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WO1996027010A1 - Murine/human chimeric monoclonal antibody or a fragment thereof specific for the egf receptor - Google Patents

Murine/human chimeric monoclonal antibody or a fragment thereof specific for the egf receptor Download PDF

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Publication number
WO1996027010A1
WO1996027010A1 PCT/EP1996/000805 EP9600805W WO9627010A1 WO 1996027010 A1 WO1996027010 A1 WO 1996027010A1 EP 9600805 W EP9600805 W EP 9600805W WO 9627010 A1 WO9627010 A1 WO 9627010A1
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WIPO (PCT)
Prior art keywords
chmint5
seq
antibody
fragment
ser
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PCT/EP1996/000805
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French (fr)
Inventor
Antonio Mele
Rita De Santis
Cristina Ferrer Marsal
Anna Maria Anastasi
Anna Maria Di Massimo
Maria Ines Colnaghi
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Ministero Universita' Ricerca Scientifica E Tecnologica
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Application filed by Ministero Universita' Ricerca Scientifica E Tecnologica filed Critical Ministero Universita' Ricerca Scientifica E Tecnologica
Priority to AU47191/96A priority Critical patent/AU4719196A/en
Publication of WO1996027010A1 publication Critical patent/WO1996027010A1/en

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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
    • C07K16/2863—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 growth factors, growth regulators
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to a murine/human chimeric monoclonal antibody, termed chMint5. which is specific for the EGF-receptor. Particularly, the nucleotide sequences and the amino acid sequences encoded by them of the variable regions (VH and VL) of such chimeric antibody, chMint5, are disclosed.
  • VH and VL variable regions
  • Anterior Art Mint5 is a monoclonal antibody specific for the EGF-receptor (EGF-R), obtained by immunization of mice with A431 cell line.
  • Mint5 has got an inhibitory effect on growth of cells having high levels of EGF-R expression in experiments either in vivo or in vitro, but it has got no effect on cells having normal levels of receptor expression.
  • Mint5 is efficiently internalized. Moreover, capability of inhibiting growth of tumor cells which were transplanted in athymic mice was shown.
  • the cell line of hybridoma producing Mint5 antibody was filed in 1993. the seventh of September, by DSM (Deutsches Sammlung von Mikroorganismen und Zellkulturen GmbH) with number ACC2150.
  • the variable domains are determining for the antigenic identification of the tumor cell.
  • the murine origin of such antibody limits its use in clinical therapy owing to the immune response of the host receiver.
  • chMint5. a novel chimeric monoclonal antibody which could show a lower immnunogenicity when administered to humans.
  • this invention relates to a chimeric monoclonal chMint5 antibody or to a fragment thereof comprising a certain amino acid sequence of the variable regions of the heavy chain (VH) and light chain (VL) of the monoclonal antibody obtained by the hybridoma deposited as DSM ACC2150 (murine Mint5).
  • This invention relates, therefore, to nucleotide sequences coding the variable regions of the heavy chains (VH) and light chains (VL) of chMint5 antibody, to the correspondent amino acid sequences and it also relates to chMint5 antibody and to the fragments thereof comprising said sequences of the variable regions, and comprising whole constant regions or parts thereof of the heavy and light chains obtained from human i munoglobulins.
  • chMint5 antibody can be used alone or combined to cytotoxic molecules according to the present invention in the antitumor therapy of patients affected by tumors characterized by a high level of EGF-R expression in a diagnostic assay specific for the determination of the EGF-R.
  • This diagnostic method comprises a ligand or a conjugate (e.g. another antibody), eventually labeled or bound to a chromogen, which recognize the variable regions of said chMint5. Description of Figures and List of the Sequences
  • FIG. 1 shows a schematic illustration of the cDNA amplification of the heavy chain (H) of chMint5- - 3 -
  • FIG. 2 shows a schematic illustration of the procedure of chMint5 expression in CHO cells.
  • FIGS. 3A and 3B show FACS analysis (Fluorescence Activated Cell Sorter) of chMint5 on EGF-R + and EGF-R " cell lines.
  • the cells (1X10 6 ) were incubated into ice for 30 minutes, with chMint5 or PBS/13.FCS (negative control). The cells were then washed with PBS/l ⁇ FCS and incubated with FITC-conjugated anti-human-IgGl murine antibody
  • FIG. 4a shows the affinity constant of murine Mint5 bound on A431 cells. The characteristics are the following:
  • - SEQ ID N0:1 sequence shows the nucleotide and amino acid sequences of the variable region of the chM ⁇ nt5 antibody heavy chain.
  • - SEQ ID NO:2 sequence shows the amino acid sequence (deduced from the nucleotide sequence indicated in SEQ ID N0:1) of the variable region of the chMint5 antibody heavy chain.
  • SEQ ID NO:3 sequence shows a N-terminal fragment of the amino acid sequence of the variable region of the heavy chain. Said sequence was determined by means of Edman degradation. Only No. 1,3.5 and 6 amino acids differ from the deduced amino acid chain indicated in SEQ ID N0:2. Amino acids indicated as Xaa were not correctly defined. As these didn't influence the identification of the variable region, the antibody was not subjected to further analysis of the proteic sequence.
  • sequence shows the nucleotide and the amino acid sequences of the variable region of the chMint5 antibody light chain.
  • the nucleotide sequence of eight codons (24 nucleotides) at 5'- and 3'- ends were established by primers used in the cDNAs amplification by means of PCR. Sequences indicated as CDR1, CDR2 and CDR3 refer to hypervariable regions.
  • - SEQ ID NO: sequence shows the amino acid sequence (deduced by the nucleotide sequence indicated in SEQ ID NO: ) of the variable region of the chMint5 antibody light chain.
