Here, we used a PE expression vector that recognizes only EGF receptor-bearing cells. This will be explained using TGFα, which is a recognition molecule. Use these cloning sites Then, TGFα was added to the last amino acid of PE after insertion (Table A ), a highly active molecule that killed EGF receptor-bearing cells was released. It was inserted near the carboxyl terminal of the generated PH. The details of this operation are explained below. I will clarify.

Results The role of the sequence at the carboxyl terminal of PE is 1, 2, 3, 7, 8, 1 A series of carboxyl-terminal deletion mutations that removed 1, 14 and 24 amino acids. Strains were produced and determined. Removal of two or more amino acids reduces ADP ribosylation activity. The cytotoxic effect disappeared without affecting the sex (see Table 1 and Figure 1). actually, Even 11 amino acids (603-613) caused loss of ADP ribosylation activity. I was able to remove it without causing any damage. However, 14 amino acids (600-613 ), proteins with low but measurable ADP-ribosylation activity are removed. When fermentation occurred and removed 24 amino acids (590-613), An essentially inactive protein was produced. These results indicate that the carboxyl terminus of PE A specific sequence in the toxin is not required for ADP-ribosylation activity. This shows that the company is fulfilling its role.

The role of the carboxyl-terminal sequence in toxin action has been determined by a series of internal deletions and substitutions. This was revealed by causing a reaction (see Table 2). These mutations are amino starts at acid 602, does not affect ADP riboxylation activity, and continues until position 611. It was extended. Surrounding amino acids 601-604 and 606-608 A few small deletions did not reduce the cytotoxic effect. In addition, 60 amino acids Two substitutions that changed amino acids 3-608 as well as within amino acids 606-608 of PE Two other substitutions also did not reduce the cytotoxic effect. Therefore, position 60 The sequence of amino acids 2-608 is not important for cytotoxic effects. It seemed to me. However, a deletion that removed the arginine at 609 (pVc4921 5 and pVC49255), the cytotoxic effect of PE was greatly reduced. these This result suggests that deletion of amino acids 612 and 613 abolishes the cytotoxic effect. When considered with the experiment in Table 1 which shows that It clustered at amino acids 609 to 613 located at the sill terminus.

The role of arginine 609 can be altered by either deleting it or modifying it by several different amino acids. This study was carried out by replacing it with a noic acid.

When arginine 609 was replaced with another basic amino acid, lysine, PE Cytotoxic activity was retained (see Table 3). However, lacking arginine 609, (pVc49115) or arginine 609 with glycine or glutamine. Substitution with acid or leucine reduced the cytotoxic effect by approximately 6-10 times. this Therefore, the basic amino acid at position 609 seems to be important.

To examine the sequence specificity of the last five amino acids of PE, we next I built some children. In two of these, at positions 610 and 611 The order of acidic amino acids was reversed, and lysine 613 was deleted (Table 4, pVC49415 and pVC49425). These molecules are rigid at position 609. It had sufficient activity regardless of whether it was arginine or arginine. Also, Molecule with leucine at position 609 and arginine at position 612 (pVC4 9435) was also produced but was inactive.

Deletion of the terminal amino acid lysine at position 613 did not affect the cytotoxic effect; Other mutations at this position shift the ligand to the peptide at the carboxyl terminus of the PE. An undesirable method due to the low activity of various chimeric toxins placed in the bond. This is thought to affect the cytotoxic effect. Therefore, lysine 613, Converted to glutamine, albaragine, or aspartate. With these mutations All produced molecules with less cytotoxic effects (see Table 5). PE carbo Addition of 6 or 11 amino acids to the xyl terminus also resulted in less cytotoxic effects. Small molecules were generated (data not shown). However, since lys is basic Substitution with the amino acid arginine did not reduce cytotoxic effects.

Therefore, both positions 609 and 613 have sufficient cytotoxic activity. Basic amino acids are required for this purpose.

At the carboxyl terminus of PH there are two other lysine residues, these at position 5 90 and 606.

Both of these lysines can act as uncharged amino acids without reducing their cytotoxic effects. It could be converted to acid glutamine. This means that positions 590 and 606 are positively charged. This indicates that additional amino acids are not required (see Table 5).

