US20040253233A1 - Ganglioside-associated recombinant antibodies and the use thereof in the diagnosis and treatment of tumors - Google Patents

Ganglioside-associated recombinant antibodies and the use thereof in the diagnosis and treatment of tumors Download PDF

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US20040253233A1
US20040253233A1 US10/473,977 US47397703A US2004253233A1 US 20040253233 A1 US20040253233 A1 US 20040253233A1 US 47397703 A US47397703 A US 47397703A US 2004253233 A1 US2004253233 A1 US 2004253233A1
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monoclonal antibody
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Cristina Del Rio
Josefa Valladares
Lourdes Roque Navarro
Alejandro Lopez Requena
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Centro de Immunologia Molecular
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Assigned to CENTRO DE IMMUNOLOGIA MOLECULAR reassignment CENTRO DE IMMUNOLOGIA MOLECULAR ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LOMBARDERO VALLADARES, JOSEFA, LOPEZ REQUENA, ALEJANDRO, MATEO DE ACOSTA DEL RIO, CRISTINA, ROQUE NAVARRO, LOURDES TATIANA
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    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
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Definitions

  • the present invention is related to the biotechnology field, in particular with new recombinant antibodies obtained by genetic engineering, specifically with chimeric and humanized antibodies obtained from the murine monoclonal antibody P3 (MAb P3) and its anti-idiotype murine monoclonal antibody 1E10 (MAbai 1E10).
  • the invention is related with antibodies that bind to gangliosides containing N-glycolylated sialic acid, but not with the acetylated forms of the gangliosides neither with neuter glycolipids.
  • Gangliosides containing N-glycolylated sialic acid are antigens widely expressed in breast cancer and melanomas.
  • the anti-tumor effect of the MAbai 1E10 has also been demonstrated in experimental models.
  • the present invention is also related with the pharmaceutical compositions that contain the previously described recombinant antibodies useful in the diagnosis and therapy of cancer, particularly breast cancer and melanomas.
  • Gangliosides are glycosphingolipids that contain sialic acid and they are present in the plasmatic membrane of cells of vertebrates (Stults et al. (1989): Glycosphingolipids: structure, biological source and properties, Methods Enzymology, 179:167-214). Some of these molecules have been reported in the literature as antigens associated to tumors or tumor markers (Hakomori et al. (1991): Possible functions of tumor associated carbohydrate antigens, Curr. Opin. Immunol., 3: 646-653), for that reason the use of anti-gangliosides antibodies has been described as useful in the diagnosis and therapy of cancer (Hougton et al.
  • Sialic acids more frequently expressed in animals are N-acetyl (NeuAc) and N-glycolyl (NeuGc) (Corfield et al. (1982): Occurrence of sialic acids, Cell. Biol. Monogr., 10: 5-50).
  • NeuGc is not expressed in normal human and chickens tissues in general, but it is broadly distributed in other vertebrates (Leeden and Yu, (1976): Chemistry and analysis of sialic acid. In: Biological Role of Sialic Acid. Rosemberg A and Shengtrund C L (Eds). Plenum Press, New York, 1-48; Kawai et al.
  • the monoclonal antibody (Mab) P3 produced by the cell line deposited with accession number ECACC 94113026 (European Patent EP 0 657 471 B1), it is a murine monoclonal antibody with IgM isotype, that was obtained when fusing murine splenocytes from a BALB/c mouse immunized with liposomes containing GM3(NeuGc) and tetanic toxoid, with the cell line P3-X63-Ag8.653; which is a murine myeloma.
  • This Mab P3 reacts strongly with gangliosides containing N-glycolylated sialic acid but not with the acetylated forms of the gangliosides neither with the neuter glycolipids. It was demonstrated by immunocytochemical and immunohistochemical studies carried out with cell lines and tissues from benign and neoplasic tumors that the Mab P3 recognizes breast cancer (Vázquez et al. (1995): Generation of a murine monoclonal antibody specific for N-glycolylneuraminic acid-containing gangliosides that also recognizes sulfated glycolipids, Hybridoma, 14: 551-556) and melanoma.
  • the anti-idiotypic Mab 1E10 (Mabai 1E10) of subtype IgG1, was obtained from a mouse BALB/c immunized with the Mab P3 coupled to KLH (U.S. Pat. No. 6,063,379, cell line deposited under accession number ECACC 97112901).
  • Mabai 1E10 recognized specifically the MAb P3 and it did not bind other IgM anti-ganglioside antibodies.
  • the MAbai 1E10 induced a strong immune response of Ab3 antibodies when mice from syngeneic or alogenic models were immunized, these Ab3 antibodies didn't exhibit the same specificity as the Mab P3 eve when they carry idiotopes similar to those carried by the Ab1 antibody (Vázquez et al. (1998): Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody, Hybridoma, 17: 527-534).
  • MAbai 1E10 induced a strong antitumor effect in syngeneic as well as alogenic mice.
  • the growth of the mammary carcinoma cell line F311 was significantly reduced by the repeated dose of the MAbai 1E10 coupled KLH in Freund's adjuvant, when BALB/c mice were vaccinated. Also the number of spontaneous lung metastasis was reduced after the vaccination.
