WO1997049726A1

WO1997049726A1 - Recombinant ribosomal inhibitor protein (rip) and use as immunoconjugate

Info

Publication number: WO1997049726A1
Application number: PCT/EP1997/003359
Authority: WO
Inventors: Antonio Mele; Rita De Santis; Dino Parente; Maria Ines Colnaghi
Original assignee: Ministero Universita' Ricerca Scientifica E Tecnologica
Priority date: 1996-06-27
Filing date: 1997-06-26
Publication date: 1997-12-31
Also published as: IT1286663B1; ITFI960155A1; AU3437597A

Abstract

The following description refers to a new RIP protein (SEQ ID No: 3) the cDNA sequence expressing same (SEQ ID No: 2), its preparation and use in the preparation of chemical and recombinant conjugates having anticancer properties.

Description

RECOMBINANT RIBOSOMAL INHIBITOR PROTEIN (RIP) AND USE AS IMMUNOCONJUGATE

Field of the invention The present invention relates to a new protein which - after being transferred into the cell by a suitable vector - is capable of inhibiting the activity of πbosomes Therefore, it can be used as an anticancer and/or antiviral agent State of the art As known, proteins extracted from filamentous fungi of the genus Aspergillus such as α-sarcin, restrictocin and mitogilhn, can inhibit protein synthesis by inactivating eukaryotic πbosomes The nucleotide sequence of genomic DNA expressing said proteins is also known

The antiviral activity of RIPs is related to the higher membrane permeability to the RIPs of virus-infected cells, with consequent injury to their πbosomes and consequent death of the infected cell It follows that viral replication is interrupted It has recently been disclosed that several RIPs inhibit HIV replication and that a RIP preparation, thπchosantin (a protein extracted from the roots of Tπchosantes kinloown) was used in phase l/ll clinical studies [ Byers, VS et al , A phase l/ll study of thπchosantin treatment of HIV diseases, AIDS, 4, 1189-1196 (1990)] With a view to obtain selectively cytotoxic molecules, several of the known RIPs were bound to proteic and non-proteic vectors capable of trasferπng them on specific target cell populations Compounds with a specific cytotoxic action are most frequently prepared with monoclonal antibodies as protein vectors (immunotoxins) However, hormones, growth factors, lectins have also been used as vectors for the treatment of cancer

The protein that has been most widely used so far for the construction of immunotoxins is πcin chain A, however, several RIPs of type 1 (gelonin, PAP, sapoπn, momordm, bryodin, barley RIP) have been recently tested in the treatment, e g , of tumours, autoimmune diseases, transplant rejections, parasitoses, etc Since tumour cells are often toxin-resistant and all toxins used so far in therapeutic treatments induce an immune response in treated patients, the identification and purification to homogeneity of new RIPs are of great importance in all therapeutic applications and in particular in the generation of new immunotoxins.

Summary of the invention

The present invention relates to a new RIP protein SEQ ID No: 3, able to inhibit protein synthesis by inactivating ribosomes. Said RIP protein is herein referred to as clavin. The present invention also concerns the nucleotide sequence SEQ ID No: 2, responsible of the expression of the new RIP protein clavin.

The present invention further includes the conjugates of the aforesaid protein with monoclonal antibodies, hormones, liposomes, growth factors, cytokines, transferrin and peptides, consisting of fragments of said proteins, obtainable by chemical conjugation or by genie recombination techniques whenever applicable.

It is another object of the present invention the Mgr6-clavin conjugate having amino acid composition SEQ ID No: 5, as well as the nucleotide sequence expressing it, corresponding to SEQ ID No: 4.

Detailed description of the invention SEQ ID No: 1 reports the complete cDNA sequence (i.e., including non-coding 3' and 5' sequences) of clavin, in which:

1 -279 = 5' UTR (5' fragment, untranslated);

280-360 = sequence encoding for hypothetical secretion sequence;

361 -813 = sequence encoding for mature protein (clavin); 814-1011 = 3' UTR (3' fragment, untranslated) + polyA.

SEQ ID No: 2 refers to the cDNA sequence encoding for mature clavin.

SEQ ID No: 3 corresponds to clavin protein sequence.