  • - SEQ ID NO:6 sequence shows a N-terminal fragment of the amino acid sequence of the variable region of the chMint5 antibody light chain. Said sequence was determined by means of Edman degradation. Only No. 3 and 8 amino acids differ from the deduced amino acid sequence indicated in SEQ ID NO:5- Amino acids indicated as Xaa were not correctly defined. As these didn't influence the identification of the variable region, the antibody was not subjected to further analysis of the proteic sequence.
  • SEQ ID NO:7 shows the nucleotide sequence of a 5' primer specific for the leader sequence of the light and heavy chains for use in PCR.
  • - SEQ ID NO:8 shows the nucleotide sequence of CKhu primer specific for the constant region of the light chain for use in PCR.
  • - SEQ ID NO: shows the nucleotide sequence of the antisensus VH1F0R primer specific for the sequence of a fragment of the constant region of the heavy chain for use in PCR.
  • - SEQ ID NO: 10 shows the nucleotide sequence of the CHhu primer specific for the sequence at the 3 '-end of the constant region fragment of the heavy chain for use in PCR.
  • Murine/human chimeric monoclonal chMint5 antibody was obtained by fusion of the genes of the variable regions of the light and heavy chains of murine Mint5 antibody at the nucleotide sequences coding for human immunoglobulin constant regions.
  • VH and VL nucleotide sequences were bound to the nucleotide sequences of human C-gammal and
  • RNA from Mint5 hybridomas was isolated and VH and VL genes were obtained by amplification by means of PCR, using consensus primers.
  • the amplified VH and VL genes were cloned in M13- derived vehicles (in M13VHPCR1 vehicle and in M13VKPCR1 vehicle respectively, courteously given by Dr.G. inter) containing the promoter and the leader sequence of the immunoglobulin genes. and then sequenced.
  • VH fragment gene was directly cloned in M13VHPCR1 vehicle and sequenced (Sequenase Version 2.0 U.S.B., USA). Instead, the VL gene (then indicated as VK, too) was at first introduced in M13mpl9 vehicle in order to remove the two inside BamHI and PvuII restriction sites, by means of site-directed mutagenesis (Oligonucleotide-Directed In vitro mutagenesis System Version 2. Amersham International pic. Little Chalfont. UK). These restriction sites would have hinder the VK insertion into M13VKPCR1 vehicle and the subcloning in the vehicle of expression for the chimeric light chain (alfa-Lysl7 vehicle). The thus modified VK gene was then cloned in M13VKPCR1 vehicle and at last sequenced.
  • VH and VK genes of Mint5 were inserted in alfa-Lys30 and alfa-Lysl7 vehicles, respectively (courteously given by Dr.G.Winter) .
  • Alfa-Lys30 plasmid contains the human Cgammal chain gene and gpt gene (xanthine-guanine phosphoribosyl transferase) as selection marker.
  • pMRSl ⁇ vehicle of expression was therefore prepared.
  • Said vehicle of expression was obtained by cloning Hindlll-BamHI fragment, obtained from M13VHPCR1 vehicle, which contains the promoter, the leader sequence and the Mint5 VH region in alfa-Lys30 vehicle, upstream from the human constant region gene.
  • Alfa-Lysl7 plasmid contains the human CK chain gene and the gene for resistance to hygromicine as selection marker.
  • pMRS17 vehicle of expression was therefore prepared.
  • Said vehicle of expression was obtained by cloning Hindlll-BamHI fragment, obtained from M13VKPCR1 vehicle which contains the promoter, the leader sequence and the Mint5 VK region in alfa-Lysl7 vehicle, upstream from the human constant region gene.
  • non- secernent-immunoglobulin murine myeloma NSO/1 cells were cotransfected with pMRSl ⁇ and pMRS17 vehicles by means of electroporation (Potter et al.. 1984, PNAS, 81:7l6l-7l65) .
  • the stable transfectans were analyzed and selected according to their mycophenolic acid resistance and hygromicine resistance and according to the chimeric antibody production by means of ELISA.
  • the transfectomas in NSO cells showed chMint5 productivity of 1-5 ng/10 cells/24 hours. These productivity levels, although they didn't allow the preparatory purification of the chimeric antibody, showed that chMint5 genes are correctly translated by transfected NSO/1 cells which can be then utilized for the isolation of cDNAs of the chMint5 heavy and light chains in order to achieve a better expression.
  • Example 2
  • the primers utilized in PCR for the amplification of the chMint5 cDNAs are indicated in the following Table.
  • a leader primer CKhu (SEQ ID N0:7) (SEQ ID N0:8)
  • A cDNA of the light chain
  • B cDNA of the fragment of the variable region of the heavy chain
  • C cDNA of the fragment of the constant region of the heavy chain ' 'VH1F0R primer was described in Orlandi et al., (as above). PCR was performed by using leader primers complementary to the 5'- leader region of the heavy and light chains by inserting a Xbal site. For human heavy and light constant 3' regions, CHhu and CKhu primers respectively were used, primers which introduce a Smal site.
  • the cDNA of the chimeric heavy chain was amplified in two phases (according to the scheme of Figure 1): a) the leader and variable regions were amplified by using a leader primer at the 5'-end and VHIFOR primer at the 3'-end; the constant region was amplified by using, at the 5'-end, antisensus VHIFOR primer (complementary to VHIFOR primer), and CHhu primer at 3'-end; b) products of such amplifications were amplified once again, all together in order to achieve the whole heavy chain.
  • pMRS ⁇ l and pMRS71 Two pSV2-derived vehicles, termed pMRS ⁇ l and pMRS71 were used to express the genes of the chMint5 heavy and light chains respectively, in CHO cells.