The importance of specific amino acids at the carboxyl terminus of PE has been shown, so five The carboxyl-terminal amino acids of the ADP-ribosylated donodomain are separated and It was found that the xin could be redifferentiated. Amino acids of intact PE as shown in Table 6 551-613.567-613 or 598-613 of PEΔ609-613 By adding to the carboxyl terminus, a fully active cytotoxic molecule can be , PEΔ609-613 (not cytotoxic). Something like this From, ADP-ribosylated donodomain position 609 to 609 ends around amino acid 600. The distance between essential amino acids at 613 is less important and reduces cytotoxic effects. I was able to increase it considerably without decreasing it. Also, in Table 6, instead of PREDLK, A PE molecule with the carboxyl terminus of RPHMPGD I LK is also shown . This molecule with altered arg and asp was not cytotoxic. deer While attaching the last 16 amino acids of the intact PE molecule to RPHMPGD Obtaining the carboxyl terminal consisting of PDYASQPGKPPREDLK, this part The cytotoxic effect of the child was restored.

Additionally, cDNA TGFα was isolated from the inactive carboxyl terminus (Table A, pVc473 15/4Ef+T) and active carboxyl terminus (Table A, pVC47355f+T and and pVC47395f+T) inserted into the carboxyl terminus of PE. It was created. EGF receptor (TGF) construct with good carboxyl terminus α binds to the EGF receptor). It was more than 50 times as large as the one with a ruboxyl end. This means that the highest cell Having a suitable carboxyl terminus is a prerequisite for obtaining a damaging effect. It clearly shows that.

The overall data here suggests that inactive due to deletions or alterations within the carboxyl terminus. The cytotoxic activity of certain PE molecules can be enhanced by attaching an intact carboxyl terminus. It is clear that it can be revived. Therefore, binding ligands such as TGFα can be by inserting the last five amino acids at position 608 within the carboxyl terminus of RED By retaining it as LK, it becomes possible to generate active chimeric molecules.

It has already been revealed that domain I of PH is a region that causes cell binding. However, why mutations at the carboxyl terminus of PH that reduced cytotoxicity It was important to show that it does not reduce binding. To test this, [3 The ability of various mutant forms of PE to compete for the uptake of H]-PE was evaluated. . As shown in Figure 2, mutations at the carboxyl terminus of PE induce cytotoxic effects. Some reduced PE mutants have a combination of authentic wild-type PE and [3H]-PE. The competition for entry was exactly the same. In this competition assay, domain I PE40 and Peglu57 with deletions were inactive as described above ( (see Jinno et al., supra).

The results of these uptakes are determined by fluorescence measurements that measure the internalization of PE and various mutant PE molecules. This was confirmed using an assay (see Figure 3). In this assay, cytotoxic effects are Incubate with various toxins for 30 minutes to allow binding to and internalization of the internalized aqueducts. It was. Molecules with a point mutation in domain I (PEglu57), i.e. PE 40 did not internalize. In contrast, all other PE molecules have domain Regardless of whether or not it contains a mutation in the carboxyl terminus of Binds to and internalizes other elements in the trans-Golgi system in the perinuclear region of cells. (Figure 3, panels B and D). These results indicate that carboxyl The reduced cytotoxic effect of terminal mutants is due to decreased receptor binding and cellular uptake of PE molecules. This clearly shows that this is not due to small numbers.

In short, the results here suggest that mutations at the carboxyl terminus of PE and in particular PE Mutations in the last five amino acids of have sufficient ADP-ribosylation activity It is clear that this results in a molecule with greatly reduced cytotoxic effects. Is it data? et al., the amino acid sequence at the carboxyl terminus of PH is Arg, GluSAsp, L It is clear that euSLys (REDLK, Table 2). position 6 Arginine in 09 can be replaced with lysine, but non-basic amino acids are not tolerated (See Table 3). Lysine at position 613 is not essential and deletion causes cytotoxicity. There is no loss of activity (see Table 1), but non-basic amino acids cannot be substituted (Table 1). (see 5). That is, ArgGluAspLeu or LysGluAspLeuL Molecules that have sufficient cytotoxic effects by having ys at the carboxyl terminus was generated (see Table 4). present in other molecules and have specific biological effects. A literature review of similar sequences revealed that newly formed within the endoplasmic reticulum. The sequence carrying the protein was revealed to be LysAspGluLeu. Or it becomes. [Munro et al., 1987, Ce1l, 4 8.899-907]. Therefore, several other mutant molecules were constructed.