SEQ ID No: 4 describes the nucleotide sequence encoding for the Mgr6-clavin immunotoxin produced in pRSET, in which: 1 -108 = pRSET sequence containing the 6 histidines and the cleavage site for enterokinase; 109-861 = sequence encoding for ScFv (single Fv) of Mgr6

(109-462 = variable sequence of heavy chain, Bankit I.D. (Gene Bank) 54241 , access No. U 61494)

(463-507 = linker sequence) (508-861 = variable sequence of light chain, Bankit I.D. (Gene Bank) 54263, access No. U 61495)

(862-1311 = sequence encoding for clavin).

SEQ ID IN No: 5 corresponds to the protein sequence of Mgr6-claim immunotoxin produced in pRSET The purified protein is >95% pure as shown by SDS/PAGE analysis, N-terminal sequencing and reversed-phase HPLC. The protein molecular weight is approx.

17kDa.

The following examples are conveyed for a better understanding of the protein purification process according to the invention. cDNA isolation and sequencing

Total RNA was extracted from Aspergillus clavatus IFO 8605 (Institute of

Fermentation, Osaka). mRNA was purified using the kits for total RNA and, respectively, mRNA purification (Clontech). The two following primers were synthesized: 3' α-primer: 5'-ACGTAAGCTTCTAATGAGAGCAGAGCTT-3' (SEQ ID No: 6)

5' α-primer: 5 -ACGTCTGCAGTGACCTGGACCTGCTTGAACG-3' (SEQ ID No: 7)

Primers were drawn on the basis of the known α-sarcin sequence by assuming a high amino acid sequence homology with the toxin of Aspergillus clavatus.

The synthesis of cDNA with 5 μg mRNA and 3' α-primer was carried out using an appropriate synthesis kit (BRL). Part of the product obtained was added to a reaction mixture for PCR containing Taq polymerase (USB) and the two aforesaid primers; cDNA amplification for a total of 30 cycles was performed using a thermal cyclizer for DNA (Perkin-Elmer).

Each cycle consisted of 1-min denaturation at 94°C, 1 -min annealing at 42°C and 2-min extension at 72°C; in the final cycle extension at 72°C lasted 8 min. Amplified cDNA corresponded to the cDNA of clavin, but contained the sequences imposed by 3 α- and 5'α-prιmers Isolation of complete native cDNA of clavin was carried out by the RACE method

On 3 end, RACE was performed according to Frohman, using the following -> primer

5'-GACTCGAGTCGACATCGA(T)i7-3' (SEQ ID No 8), and the adjustment primer

5 -GACTCGAGTCGACATCG-3' (SEQ ID No 9)

Primer 5'-ACGTGGATCCTCTACAACCAGAAC-3' (SEQ ID No 10), which refers to the codons for ammo acids 23-29 of mature protein and bearing a restriction io site BamHI, was used as a gene-specific primer

On 5' end, RACE was performed using the 5'-AmplιFINDER RACE Kit (Clontech) and primers

5 -TGAACCAGTGAGGATAG-3' (SEQ ID No 11 )

5'-ACGTCTGCAGGCGCTTGTTCTCATA-3' (SEQ ID No 12) iτ referring to the codons for ammo acids 47-53 and 18-23 of mature protein were used as gene-specific primers, the latter primer also contains a restriction site

Pstl

The various PCR products were purified digested and subcloned in pUC19

Sequeces were analysed using PC GENE software (Intelligenetics) 20 The complete cDNA sequence obtained is shown in Fig 1 Said sequence contains an ORF encoding for a 177 ammo acid polypeptide chain The first 27 ammo acids represent a signal peptide involved in secretion, while mature protein consists of the 150 ammo acids shown in the figure

Recombinant clavin heterologous expression 25 Vector pEZZ18 (Pharmacia) was used for recombinant clavin heterologous expression Said vector directs the expression of fused proteins with a linking synthetic domain IgG (ZZ) based on staphylococcus protein A (Nilsson et al ,

1987) Clavin cDNA obtained by PCR with primers based on α-sarcin, as previously described, was re-amplified with 3' primer 30 5 -GATCCTGCAGCGACCTGGACTTGCATGAACGAGCAGAAGAACCCAAAG-