  • pMRS ⁇ l was obtained from pMCMVbeta51-2. This vehicle was digested with AccI and Hindlll, thus eliminating the immediate early promoter of murine Cytomegalovirus. At Accl-Hindlll sites the immediate early promoter of human cytomegalovirus was cloned, with Accl-Hindlll end (promoter which was disclosed in Int. Appln. No. W095/H982 filed on October 24. 1994. In this way, pUthCMVbeta vehicle was obtained.
  • pMRS71 was obtained from pMCMVbeta51-2 vehicle.
  • pMCMVbeta51-2 vehicle was linearized with BamHI and treated with alkaline fosphatase from intestinal calf ucosa (CIP); in the thus treated vehicle, the transcription VIDHFR unit was inserted.
  • CIP intestinal calf ucosa
  • pMCMVbeta51-2 vehicle and the transcription VIDHFR unit are mentioned in Int. Appln. No. W095/H982 filed on October 24, 1994.
  • pMRS ⁇ l is, therefore, a plasmid containing the early promoter of human Cytomegalovirus, the polyadenylation signal of the 1-betaglobin of rabbit and the gtp gene as selection marker.
  • cDNA of the chMint5 heavy chain was cloned in Xbal-Smal cloning site as Xbal-blunt fragment owing to the presence of an inside Smal site in the human constant region.
  • the thus obtained vehicle was termed pMRS ⁇ 9- cDNA of the chimeric light chain, amplified from transfected NSO/1 cells, was cloned as a Xbal-Smal fragment in pMRS71 plasmid containing the early promoter of murine Cytomegalovirus, the polyadenylation signal of 1-betaglobin of rabbit and the gene of dihydrofolate reductase (dhfr) as amplificable selection marker.
  • CHO dhfr " cell line was cotransfected with pMRS ⁇ 9 and pMRS95 vehicles (see Figure 2) by lipofection (Feigner P. et al., 19 ⁇ 7. PNAS, ⁇ 4, 7413-7417).
  • the transfectants were selected according to the dhfr + /gpt double resistance phenotype.
  • the single clones were moved to 24-well plates and after one week of growth, the supernatants of culture were tested for production of chMint5 by ELISA.
  • Such transfectant clones secreted amounts of chMint5 from 0 to 100 ng/10° cells/24 hours.
  • Mint5 antibody was purified from supernatants of cultures of a CHO transfectant single clone, by means of affinity chromatography on A Protein-Sepharose. The binding activity of the chimeric antibody was analyzed by means of flow cytometry. A 31 cells of human epider oid carcinoma and human Jurkat T EGF-R negative cells were used in the assay. The results, illustrated in Figure 3.
  • chMint5 specifically recognizes the A431 cells (EGF-R + ), but it doesn't bind the Jurkat T cells (EGF-R " ).
  • the binding affinity constants for murine and chimeric antibodies were determined by means of the titration curves of the monoclonal antibody, obtained by IRMA on A431 target cells.
  • Figures 4a and 4b show a Kaff value (affinity constant) of 6.76X10 ⁇ M " for chMint5. whereas Mint5 value was 2.05X10 M ⁇ , thus indicating that chMint5 substantially keeps the binding affinity properties of the murine Mint5-
  • chMint5 antibody showed to keep the specificity and the binding affinity properties of the murine Mint5 antibody. chMint5 also shows a greater utilization in the human tumor therapy.
  • chMint5 antibody can be used for the preparation of a pharmaceutical composition, useful in human therapy, comprising a pharmaceutically acceptable carrier and/or excipient. chMint5 can be also used in the preparation of immunotoxins or immunocytokines.
  • Such immunotoxins or immunocytokines can be obtained by means of chemical conjugation or by recombination of genes which correspond to chMint5 or to parts of it and by recombination of genes coding for the toxins or the cytokines which will be in the immune-complex. Further this invention relates to the use of such immunotoxins or immunocytokines for the preparation of pharmaceutical compositions useful in the human antitumor therapy, combined to a pharmaceutical acceptable carrier and/or excipient. chMint5 can be also used in a diagnostic test specific for the EGF-R.
  • Such diagnostic method comprises the chMint5 antibody and a ligand or a conjugate (for example another antibody), eventually labeled or bound to a chromogen, which recognize the variable regions of said chMint5.
  • a ligand or conjugate for example another antibody
  • such ligand or conjugate will comprise a sequence complementary to at least one of the sequences indicated in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5.
  • GCA TAC ATT AGT AAT GGT GGT GGT AGC ACC TAT TAT CCA GAC ACT GTA 1 Ala Tyr lie Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60
  • Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
  • MOLECULE TYPE DNA
  • ANTI-SENSE YES
  • FEATURE VHIFOR anti-sense primer specific for the sequence of a fragment of the constant region of the heavy chain for use in PCR

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Abstract

A murine/human chimeric monoclonal antibody (termed chMint5), obtained by the murine monoclonal chMint5, which is specific for the EGF-receptor, is described. Particularly, the nucleotide sequences and the amino acid sequences encoded by them of the variable regions (VH and VL) of such chMint5 antibody, are disclosed.

Description

MURINE/HUMAN CHIMERIC MONOCLONAL ANTIBODY OR A FRAGMENT THEREOF
SPECIFIC FOR THE EGF RECEPTOR.
Field of the Invention
This invention relates to a murine/human chimeric monoclonal antibody, termed chMint5. which is specific for the EGF-receptor. Particularly, the nucleotide sequences and the amino acid sequences encoded by them of the variable regions (VH and VL) of such chimeric antibody, chMint5, are disclosed.
Anterior Art Mint5 is a monoclonal antibody specific for the EGF-receptor (EGF-R), obtained by immunization of mice with A431 cell line.