One of these is the accumulation of proteins in the lumen of the endoplasmic reticulum. It contains the exact sequences explained above that contribute to retention (Table 1). (See 4) o There are ten molecules that terminate with LysAspGluLeu (KDEL). It was found that it has a significant cytotoxic effect. Also, LeuAspGluArg Because it terminates with ArgAspGluLeu (RDEL) instead of (LDER) The offspring also had sufficient activity. These findings suggest that PE is an internal compartment. To successfully invade the side sills, cells within the endoplasmic reticulum Requires interaction with similar cellular components that help retain manufactured proteins It shows that there is. These findings also suggest that the arrangement at the carboxyl terminus of PH columns to facilitate the transition of PE from the internal compartment to the side sills. It has also been suggested that this protein may play a role as a kind of recognition sequence. performs similar functions. Other sequences will increase activity as well.

A further important finding is that cell-targeting ligands can be linked to two clones in the PE molecule. (near the amino terminus described above or the alkyl terminus described herein) The same or different targeting ligands can be inserted into these two regions. can increase cell binding, cytotoxicity, or both. two cronins Different targeting molecules in each of the coding regions allow chimeric toxins to be present on the same cell. can bind to two different types of receptors. target cells are fully internalized. Therefore, the above is insufficient as a target for immunotoxins. is important. However, if the toxin also binds to another antigen enough to be internalized If so, specific cell killing would be greatly increased.

Additionally, during the study of temporary mutations at the carboxyl terminus of PEO, if REDL When the K (single letter amino acid code) sequence is replaced with KDEL, the resulting molecule is An almost two-fold increase in activity was found. Even more impressively, REDL Molecules with three repeats of KDEL instead of K were found to be three times more active. (see Table B). This means adding KDEL or an equivalent repeat sequence. demonstrated that chimeric toxins with enhanced cytotoxicity can be produced by are doing.

In summary, the present invention clarifies the following for the first time.

1. Appropriate carboxyl terminal sequence is absolutely necessary for the cytotoxic effect of PE .

2. Deletion of two minor amino acids from the carboxyl terminus of PE results in ADP The result is a molecule with sufficient ribosylation and receptor binding activity, but is non-toxic to target cells. (see Table 1).

3. From mutational analysis, PE has a positively charged amino acid at position 609, positions 610 and 6 11 has a negatively charged amino acid and position 612 has a leucine. I found out that it doesn't happen.

4. Lysine at position 613 may be deleted, but some other amino acid residues may be deleted. Cannot be substituted with groups.

5. Adding any amino acid residue to the carboxyl terminus of PEO makes it relatively inactive. molecules (data not shown).

6. PE molecules have no cytotoxic effect due to mutations in the carboxyl terminus. Addition of at least 10 carboxyl-terminal amino acids of sexuality is restored (see Table 4).

7. Having different target ligands at different termini (amino and carboxyl) can lead to binding This provides the flexibility to generate synthetic and cytotoxic PE molecules.

8. Repetitive “cytotoxic sequence (cytotoxic sequence) J” , the cytotoxic effect is multiplied in appropriate cases.

Appropriate recognition molecules, including modified recombinants such as TGFα-PE40, Other target-specific methods similar to those described above can be achieved by using cytotoxic sequences and cytotoxic sequences. Needless to say, target immunotoxins are created.