ACC-3' (SEQ ID No 13) and with 3' primer 5'-ACGTAAGCTTCTAATGAGAGCAGAGCTT-3' (SEQ ID No 14) to obtain the mature clavin native sequence, and cloned at restriction sites Pstl- Hindlll of vector pEZZ18 To obtain pMRS116, fragment EcoRI-Pstl was replaced T by linker B, which contains a sequence encoding for the cleavage site of factor Xa lle-Glu-Gly-Arg, in addition to a restdue Thr, inserted to preserve restriction site Pstl Linker B was obtained by annealing of the two oligonucleotides α-28 5'-AATTCGATCGAAGGTCGTACTGCA-3' (SEQ ID No 15) α-29 5'-GTACGACCTTCGATCG-3' (SEQ ID No 16) o For clavin production, construction pMRS116 was propagated in Escheπchia coli HB 101 [supE44, hsdS20(r_B ^"m_B ^")recA13, ara-14, proA2, lacY1 , galK2, rpsL20, xyl- 5 mtl-1 ] and cultured according to the producer's directions (Pharmacia) Recombinant clavin purification Culture supernatants were brought to pH 7 6 and added with 1 mM 5 phenylmethylsulfonyl fluoride Supernatants were injected into an IgG Sepharose Fast Flow column (Pharmacia) equalized with buffer (50 mM Tπa/HCI, 150 mM NaCI and 0 05% Tween-20, pH 7 6) The column was first washed with said buffer and then with 5 mM ammonium acetate, pH 5 1 , the fusion protein was eluted with 0 5 M ammonium acetate, pH 3 4, freeze-dned, dissolved to 2-8 mg/ml in 20 mM Tns/HCI, 100 mM NaCI, 1 mM CaCI₂, pH 8 0 and digested with Xa factor (Boehπnger) at 23°C for 18-24 h in an enzyme/substrate ratio equal to 1 100 Clavin was purified using a two-phase chromatographic process In the first phase, the digestion mixture was injected into an S Sepharose Fast Flow column (Pharmacia) equalized with 20 mM sodium phosphate, pH 5 8, and eluted in NaCI τ gradient, the fusion protein left undigested and clavin were eluted in a single peak The second phase was still performed on IgG Sepharose Fast Flow column clavin was collected with the eluent, while the non-digested protein was eluted from the colum with 0 5 M ammonium acetate, pH 3 4 immunotoxin synthesis and purification (chemical conjugate) Clavin was denvatized with 40-molar excess ethyl S-acetyl-3-propιonthιoιmιdate at 4°C for 1 h to obtain an average molar ratio of ethyl S-acetyl-3- propioπthioimidate groups to toxin molecule equal to 1 2 s Monoclonal antobody Mgr6, directed against the extracellular domain of ErbB2 and produced from hybπdoma Mgr6-C4 MCB c # 762, deposited in the Interlab Cell Line Collection bank (CBA), an international deposit authority, was purified from ascitic liquid as described in Centis et al , 1992 It was then denvatized with 10-molar excess 2-ιmιnothιolane in ethanol at room temperature for 30 mm, added o with 5,5'-dιthιo-bιs(2-nιtrobenzoιc acid) to block the free -SH groups and obtain a groups/toxin molecule molar ratio equal to 1 6 The mixture was applied to a BioGel P-6DG column to remove all reagents

Denvatized products were mixed using a 5-molar excess clavin, concentrated to a final volume of 1 ml, and added with 100 ul of 0 5 M hydroxylamine, 12 5 mM 5 EDTA, pH 7 2 The solution was stirred at 22°C for 14 h and at 4°C for additional 18 h The reaction was interrupted by addition of 20 ul of 200 mM N- ethylmaleimide

The immunoconjugate was purified to homogeneity by ion exchange chromatography 0 Recombinant immunotoxin Mgrβ-clavin Genie construction

The gene encoding for variable regions of monoclonal antobody Mgr6 was obtained by the "Recombinant Phase Antibody System" kit (Pharmacia) from mRNA of the antibody-producing hybπdoma Said procedure allows the τ obtamment of DNA encoding for ScFv (Single chain Fv), in which the sequences for the variable regions of heavy and light chains are joined by a linker sequence The DNA for ScFv was then linked to clavin DNA to obtain the gene for immunotoxin, cloned in commercial vector pRSET (Introvigen). Figs 4 and 5 show the nucleotide and, respectively, the ammo acid sequence of the immunotoxin o inserted in pRSET