The major feature of chMint5 is its capability of discriminating between cells having normal levels of EGF-R expression and cells having high levels of such expression. Mint5 has got an inhibitory effect on growth of cells having high levels of EGF-R expression in experiments either in vivo or in vitro, but it has got no effect on cells having normal levels of receptor expression.
Once bound to A431 cells, Mint5 is efficiently internalized. Moreover, capability of inhibiting growth of tumor cells which were transplanted in athymic mice was shown.
The cell line of hybridoma producing Mint5 antibody was filed in 1993. the seventh of September, by DSM (Deutsches Sammlung von Mikroorganismen und Zellkulturen GmbH) with number ACC2150. The variable domains are determining for the antigenic identification of the tumor cell. However, the murine origin of such antibody limits its use in clinical therapy owing to the immune response of the host receiver.
Summary of the Invention
Authors of this invention prepared a novel chimeric monoclonal antibody, termed chMint5. which could show a lower immnunogenicity when administered to humans.
Particularly, this invention relates to a chimeric monoclonal chMint5 antibody or to a fragment thereof comprising a certain amino acid sequence of the variable regions of the heavy chain (VH) and light chain (VL) of the monoclonal antibody obtained by the hybridoma deposited as DSM ACC2150 (murine Mint5).
This invention relates, therefore, to nucleotide sequences coding the variable regions of the heavy chains (VH) and light chains (VL) of chMint5 antibody, to the correspondent amino acid sequences and it also relates to chMint5 antibody and to the fragments thereof comprising said sequences of the variable regions, and comprising whole constant regions or parts thereof of the heavy and light chains obtained from human i munoglobulins. chMint5 antibody can be used alone or combined to cytotoxic molecules according to the present invention in the antitumor therapy of patients affected by tumors characterized by a high level of EGF-R expression in a diagnostic assay specific for the determination of the EGF-R. This diagnostic method comprises a ligand or a conjugate (e.g. another antibody), eventually labeled or bound to a chromogen, which recognize the variable regions of said chMint5. Description of Figures and List of the Sequences
- Figure 1 shows a schematic illustration of the cDNA amplification of the heavy chain (H) of chMint5- - 3 -
- Figure 2 shows a schematic illustration of the procedure of chMint5 expression in CHO cells.
- Figures 3A and 3B show FACS analysis (Fluorescence Activated Cell Sorter) of chMint5 on EGF-R+ and EGF-R" cell lines. The cells (1X106) were incubated into ice for 30 minutes, with chMint5 or PBS/13.FCS (negative control). The cells were then washed with PBS/l^FCS and incubated with FITC-conjugated anti-human-IgGl murine antibody
(fluorescein isothiocyanate; Sigma), then washed again and analyzed by using a FACS (FACSort, Beeton-Dickinson) . - Figure 4a shows the affinity constant of murine Mint5 bound on A431 cells. The characteristics are the following:
Molecular weight: 155000; specific activity (Ci/g):10.7; counter efficiency: 0.84; volume of the sample: 0.1 ml; non-specific calculations: 1061. - Figure 4b shows the affinity constant of chMint5 bound on A431 cells.
The characteristics are the following:
Molecular weight : 155000 ; specific activi ty ( Ci /g ) ; 19 - 56 ; counter efficiency : 0. 84 ; volume of the sample : 0 . 1 ml ; non-speci fi c calculations : 0.
In Figures 4a and 4b , the affinity constant was determined by means of
SAB-method as described in Antoni G . and Mariani M . , 1985. J - Immunol .
Methods . 83 : 61-68.
- SEQ ID N0:1 sequence shows the nucleotide and amino acid sequences of the variable region of the chMιnt5 antibody heavy chain. The nucleotide sequences of 8 codons at '-end and of 11 codons at 3'-end were established by primers used in the cDNAs amplification by means of PCR. The primers were selected according to works by Orlandi et al., PNAS, 86:3833-3837 (1989)- Sequences indicated as CDR1, CDR2 and CDR3 (CDR= Complementarity Determinant Region) refer to hypervariable regions. - SEQ ID NO:2 sequence shows the amino acid sequence (deduced from the nucleotide sequence indicated in SEQ ID N0:1) of the variable region of the chMint5 antibody heavy chain.
- SEQ ID NO:3 sequence shows a N-terminal fragment of the amino acid sequence of the variable region of the heavy chain. Said sequence was determined by means of Edman degradation. Only No. 1,3.5 and 6 amino acids differ from the deduced amino acid chain indicated in SEQ ID N0:2. Amino acids indicated as Xaa were not correctly defined. As these didn't influence the identification of the variable region, the antibody was not subjected to further analysis of the proteic sequence.
- SEQ ID N0:4 sequence shows the nucleotide and the amino acid sequences of the variable region of the chMint5 antibody light chain. The nucleotide sequence of eight codons (24 nucleotides) at 5'- and 3'- ends were established by primers used in the cDNAs amplification by means of PCR. Sequences indicated as CDR1, CDR2 and CDR3 refer to hypervariable regions.
- SEQ ID NO: sequence shows the amino acid sequence (deduced by the nucleotide sequence indicated in SEQ ID NO: ) of the variable region of the chMint5 antibody light chain. - SEQ ID NO:6 sequence shows a N-terminal fragment of the amino acid sequence of the variable region of the chMint5 antibody light chain. Said sequence was determined by means of Edman degradation. Only No. 3 and 8 amino acids differ from the deduced amino acid sequence indicated in SEQ ID NO:5- Amino acids indicated as Xaa were not correctly defined. As these didn't influence the identification of the variable region, the antibody was not subjected to further analysis of the proteic sequence.