The examples and embodiments herein are set forth solely for the purpose of illustrating the invention. Various modifications and changes are possible from the perspective of the present invention, and those modifications It is understood that modifications and changes are within the spirit and scope of this application and the claims. It is a place where

Table 1 Deletion analysis of the carboxyl terminus of PE Mutant strain Cytotoxic effect ADP ribbonlation activity Existing deletion Amino acid Amino acid 1~589 590~613 <0.1' 01~599 600~613 < 0.1 201-602 603-613 <0.1 1001-605 60 6-613 <0.1 1001-606 607-613 <0.1 100 1~610 611~61.3 <0.1 1001~611 612.613 <0.1 1001~612 613 100 100 1-613 100 remarks: The mutant PE protein is derived from the vector C metade and model based on the T7 promoter. (Studierand Moffatt, 1986) It was expressed in enterobacteria and purified from the periplasm. All proteins are OmpA Three extended amino acids remain at the amino terminus after processing of the signal sequence. Contains acid (alaasn Ieu). These amino acids are It was not taken into account when assigning residue numbers to proteins. Cytotoxicity is Swiss 3 This was determined by assaying inhibition of protein synthesis on T3 mouse cells.

All results are expressed as a percentage of the activity obtained using recombinant full-length PE molecules. There is. All assays were performed in duplicate and at least two separate clones were tested. I tried it.

Table 2 Internal deletions and substitutions at the carboxyl terminus of PE Position of amino acids in PE Plus 6666666666666 Cell Ring Mid 000000000111 1 Harmful effect pVC1234567890123 45A S Q P G K P P RE D L K 10049215 A L K <0.1 49235 A G K P P, RE D L K 10049245 A S Q P G RE D L K 10049255 A S Q P G E　D　K　K　O13 mutant PE protein was expressed in E. coli and transferred to the periplasm. Purified from.

ADP-ribosylation activation of all mutant strains ADP-ribosylation activation of full-length PE I couldn't tell.

The amino acids within the carboxyl terminus of PE (601-613) are replaced with the - letter code. This is the expression. Replaced parts are underlined.

Table 3 Mutation at position 609 of PE Plasmid cytotoxic effect (pVC) (% of PE) 49115 609 12 to PE 49125 PELys6°9 1o.

4915 PEgly609 1゜ 49135 PE1eu609 16 49155 PE1eu609 15 remarks: Mutant PE proteins were expressed in E. coli and purified from the periplasm. The replacement part is , are shown as substituted amino acids (see also Tables 1 and 2).

Table 4 Sequence specificity of the last 5 amino acids of PE Plasmid Sequence specificity of the last 5 amino acids of PE Position Cytotoxic effect (pVC) (% of PE) 45REDLK100 49125 K E D L K 1004215 RE D L' 100 49415 K D E L 100 49425 RD EL 100 49435 L　D　E　R<0.03 For details, see the notes in Tables 1 and 2. See.

Table 5 Lysine residues 590.606 and 613 in the carboxyl-terminal domain of PE mutation of Mutant strain Cytotoxic effect ADP ribosylation PEarg613 100’ 10 0゛PEgln””　1　100 PEgl100PE 100 PEasn613 1 110 0PE 1 n606100 100 100PE""0 100 100 P　E　gl　n590,606,613　1　100590.606　613 PEgln arg 100 100 remarks: The analysis was conducted according to the methods described in Tables 1 and 2.

Table 6 Addition of various parts of PE carboxyl terminus from 609 to 613 to PE plasmid Mutant protein Cell damage ADP ribo (pVC) action Sylation activity (% of PE) (% of PE) 4905 To PE 609~613 <0.1 1004975 To PE 609~ 613+551~613 100 1004985 to PE609~613+ 567-613 100 100 4995 to PE609-613+ 598-613 100 100 4715 PE△609~613 RPHMPGDILK <0.1 10047195 PE△608~613 RPHMPGD+598~613 50 100 Notes: Plasmid pVc4 with Sma1 site between codons 608 and 609 of PE 915 and append various portions of the carboxyl terminus after codon 608. I arrived.

pVC4995 was constructed using synthetic oligonucleotides. The last one of PE Six amino acids (598-613) are PDYASQPGKPPREDLK ( (See also Tables 1 and 2). △ indicates that the following amino acid is missing. It means being lost.