The resulting plasmid has the following characteristics a) the fusion protein gene is under the control of the T7 polymerase control; b) the resulting protein has at its N-terminal site an extension containing 6 histidines, usable for the purification with IMAC (immobilized metal affinity chromatography) and a cleavage site for enterokinase K. Recombinant immunotoxin expression in E. coli

Competent cells B834(DE3)pLys were transformed, plated on 10 LB agar plates containing ampicillin (100 μg/ml), and incubated at 37°C overnight. Colonies were recovered in 500 ml LB culture medium containing ampicillin (100 μg/ml), glucose (0.5%) and MgSO₄ (1 ,62 mM), cultured at 37°C under stirring up to ODeoo = 2.3/2.5, and, after addition of 1 I culture medium (LB, ampicillin, glucose, MgSO₄), amplified at 37°C under stirring up to ODeoo = 1.2. Cells were centrifuged and resuspended in 1.5 I culture medium LB supplemented with ampicillin and induced by addition of IPTG (final 1 mM) under stirring at 37°C for 1.5 h. The cell pellet was recovered by centrifugation. Recombinant immunotoxin purification

The pellet from 1.5 I culture medium was resuspended in 150 ml of 50 mM Tris- HCI, pH 8, and frozen. 30 ml aliquots were thawed out, sonicated (3 x 20 sec), and centrifuged at 160,00 rpm at 4°C for 30 min. The resulting pellet was resuspended in 50 ml STET buffer (50 mM Tris-HCI, pH 8.5, 8% saccharose, 5% triton X-100, 50 mM EDTA) and the suspension was sonicated (3 x 45 sec) and centrifuged at 30,000 rpm for 20 min. The described washing procedure was repeated twice and twice again with 50 ml of 50 mM Tris- HCI, pH 8.5, and 100 mM NaCI. Denaturation The sample was resuspended in 30 ml buffer A (50 mM Tris-HCI, pH 8.0, 6 M Gu- HCI, 5 mM imidazole) and incubated at room temperature for at least 2 h. Immunotoxin purification by IMAC

The sample was centrifuged at 120,00 rpm for 30 min. The supernatant was analysed by chelated metal affinity chromatography (IMAC) using 20 ml Ni⁺⁺-filled chelatiπg sepharose FF resin (Pharmacia). The column was washed with 5 vol water, loaded with 5 vol of 0 1 M NιSO₄, washed with 5 vol water and equalized with 5 vol buffer A

Once the sample had been injected, the column was washed with 5 vol buffer A

Adsorbed proteins were eluted in step of pH using the following buffers T 50 mM Tπs-acetate, pH 5 5, 6 M Gu-HCI (Buffer B),

50 mM Tπs-acetate, pH 4 0, 6 M Gu-HCI (Buffer C)

The immunotoxin was eluted in buffer C

Immunotoxin reduction

The sample obtained from IMAC (in a concentration of 1-2 mg/ml) was brought to io pH 8 3 with 1 M Tπs base 2 mM EDTA and final 300 mM DTT were added The resulting product was incubated at room temperature for 3 h

Immunotoxin refolding

The reduced sample was rapidly diluted (1 100) in the refolding buffer (50 mM

Tris-HCI, pH 8 3, 0 5 M L-Arg, 2 mM EDTA, 4 mM GSSG, 2 mM DTT) and i T incubated at 10°C for 60 h

Immunotoxin concentration and dialysis

The sample (ca 500 ml) was added with Tween-20 (final 0 005%) and concentrated by ultrafiltration through membrane Amicon YM 10

Dialysis was carried out using a 10,000-cut-off membrane vs dialysis buffer (50 0 mM Tris-HCI, pH 8 0, 0 2 M NaCI, 0,005% Tween-20, 10% glycerol)

The resulting product was centrifuged at 120,00 rpm for 30 mm and the supernatant containing the immunotoxin was recovered

Inhibition of cell protein synthesis (chemical immunoconjugate)