- SEQ ID NO:7 shows the nucleotide sequence of a 5' primer specific for the leader sequence of the light and heavy chains for use in PCR.
- SEQ ID NO:8 shows the nucleotide sequence of CKhu primer specific for the constant region of the light chain for use in PCR. - SEQ ID NO: shows the nucleotide sequence of the antisensus VH1F0R primer specific for the sequence of a fragment of the constant region of the heavy chain for use in PCR.
- SEQ ID NO: 10 shows the nucleotide sequence of the CHhu primer specific for the sequence at the 3 '-end of the constant region fragment of the heavy chain for use in PCR.
Detailed Description of the Invention
Murine/human chimeric monoclonal chMint5 antibody was obtained by fusion of the genes of the variable regions of the light and heavy chains of murine Mint5 antibody at the nucleotide sequences coding for human immunoglobulin constant regions.
Particularly, for the chMint5 preparation, the VH and VL nucleotide sequences were bound to the nucleotide sequences of human C-gammal and
CK genes.
The realization of this invention will be clarified on the basis of the following examples.
Example 1
Preparation of chMint5 antibody Genes for chimeric (murine/human) light and heavy chMint5 chains were essentially prepared as described by Orlandi et al. , 1989. PNAS,
86:3833-3837-
Total RNA from Mint5 hybridomas (DSM ACC2150) was isolated and VH and VL genes were obtained by amplification by means of PCR, using consensus primers. The amplified VH and VL genes were cloned in M13- derived vehicles (in M13VHPCR1 vehicle and in M13VKPCR1 vehicle respectively, courteously given by Dr.G. inter) containing the promoter and the leader sequence of the immunoglobulin genes. and then sequenced.
The VH fragment gene was directly cloned in M13VHPCR1 vehicle and sequenced (Sequenase Version 2.0 U.S.B., USA). Instead, the VL gene (then indicated as VK, too) was at first introduced in M13mpl9 vehicle in order to remove the two inside BamHI and PvuII restriction sites, by means of site-directed mutagenesis (Oligonucleotide-Directed In vitro mutagenesis System Version 2. Amersham International pic. Little Chalfont. UK). These restriction sites would have hinder the VK insertion into M13VKPCR1 vehicle and the subcloning in the vehicle of expression for the chimeric light chain (alfa-Lysl7 vehicle). The thus modified VK gene was then cloned in M13VKPCR1 vehicle and at last sequenced.
For the preparation of the chimeric heavy and light chains genes, the VH and VK genes of Mint5 were inserted in alfa-Lys30 and alfa-Lysl7 vehicles, respectively (courteously given by Dr.G.Winter) . Alfa-Lys30 plasmid contains the human Cgammal chain gene and gpt gene (xanthine-guanine phosphoribosyl transferase) as selection marker. pMRSlδ vehicle of expression was therefore prepared. Said vehicle of expression was obtained by cloning Hindlll-BamHI fragment, obtained from M13VHPCR1 vehicle, which contains the promoter, the leader sequence and the Mint5 VH region in alfa-Lys30 vehicle, upstream from the human constant region gene. Alfa-Lysl7 plasmid contains the human CK chain gene and the gene for resistance to hygromicine as selection marker. pMRS17 vehicle of expression was therefore prepared. Said vehicle of expression was obtained by cloning Hindlll-BamHI fragment, obtained from M13VKPCR1 vehicle which contains the promoter, the leader sequence and the Mint5 VK region in alfa-Lysl7 vehicle, upstream from the human constant region gene.
In order to obtain the stable expression of Mint5 antibody, non- secernent-immunoglobulin murine myeloma NSO/1 cells were cotransfected with pMRSlδ and pMRS17 vehicles by means of electroporation (Potter et al.. 1984, PNAS, 81:7l6l-7l65) . The stable transfectans were analyzed and selected according to their mycophenolic acid resistance and hygromicine resistance and according to the chimeric antibody production by means of ELISA.
The transfectomas in NSO cells showed chMint5 productivity of 1-5 ng/10 cells/24 hours. These productivity levels, although they didn't allow the preparatory purification of the chimeric antibody, showed that chMint5 genes are correctly translated by transfected NSO/1 cells which can be then utilized for the isolation of cDNAs of the chMint5 heavy and light chains in order to achieve a better expression. Example 2
Isolation of cDNAs of the chMint5 heavy and light chains and expression thereof in CHO cells. o From 10 cells selected from the mostly productive transfected NSO/1 clones, total RNA was extracted and the genes of the chimeric chains were amplified by reverse transcription followed by PCR, as indicated in the schematic illustration of Figure 1.
The primers utilized in PCR for the amplification of the chMint5 cDNAs are indicated in the following Table.
DNA fragment '-end 3'-end
A leader primer CKhu (SEQ ID N0:7) (SEQ ID N0:8)
B leader primer VH1F0R(1) (SEQ ID NO:7)
C antisensus VH1F0R CKhu (SEQ ID N0:9) (SEQ ID NO:10)
A = cDNA of the light chain
B = cDNA of the fragment of the variable region of the heavy chain C = cDNA of the fragment of the constant region of the heavy chain ' 'VH1F0R primer was described in Orlandi et al., (as above). PCR was performed by using leader primers complementary to the 5'- leader region of the heavy and light chains by inserting a Xbal site. For human heavy and light constant 3' regions, CHhu and CKhu primers respectively were used, primers which introduce a Smal site. The cDNA of the chimeric heavy chain was amplified in two phases (according to the scheme of Figure 1): a) the leader and variable regions were amplified by using a leader primer at the 5'-end and VHIFOR primer at the 3'-end; the constant region was amplified by using, at the 5'-end, antisensus VHIFOR primer (complementary to VHIFOR primer), and CHhu primer at 3'-end; b) products of such amplifications were amplified once again, all together in order to achieve the whole heavy chain.