Table A pVc47315/PEI~607 RPHMA (TGFα)4Ef +T AHMPGDILK>25pVC47395/PEI ~607RPH MA (TGFα)4Ef +T AHMPGIPDYASQPGKPPRED LK 0.5pVC47355/PEI~607 RPHMA (TGFα )4Ef +T AHMPGKPPREDLK O, 5 notes) a: The fusion protein was partially purified from the periplasm.

The purity of the fusion protein was 20-30% by 5DS-PAGE. PH Consistently occurring residues are underlined.

b: ID5o is the protein Concentration of fusion protein required to inhibit synthesis by 50% (estimated as total protein concentration) (specified). Protein synthesis was measured by 3H-leucine incorporation. .

Table B Cytotoxic activity of various PE derivatives against Swiss 3T3 cells Plasmid protein ID5゜ pVC45f+T PEl-608REDLK 1.6pVc49415f+T PEl-608KDEL 0.76pSS49445f+T PEl-608 KDELKDELKDEL O, 55 notes) The a+PE protein was purified using a MonoQ column. Purity was approximately 90%.

b: Same as Table A.

2] Buff (nri/rat) #BA and related figures (/,) Engraving (no changes to the content) Summary book Target-specific cytotoxic recombinant Pseudomonas exotoxins are disclosed . Such toxins contain at least the domain ■ at the carboxyl terminus of the PE molecule. produced by inserting a specific recognition molecule into the specific cloning site of be done. By variously changing the carboxyl terminus of the PE molecule, cytotoxic effects can be achieved. It has also been disclosed to enhance the efficacy of the drug.

Handbook Supplementary Book 1992 10 month 7 days prisoner

Claims (18)

[Claims] 1. Carboxyl-terminal domain II of Pseudomonas exotoxin (PE) has a first recognition molecule inserted into I, and the resulting PE is recognized by the recognition molecule. A cytotoxic group characterized by showing a selective cytotoxic effect on target cells. Recombinant Pseudomonas exotoxin. 2. Claim wherein the second recognition molecule is inserted in a region other than the carboxyl terminus. PE according to 1. 3. 3. The method according to claim 2, wherein the first recognition molecule and the second recognition molecule are the same or different. PE listed. 4. Claim wherein the recognition molecule is an antibody or a portion of an antibody that recognizes the target cell. PE according to item 1. 5. The recognition molecules may be selected from growth factors, lymphokines, cytokines and hormones. PE according to claim 1, which is selected from the group consisting of: 6. P according to claim 1, wherein the recognition molecule is inserted after amino acid 607. E. 7. The carboxyl terminus is inserted into positions 607 and 608-61. 2, having only the last 10 amino acid residues following a deletion of 3. P.E. 8. The amino acids after residue 608 are the cytotoxic sequence KEDL, RDEL or PE according to claim 1, comprising repeats of. 9. The amino acids after residue 608 do not contain the cytotoxic sequence KDELKDEL. 9. The PE according to claim 8. 10. The cytotoxic sequence after residue 608 is KDELKDELKDEL. PE according to claim 8. 11. Repeated cytotoxic sequences have an increased cytotoxic effect compared to molecules without repeats. 9. PE according to claim 8, resulting in a strengthened rPE molecule. 12. PE according to claim 1 in an amount that produces a cytotoxic effect, and a pharmaceutically acceptable A composition comprising a carrier. 13. The target cells to be killed and the amount that causes a cytotoxic effect according to claim 1. A method of killing a target cell, the method comprising contacting a PE with a PE. 14. The first target is specific to at least domain III of the carboxyl terminus of the PE molecule. Production of a target-specific cytotoxic PE molecule comprising inserting a target recognition molecule Method. 15. Replace the amino acid residue after position 608 with the sequence KDEL or its repeats. 15. The method of claim 14, further comprising the step of: 16. A process for inserting a second recognition molecule into a region other than the carboxyl terminal of the PE molecule 16. The method of claim 15, further comprising the steps of: 17. 17. The second recognition molecule is the same as or different from the first recognition molecule. Method described. 18. 18. The second recognition molecule is inserted at the amino terminus of the PE molecule. How to put it on.