The capacity of clavin and of immunotoxin Mgr6-clavιn for inhibiting the protein 25 synthesis was measured on SKBr3 (ErbB2⁺ cells) and on MeWo (ErbB2^" cells) cultured in RPMI 1640 containing 10% FCS The test was carried out essentially as described by Casahni et al , 1993

The immunotoxin, the toxin or the monoclonal antobody were diluted in turn in the culture medium Cells (1 2 x 10⁶) were incubated at 4°C for 3 h in polypropylene 30 test tubes, in 800 ul culture medium containing the appropriate concentrations of immunotoxin, toxin or monoclonal antibody alone Control cells were incubated only with the culture medium. Cells were then centrifuged, resuspended in a fresh culture medium and seeded in triplicate in 96-well plates (3 x 10⁵ cells/well). After incubation at 37°C for 48 h, the culture medium was removed and a fresh culture medium containing [³H] proline (luCi/well) was added. 48 h later, cells were washed and the amount of [³H] proline incorporated was determined.

In various tests, clavin shows a dose/response effect with IC50 values ranging from 0.1 to 1 μM.

The cytotoxicity of the Mgr6-clavin conjugate is similar to that of ricin A bound to the same monoclonal antibody. Inhibition of cell protein synthesis (recombinant immunoconjugate)

The capacity of recombinant immunotoxin Mgr6-clavin for inhibiting the protein synthesis was measured as already described for the chemical immunoconjugate. The IC50 of recombinant immunotoxin ranges from 0.1 to 1 μM, whereas antibody Mgr6 does not produce any effect.Therefore, clavin is a promising candidate for immunotoxin production.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(1) APPLICANT:

(A) NAME: MINISTERO UNIVERSITA' RICERCA SCIENTIFICA E

TECNOLOGICA

(B) STREET: c/o NOTARBARTOLO 4 GERVASI S.P.A. Corso d_l

Porta Vittoria, 9

(C) CITY: MILAN

(D) STATE: MI

(E) COUNTRY: ITALY

(F) POSTAL CODE (ZIP): 20122

(G) TELEPHONE: 02-5417991 (H) TELEFAX: 02-54179920

(ii) TITLE OF INVENTION: A protein capable of inhibiting ribosomal activity, its preparation and use as a chemical or recombinant immunoconjugate, and the cDNA sequence expressing said protein.

(m) NUMBER OF SEQUENCES: 16

(IV) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: IT FI96A000155

(B) FILING DATE: 27-JUN-1996

(2) INFORMATION FOR SEQ ID NO: 1:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1011 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus clavatus

(B) STRAIN: IFO8605 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AACCAGAAAC AAAGGATATG TGGTGAGATT TGTGAGAAAC CAGAACGCTT GGAAAAGAAA 60