Two pSV2-derived vehicles, termed pMRSδl and pMRS71 were used to express the genes of the chMint5 heavy and light chains respectively, in CHO cells. pMRSβl was obtained from pMCMVbeta51-2. This vehicle was digested with AccI and Hindlll, thus eliminating the immediate early promoter of murine Cytomegalovirus. At Accl-Hindlll sites the immediate early promoter of human cytomegalovirus was cloned, with Accl-Hindlll end (promoter which was disclosed in Int. Appln. No. W095/H982 filed on October 24. 1994. In this way, pUthCMVbeta vehicle was obtained. In this vehicle, the 2.25 kbp fragment with BamHI-end, which was obtained from pMSG vehicle (Pharmacia) and containing a transcription unit for gpt gene, was cloned. In pMRSδl vehicle the two eukaryotic transcription units were oriented in the same direction. pMRS71 was obtained from pMCMVbeta51-2 vehicle. For such purpose, pMCMVbeta51-2 vehicle was linearized with BamHI and treated with alkaline fosphatase from intestinal calf ucosa (CIP); in the thus treated vehicle, the transcription VIDHFR unit was inserted. In pMRS71 vehicle, the two eukaryotic transcription units are oriented in the same direction. pMCMVbeta51-2 vehicle and the transcription VIDHFR unit are mentioned in Int. Appln. No. W095/H982 filed on October 24, 1994. pMRSδl is, therefore, a plasmid containing the early promoter of human Cytomegalovirus, the polyadenylation signal of the 1-betaglobin of rabbit and the gtp gene as selection marker. cDNA of the chMint5 heavy chain was cloned in Xbal-Smal cloning site as Xbal-blunt fragment owing to the presence of an inside Smal site in the human constant region. The thus obtained vehicle was termed pMRSδ9- cDNA of the chimeric light chain, amplified from transfected NSO/1 cells, was cloned as a Xbal-Smal fragment in pMRS71 plasmid containing the early promoter of murine Cytomegalovirus, the polyadenylation signal of 1-betaglobin of rabbit and the gene of dihydrofolate reductase (dhfr) as amplificable selection marker. The thus obtained vehicle was termed pMRS95-
CHO dhfr" cell line was cotransfected with pMRSδ9 and pMRS95 vehicles (see Figure 2) by lipofection (Feigner P. et al., 19δ7. PNAS, δ4, 7413-7417). The transfectants were selected according to the dhfr+/gpt double resistance phenotype. The single clones were moved to 24-well plates and after one week of growth, the supernatants of culture were tested for production of chMint5 by ELISA. Such transfectant clones secreted amounts of chMint5 from 0 to 100 ng/10° cells/24 hours. After amplification with methotrexate (MTX) (up to 600 nM) , the productivity appeared increased to 1-2 microg/10 cells/24 hours. Example 3 Characterization of chMint5 The Mint5 antibody was purified from supernatants of cultures of a CHO transfectant single clone, by means of affinity chromatography on A Protein-Sepharose. The binding activity of the chimeric antibody was analyzed by means of flow cytometry. A 31 cells of human epider oid carcinoma and human Jurkat T EGF-R negative cells were used in the assay. The results, illustrated in Figure 3. show that chMint5 specifically recognizes the A431 cells (EGF-R+), but it doesn't bind the Jurkat T cells (EGF-R"). The binding affinity constants for murine and chimeric antibodies were determined by means of the titration curves of the monoclonal antibody, obtained by IRMA on A431 target cells. Figures 4a and 4b show a Kaff value (affinity constant) of 6.76X10^ M" for chMint5. whereas Mint5 value was 2.05X10 M~ , thus indicating that chMint5 substantially keeps the binding affinity properties of the murine Mint5-
The monoclonal chMint5 antibody showed to keep the specificity and the binding affinity properties of the murine Mint5 antibody. chMint5 also shows a greater utilization in the human tumor therapy. chMint5 antibody can be used for the preparation of a pharmaceutical composition, useful in human therapy, comprising a pharmaceutically acceptable carrier and/or excipient. chMint5 can be also used in the preparation of immunotoxins or immunocytokines.
Such immunotoxins or immunocytokines can be obtained by means of chemical conjugation or by recombination of genes which correspond to chMint5 or to parts of it and by recombination of genes coding for the toxins or the cytokines which will be in the immune-complex. Further this invention relates to the use of such immunotoxins or immunocytokines for the preparation of pharmaceutical compositions useful in the human antitumor therapy, combined to a pharmaceutical acceptable carrier and/or excipient. chMint5 can be also used in a diagnostic test specific for the EGF-R. Such diagnostic method comprises the chMint5 antibody and a ligand or a conjugate (for example another antibody), eventually labeled or bound to a chromogen, which recognize the variable regions of said chMint5. Preferably, such ligand or conjugate will comprise a sequence complementary to at least one of the sequences indicated in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5. SEQ ID NO:6 or to fragments thereof. This invention hence relates to the use of chMint5 in diagnostic test,too, as described above.
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: MINISTERO UNIVERSITA' RICERCA SCIENTIFICA
E TECNOLOGICA (E STREET: Piazzale Kennedy, 20 (C) CITY: Rome EUR
(E) COUNTRY: ITALY
(F) POSTAL CODE (ZIP): 00144
(ii) TITLE OF THE INVENTION: Murine/human chimeric monoclonal antibody or a fragment thereof specific for the EGF receptor
(iii) NUMBER OF SEQUENCE: 10
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatibile
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Versione #1.25 (EPO)
(v) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: IT FI 95 A 000036
(B) FILING DATE: Ol-MAR-1995
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 360 base pairs (B) TYPE: nucleic acid
(C) CHAIN: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..360
( ix) FEATURE:
(A) NAME/KEY: CDR1
(B) LOCATION: 91..105 ( ix ) FEATURE :
(A) NAME/KEY: CDR2
(B) LOCATION: 150..198
(ix) FEATURE:
(A) NAME/KEY: CDR3
(B) LOCATION: 295..327
(ix) FEATURE:
(B) LOCATION: 1..24
(D) OTHER INFORMATION: note= "This sequence was imposed by the primer used in the cDNA amplification by means of PCR"
(ix) FEATURE:
(B) LOCATION: 328..360
(D) OTHER INFORMATION; note= "This sequence was imposed by the primer used in the cDNA amplification by means of PCR"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CAG GTC CAA CTG CAG CAG TCT GGA GGA GCC TTA GTG CAG CCT GGA GGG Gin Val Gin Leu Gin Gin Ser Gly Gly Ala Leu Val Gin Pro Gly Gly 1 5 10 15
TCC CTG AAA CTC TCC TGT GCA ACC TCT GGA TTC ACT TTC AGT GAC TAT Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30
TAC ATG TAT TGG GTT CGC CAG ACT CCA GAG AAG AGG CTG GAG TGG GTC 1 Tyr Met Tvr Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45
GCA TAC ATT AGT AAT GGT GGT GGT AGC ACC TAT TAT CCA GAC ACT GTA 1 Ala Tyr lie Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60
AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT GCC AAG AAC ACC CTG TAC 2 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
CTG CAA ATG AGC CGT CTG AAG TCT GAG GAC ACA GCC ATG TAT TAC TGT 2 Leu Gin Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
GCA AAC TCT CTC TAC TTT GAT TTC GAC GAT CTC TCT TAC TGG GGC CAA 3 Ala Asn Ser Leu Tyr Phe Asp Phe Asp Asp Leu Ser Tyr Trp Gly Gin 100 105 110
SUBSTITUTE SHEET (RULE 26J GGG ACC ACG GTC ACC GTC TCC TCA 36
Gly Thr Thr Val Thr Val Ser Ser
115 120
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gin Val Gin Leu Gin Gin Ser Gly Gly Ala Leu Val Gin Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30
Tyr Met Tyr Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45
Ala Tyr lie Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Asn Ser Leu Tyr Phe Asp Phe Asp Asp Leu Ser Tyr Trp Gly Gin 100 105 110
Gly Thr Thr Val Thr Val Ser Ser 115 120
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(C) CHAIN: singlE
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
( ix) FEATURE: Xaa rapresents an amino acid not exactly characterized
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Glu Val Lys Leu Val Glu Ser Gly Gly Ala Leu Val Glu Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Xaa Ala Thr Ser Gly Phe Thr Phe Ser Xaa Tyr 20 25 30
Tyr Met Xaa Xaa Val 35
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 base pairs (B) TYPE: nucleic acid
(C) CHAIN: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..324
(ix) FEATURE:
(A) NAME/KEY: CDR1
(B) LOCATION: 70..102
(ix) FEATURE:
(A) NAME/KEY: CDR2
(B) LOCATION: 148..168
(D) OTHER INFORMATION: CDR2
(ix) FEATURE:
(A) NAME/KEY: CDR3
(B) LOCATION: 265..291 (ix) FEATURE:
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /note= "This sequence was imposed by the primer used in the cDNA amplification by means of PCR"
(ix) FEATURE:
(B) LOCATION: 301..324
(D) OTHER INFORMATION: /note= "This sequence was imposed by the primer used in the cDNA amplification by means of PCR"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GAC ATC CAG CTG ACC CAG TCT CCA GCC ACC CTG TCT GTG ACT CCA GGA 4 Asp lie Gin Leu Thr Gin Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15
GAT AGC GTC AGT CTT TCC TGT AGG GCC AGC CAA AGT ATT AGC AAC AGC 9 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gin Ser lie Ser Asn Ser 20 25 30
CTA CAC TGG TAT CAA CAA AAA TCA CAT GAG TCT CCA AGG CTT CTC ATC 14 Leu His Trp Tyr Gin Gin Lys Ser His Glu Ser Pro Arg Leu Leu lie 35 40 45
AAG TAT GTT TCC CAG TCC ATC TCT GGG ATC CCC TCC AGG TTC AGT GGC 192 Lys Tyr Val Ser Gin Ser lie Ser Gly lie Pro Ser Arg Phe Ser Gly 50 55 60
AGT GGA TCA GGG ACA GAT TTC ACT CTC ACT ATC AAC AGT GTG GAG ACT 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Asn Ser Val Glu Thr 65 70 75 80
AAA GAT TTT GGA ATG TAT TTC TGT CAA CAG AGT GAC AGT TGG CAG TGG 288 Lys Asp Phe Gly Met Tyr Phe Cys Gin Gin Ser Asp Ser Trp Gin Trp
85 90 95
ACG TTC GGT GGA GGG ACC AAG CTG GAG ATC AAA CGT 324
Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg 100 105
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Asp lie Gin Leu Thr Gin Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15
Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gin Ser lie Ser Asn Ser 20 25 30
Leu His Trp Tyr Gin Gin Lys Ser His Glu Ser Pro Arg Leu Leu lie 35 40 45
Lys Tyr Val Ser Gin Ser lie Ser Gly lie Pro Ser Arg Phe Ser Gly 50 55 60
Ser Glv Ser Gly Thr Asp Phe Thr Leu Thr lie Asn Ser Val Glu Thr 65 70 75 80
Lys Asp Phe Gly Met Tyr Phe Cys Gin Gin Ser Asp Ser Trp Gin Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg 100 105
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) CHAIN: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ANTI-SENSE: NO iv ) FRAGMENT TYPE : N-terminal
(ix) FEATURE: Xaa rapresents an amino acid not exactly characterized