ACAAAAGAGA GAAAAGTAAT CACCATCGAT GAGGATATTG TCTGACTCAG AGATCCAACG 120

AAATAATAGT CAACTTCGGA ATGCTTCAAG TCGCCCACAT CGAGCTGGGT CAATGGAGTC 180

7CTCGAGTCA GCCAGAGCAC ATATAAAAGC TGCTAGATCC TCGCGGTTCT CCCAGGAAAA 240

CCCAAGATC3 TGATCTCAAG CATCTTAACC ACATCCAAAA TGGTCGCAAT CAAGAACCTC 300

GTCCTGGTGG CCCTCACGGC CGTGACCGCC CTTGCGATGC CTTCGCCTCT CGAGGAGCGC 360

GCGGCGACCT GGACTTGCAT GAACGAGCAG AAGAACCCAA AGACCAACAA GTATGAGAAC 420

AAGCGCCTCC TCTACAACCA GAACAATGCC GAGAGCAACG CCCACCACGC GCCTCTCTCC 480

GACGGCAAGA CCGGTAGCAG CTATCCTCAC TGGTTCACCA ACGGCTACGA CGGCGATGGA 540

AAGATCCTCA AGGGCCGCAC GCCCATCAAG TGGGGAAATT CGGACTGCGA CCGCCCTCCC 600

AAGCACAGCA AGAATGGTGA TGGCAAGAAT GACCATTACC TGCTGGAGTT CCCAACATTC 660

CCCGATGGAC ACCAGTATAA TTTCGACTCG AAGAAGCCCA AGGAGGACCC CGGCCCGGCA 720

CGGGTCATCT ACACCTATCC TAACAAGGTG TTCTGCGGCA TTGTTGCCCA CACGAGGGAG 780

AACCAGGGTG ACCTGAAGCT CTGCTCTCAT TAAATGGGCT TGCACAGGGA TATAGTTTGC 840

CATTGGTCGT TCTTCAACCA CGGCTGATAC TATATCGCAT TGGGAAGTGG GGGAGGGAGC 900

TGAATGTTTC ACATATGTTG GTGCAGAACT TGTTCTATGT TATCTAGTCA ATCCCAGTCT 960

CTCGCTTTGA TATCTATGCA TATTGCACTT CATTGCAAAA AAAAAAAAAA A 1011 ' 2 ) INFORMATION FOR SEQ ID NO: 2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 453 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus clavatus

(B) STRAIN: IFO8605 (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GCGGCGACCT GGACTTGCAT GAACGAGCAG AAGAACCCAA AGACCAACAA GTATGAGAAC 60

AAGCGCCTCC TCTACAACCA GAACAATGCC GAGAGCAACG CCCACCACGC GCCTCTCTCC 120

GACGGCAAGA CCGGTAGCAG CTATCCTCAC TGGTTCACCA ACGGCTACGA CGGCGATGGA 180

.AAGATCCTCA AGGGCCGCAC GCCCATCAAG TGGGGAAATT CGGACTGCGA CCGCCCTCCC 240

AAGCACAGCA AGAATGGTGA TGGCAAGAAT GACCATTACC TGCTGGAGTT CCCAACATTC 300

CCCGATGGAC ACCAGTATAA TTTCGACTCG AAGAAGCCCA AGGAGGACCC CGGCCCGGCA 360

CGGGTCATCT ACACCTATCC TAACAAGGTG TTCTGCGGCA TTGTTGCCCA CACGAGGGAG 420

AACCAGGGTG ACCTGAAGCT CTGCTCTCAT TAA 453 (2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 150 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ill) HYPOTHETICAL: MO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus clavatus

(B) STRAIN: IFO8605

(>:ι) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Ala Ala Thr Trp Thr Cys Met Asn Glu Gin Lys Asn Pro Lys Thr Asn 1 5 10 15

Lvs Tyr Glu Asn Lys Arg Leu Leu Tyr Asn Gin Asn Asn Ala Glu Ser 20 25 30

Asn Ala His His Ala Pro Leu Ser Asp Gly Lys Thr Gly Ser Ser Tyr 35 40 ^' 45

Pro His Trp Phe Thr Asn Gly Tyr ASD Gly Asp Gly Lys lie Leu Lys 50 55 ^" 60

Gly Arg Thr Pro lie Lvs Trp Gly Asn Ser Asp Cys Asp Arg Pro Pro 65 70 75 ^' 80

Lys His Ser Lys Asn Gly Asp Gly Lys Asn Asp His Tyr Leu Leu Glu 85 ^* 90 95

Phe Pro Thr Phe Pro Asp Gly His Gin Tyr Asn Phe Asp Ser Lys Lys 100 105 110

Pro Lvs Glu Asp Pro Gly Pro Ala Arg Val lie Tyr Thr Tyr Pro Asn 115 120 ^' 125

Lys Val Phe Cys Gly lie Val Ala His Thr Arg Glu Asn Gin Gly Asp 130 135 140

Leu Lys Leu Cys Ser His 145 150 (2) INFORMATION FOR SEQ ID NO: 4:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1314 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus clavatus