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Asp lie Val Leu Thr Gin Ser Xaa Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
ASD Ser Val Ser Leu Xaa Xaa Arg Ala Ser Gin Ser 20 25 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) CHAIN: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ANTI-SENSE: NO
(ix) FEATURE: 5' primer 5' specific for the leader sequence of light ang heavy chains for use in PCR
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CACAGGTCTA GACCATGGGA TGGAGCTGTA TC 32
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) CHAIN: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ANTI-SENSE: NO
(i ) FEATURE: CKhu primer specific for the constant region of the light chain for use in PCR
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TTAACTCCCG GGTTAACACT CTCCCCTGTT GAAGCTCTT 38
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) CHAIN: singol
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) ANTI-SENSE: YES (ix) FEATURE: VHIFOR anti-sense primer specific for the sequence of a fragment of the constant region of the heavy chain for use in PCR
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CTGGGGCCAA GGGACCACGG TCACCGTCTC CTCA
(2) INFORMATION FOR SEQ ID NO: 10:
(i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) CHAIN: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ANTI-SENSE: NO
( i ) FEATURE: CHhu primer specific for the sequence at the
3' end of a fragment of the constant regiion of the heavy region for use in PCR
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
TTAACTCCCG GGTTATCCCG GAGACAGGGA GAGGCT

Claims

Claims 1 . Murine/human chimeric monoclonal antibody, termed chMint5. or a fragment thereof specific for the EGF receptor (EGF-R) . 2. Monoclonal chMint5 antibody or a fragment thereof , according to claim 1 , characterized in that it comprises the variable regions of the heavy ( VH ) and light ( VL ) chains of the monoclonal antibody produced by DSM ACC2150 hybridoma. 3 - Monoclonal chMint5 antibody or a fragment thereof , according to claim 1 , characterized in that it comprises the variable regions of the heavy ( VH ) and light ( VL ) chains of the monoclonal antibody produced by DSM ACC2150 hybridoma and the constant regions of the heavy and light chains of a human immunoglobulin or a fragment thereof . 4. Monoclonal chMint5 antibody or a fragment thereof, according to claim 3. comprising Cgammal ard CK regions of human immunoglobulins or fragments thereof. 5- Monoclonal chMint5 antibody or a fragment thereof, according to claims 2-4, comprising the amino acid sequences indicated in SEQ ID NO:2 and SEQ ID NO:5 sequences. 6. Nucleotide sequences, indicated in SEQ ID N0:1 and SEQ ID NO:4, coding for the variable regions of the heavy (VH) and light (VL) chains of the chMint5 antibody described in claims 1-5. 7. Amino acid sequences indicated in SEQ ID NO:2 and SEQ ID NO:5. δ. Amino acid sequence indicated in SEQ ID N0:3- 9- Amino acid sequence indicated in SEQ ID NO:6. 10. Diagnostic method specific for the determination of the EGF-R, comprising the chMint5 antibody , according to claims 1-5 and a ligand or a conjugate, optionally labeled or bound to a chromogen, which recognize the variable regions of said chMint5- 11. Diagnostic method, according to claim 10, wherein said ligand or conjugate comprises a sequence complementary to at least one of the sequences indicated in SEQ ID N0:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 or to fragments thereof. 12. Use of the chMint5 antibody, according to claims 1-5 in a diagnostic method according to claims 10-11. 13. Use of the chMint5 antibody, according to claims 1-5, for the preparation of a pharmaceutical composition, useful in therapy, comprising a pharmaceutically acceptable carrier and/or excipient. 14. Immunotoxin or immunocytokin comprising the chMint5 antibody, according to claims 1-5, and one toxin. 15. Immunotoxin or immunocytokin, according to claim 14, obtained by means of chemical conjugation or by recombination of genes which correspond to chMint5 or to parts of it and by recombination of genes coding for a toxin. 16. Use of the immunotoxin or immunocytokin, according to claims 14- 15, for the preparation of pharmaceutical compositions useful in the antitumor therapy, combined to a pharmaceutically acceptable carrier and/or excipient.
PCT/EP1996/000805 1995-03-01 1996-03-01 Murine/human chimeric monoclonal antibody or a fragment thereof specific for the egf receptor WO1996027010A1 (en)

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IT95FI000036A IT1277827B1 (en) 1995-03-01 1995-03-01 CHIMERIC MURINE/HUMAN MONOCLONAL ANTIBODY OR ITS FRAGMENT SPECIFIC FOR THE EGF RECEPTOR (EGF-R)
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US8784816B2 (en) 1999-07-14 2014-07-22 Life Sciences Research Partners Vzw Antibodies which bind factor VIII
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WO2016062851A1 (en) 2014-10-23 2016-04-28 Innate Pharma Treatment of cancers using anti-nkg2a agents
EP3659625A1 (en) 2014-10-23 2020-06-03 Innate Pharma Treatment of cancers using anti-nkg2a agents
WO2020136147A1 (en) 2018-12-26 2020-07-02 Innate Pharma Compounds and methods for treatment of head and neck cancer
CN111647564A (en) * 2020-05-18 2020-09-11 李欣 Monoclonal antibody of anti-EB virus LMP1, cell strain and application thereof
CN111647564B (en) * 2020-05-18 2023-07-04 李欣 Anti-Epstein-Barr virus LMP1 monoclonal antibody and its cell line and application

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AU4719196A (en) 1996-09-18
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IT1277827B1 (en) 1997-11-12

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