(B) STRAIN: IFO8605

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATGCGGGGTT CTCATCATCA TCATCATCAT GGTATGGCTA GCATGACTGG TGGACAGCAA 60 ATGGGTCGGG ATCTGTACGA CGATGACGAT AAGGATCGAT GGGGATCCCA GGTGCAGCTG 120 CAGGAGTCTG GGGCAGAGCT TGTGAAGCCA GGGGCCTCAG TCAAGTTGTC CTGCACAGCT 180 TCTGGCTTCA ACATTAAAGA CACCTATATG CACTGGGTGA AGCAGAGGCC TGAACAGGGC 240 CTGGAGTGGA TTGGAAGGAT TGATCCTGCG AATGGTAATA CTAAATATGA CCCGAAGTTC 300 CAGGGCAAGG CCACTATAAC AGCAGACACA TCCTCCAACA CAGCCTACCT GCAGCTCAGC 360 AGCCTGACAT CTGAGGACAC TGCCGTCTAT TACTGTGCTA GAGGAGAATA TGATTATCCT 420 TTTCCTTACT GGGGCCAAGG GACCTCGGTC ACCGTCTCCT CAGGTGGAGG CGGTTCAGGC 480 GGAGGTGGCT CTGGCGGTGG CGGATCGTAC ATCGAGCTCA CTCAGTCTCC AGCTTCCTTA 540 GCTGTATCTC TGGGGCAGAG GGCCACCATC TCATGCAGGG CCAGCCAAAG TGTCAGTACA 600 TCTAGGTATA GTTATATGCA CTGGTACCAA CAGAAACCAG GACAGCCACC CAAACTCCTC 660 ATCAAGTATG CATCCAACCT AGAATCTGGG GTCCCTGCCA GGTTCAGTGG CAGTGGGTCT 720 GGGACAGACT TCACCCTCAA CATCCATCCT GTGGAGGAGG AGGATACTGC AACATATTAC 780 TGTCAGCACA GTTGGGAGAT TCCTCGGACG TTCGGTGGAG GGACCAAGCT GGAGCTGAAA 840 CGGGCGGGAT CCCCGGAATT CGCAGCGACC TGGACTTGCA TGAACGAGCA GAAGAACCCA 900 AAGACCAACA AGTATGAGAA CAAGCGCCTC CTCTACAACC AGAACAATGC CGAGAGCAAC 960 GCCCACCACG CGCCTCTCTC CGACGGCAAG ACCGGTAGCA GCTATCCTCA CTGGTTCACC 1020 AACGGCTACG ACGGCGATGG AAAGATCCTC AAGGGCCGCA CGCCCATCAA GTGGGGAAAT 1080 TCGGACTGCG ACCGCCCTCC CAAGCACAGC AAGAATGGTG ATGGCAAGAA TGACCATTAC 1140 CTGCTGGAGT TCCCAACATT CCCCGATGGA CACCAGTATA ATTTCGACTC GAAGAAGCCC 1200 AAGGAGGACC CCGGCCCGGC ACGGGTCATC TACACCTATC CTAACAAGGT GTTCTGCGGC 1260 ATTGTTGCCC ACACGAGGGA GAACCAGGGT GACCTGAAGC TCTGCTCTCA TTAG 1314 (2) INFORMATION FOR SEQ ID NO: 5:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 437 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide !ιii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Aspergillus clavatus

(B) STRAIN: IFO8605

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15

Gly Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp ASD Lys Asp 20 25 3θ^"

Arg Trp Gly Ser Gin Val Gin Leu Gin Glu Ser Gly Ala Glu Leu Val 35 40 45

Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn 50 55 60

He Lys Asp Thr Tyr Met His Trp Val Lys Gin Arg Pro Glu Gin Gly 65 70 75 80

Leu Glu Trp He Gly Arg He Asp Pro Ala Asn Gly Asn Thr Lys Tyr 85 90 95

Asp Pro Lys Phe Gin Gly Lys Ala Thr He Thr Ala Asp Thr Ser Ser 100 105 110

Asn Thr Ala Tyr Leu Gin Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 115 120 125

Val Tyr Tyr Cys Ala Arg Gly Glu Tyr Asp Tvr Pro Phe Pro Tyr Trp 130 135 ^' 140

Gly Gin Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr He Glu Leu Thr Gin Ser 165 170 175

Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys 180 185 190

Arg Ala Ser Gin Ser Val Ser Thr Ser Arg Tyr Ser Tyr Met His Trp 195 200 205

Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Lys Tyr Ala 210 215 220

Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 225 230 235 240

Gly Thr Asp Phe Thr Leu Asn He His Pro Val Glu Glu Glu Asp Thr 245 250 255 Ala Thr Tyr Tyr Cys Gin His Ser Trp Glu He Pro Arg Thr Phe Gly 260 265 270

Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala Gly Ser Pro Glu Phe Ala 275 280 285

Ala Thr Trp Thr Cys Met Asn Glu Gin Lys Asn Pro Lys Thr Asn Lys 290 295 300

Tyr Glu Asn Lys Arg Leu Leu Tyr Asn Gin Asn Asn Ala Glu Ser Asn 305 310 315 320

Ala His His Ala Pro Leu Ser Asp Gly Lys Thr Gly Ser Ser Tyr Pro 325 330 335

His Trp Phe Thr Asn Gly Tyr Asp Gly Asp Gly Lys He Leu Lys Gly 340 345 350

Arg Thr Pro He Lys Trp Gly Asn Ser Asp Cys Asp Arg Pro Pro Lys 355 360 365

His Ser Lys Asn Gly Asp Gly Lys Asn Asp His Tyr Leu Leu Glu Phe 370 375 380

Pro Thr Phe Pro Asp Gly Kis Gin Tyr Asn Phe Asp Ser Lys Lys Pro 385 390 395 400

Lys Glu Asp Pro Gly Pro Ala Arg Val He Tyr Thr Tyr Pro Asn Lys 405 410 415

Val Phe Cys Gly He Val Ala His Thr Arg Glu Asn Gin Gly Asp Leu 420 425 430

Lys Leu Cys Ser His 435

(2) INFORMATION FOR SEQ ID NO: 6:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genoraic) (m) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACGTAAGCTT CTAATGAGAG CAGAGCTT 28 (2) INFORMATION FOR SEQ ID NO: 7:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (3) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic) (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ACGTCTGCAG TGACCTGGAC CTGCTTGAAC G 31

(2) INFORMATION FOR SEQ ID NO: 8:

(l! SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GACTCGAGTC GACATCGATT TTTTTTTTTT TTTTT 35

'2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

'ii! MOLECULE TYPE: DNA (genomic) (m) HYPOTHETICAL: NO (iv) .ANTI-SENSE: NO (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GACTCGAGTC GACATCG 17

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ACGTGGATCC TCTACAACCA GAAC 24

(2) INFORMATION FOR SEQ ID NO: 11:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (in) HYPOTHETICAL: NO (IV) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

TGAACCAGTG AGGATAG 17

(2) INFORMATION FOR SEQ ID NO: 12:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic) (m) HYPOTHETICAL: MO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ACGTCTGCAG GCGCTTGTTC TCATA 25 lo

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (ill) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GATCCTGCAG CGACCTGGAC TTGCATGAAC GAGCAGAAGA ACCCAAAGAC C 51

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACGTAAGCTT CTAATGAGAG CAGAGCTT 28

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: AATTCGATCG AAGGTCGTAC TGCA 24

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ■ (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GTACGACCTT CGATCG 16

Claims

Claims 1. Nucleotide sequence SEQ ID No: 2. 2. Protein capable of inactivating the ribosomal activity, the amino acid sequence SEQ ID No: 3. 3. Nucleotide sequence SEQ ID No: 4, encoding for the Mgr6-clavin immunotoxin. 4. Protein sequence SEQ ID No: 5 of Mgr6-clavin immunotoxin. 5. Conjugates, obtained by chemical conjugation or by genie recombination, of the protein as claimed in claim 2 with hormones, liposomes, monoclonal antibodies, growth factors, cytokines, transferrin and peptides consisting of fragments of said proteins. 6. The conjugates as claimed in claim 5, wherein the protein is conjugated with monoclonal antibodies. 7. The conjugates as claimed in claim 5, wherein the monoclonal antibody is Mgr6. 8. Pharmaceutical compositions containing, as active ingredient, the protein as claimed in claim 2 and/or the conjugates as claimed in any of claims 5 to 7, combined with suitable additives. 9. Use of the protein as claimed in claim 2 and/or of the conjugates as claimed in any of claims 5 to 7, or mixtures thereof, for the preparation of pharmaceutical preparations useful as anticancer and/or antiviral agents. A protein capable of inhibiting ribosomal activity, its preparation and use as a chemical or recombinant immunoconjugate, and the cDNA sequence expressing said protein.