  • the method comprises judiciously replacement of few amino acid residues, located in the potential immunogenic epitopes by the corresponding residues from the most homologous human sequence, the amino acids that are mainly responsible for canonical structures and also the residues in the immediate neighbourhood of the CDRs or into the Vernier zone, must be retained.
  • the present invention is related to recombinant antibodies, obtained by genetic engineering technology. Specifically, the invention is related with a chimeric antibody derived from the murine monoclonal antibody P3, produced by hybridoma cell line with deposit number ECACC 94113026.
  • MAB P3 recognizes an antigen expressed in breast tumor cells and melanomas.
  • the MAb P3 is characterized by the following sequences of the hypervariable regions (CDRs) of the heavy and light chains:
  • Heavy Chain CDR1 RYSVH
  • CDR2 MIWGGGSTDYNSALKS
  • CDR3 SGVREGRAQAWFAY
  • Cadena Ligera CDR1 KASQDVSTAVA
  • CDR2 SASYRYT
  • CDR3 QQHYSTPWT
  • the FRs sequences of the heavy and light chain are the following:
  • Heavy Chain FR1 QVQLKESGPGLVAPSQSLSITCTVSGFSLS
  • FR2 WVRQPPGKGLEWLG
  • FR3 RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR
  • FR4 WGQGTLV
  • the chimeric antibody of the present invention contains the constant region of heavy chain human IgG1 and the constant region of light chain human Ck.
  • the present invention is related with a humanized antibody derived from the Mab P3 produced by the hybridoma cell line with deposit number ECACC 94113026, characterized because it contains the constant region of human heavy chain IgG1 and the constant region of human light chain human Ck and FRs regions of the light chain contains any of the following point mutations:
  • the invention is related with a chimeric antibody derived from the murine monoclonal antibody 1E10 produced by the hybridoma cell line with deposit number ECACC 97112901, and it is an antidiotype antibody, which recognizes the MAb P3.
  • the MAbai 1E10 is characterized by the following sequences of the hypervariable regions (CDRs) of the heavy and light chains:
  • Heavy Chain FR1 QVQLQQSGAELVKPGASVKLSCKASGYTFT FR2: WVRQRPEQGLEWIG FR3: KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR FR4: WGQGTTLTV
  • the chimeric antibody of the present invention contains the constant region of heavy chain human IgG1 and the constant region of light chain human Ck.
  • Cytoplasmic RNA was extracted from about 10 6 hybridoma cells of P3 (murine IgM MAb, that recognizes to the GM3 N-glycolylated ganglioside) or of 1E10 (antidiotype anti-P3 antibody). The RNA was extracted by using the reagent TRIZOL (GIBCO BRL, Grand Island, N.Y.), according to the manufacturer's instructions.
  • variable regions VK and VH cDNAs were amplified by PCR. Shortly, 5 ⁇ l cADN of VH or VK were mixed with 25 pmoles of specific primers, 2.5 mM of each dNTP, 5 ⁇ l constituents of 10 ⁇ buffer Taq DNA polymerase and 1 unit of this enzyme. The samples were subjected to 25 thermal cycles at 94° C., 30 sec; 50° C., 30 sec; 72° C., 1 min, and a last incubation for 5 minutes at 72° C.
  • VH VK genes were excised from TA vectors by enzymatic digestion and they were cloned into the respective expression vectors (Coloma et al. (1992): Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction, J. Immunol. Meth., 152: 89-104).
  • the VH genes were excised from the TA vector by enzymatic digestion with EcoRV and NheI and cloned in an expression vector (PAH 4604) that has included a variable region human IgG1 and the histidinol resistance gene.
  • the resultant constructions were P3VH-PAH4604 and 1E10VH-PAH4604.
  • the VK genes were excised from TA vector by enzymatic digestion with EcoRV and SalI and cloned in an expression vector (PAG4622). This vector has included mycophenolic acid resistance gene and the human kappa constant region.
  • the resultant constructions were P3VK-PAG4622 and 1E10VK-PAG4622.
  • the DNAs were linearized by digestion with Pvul enzyme, precipitated with ethanol and dissolved in 50 ⁇ l of PBS. Approximately 10 7 cells were harvested by centrifugation and resupended in 0.5 ml of PBS together with the digested DNA in an electroporation cuvette. After 10 minutes on ice, the cells were given a pulse of 200 volts and 960 ⁇ F and left in ice for a further 10 minutes. The cells were distributed into 96 wells plate with D'MEM F12 plus 10% fetal calf serum. Two or four days later, it is added selective medium (D'MEM F12 with mycophenolic acid 0,45 ⁇ g/mL or histidinol 10 mM, respectively). Transfected clones were visible with the naked eyes 14 days later.
  • the wells were washed with PBS-T and peroxidase of spicy radish-conjugated goat anti-human kappa light chain or alkaline phosphatase-conjugated goat anti-human IgG (gamma chain specific), were added and incubated at 37° C. one hour.
  • the wells were washed with PBS-T and substrate buffer containing o-phenylendiamine or p-nitrophenylphosphate, respectively, was added. After half hour, absorbance at 492 or 405 nm respectively, was measured.
  • the aim of the method is to reduce immunogenicity breaking or humanizing potential immunogenic T epitopes, with a minimum of changes.
  • the method comprises judiciously replacement of few amino acid residues, located into helical amphipatic segments.
  • the amino acids which are mainly responsible for canonical structures and also the residues in the immediate neighbourhood of the CDRs or into Vernier zone, must be retained.
  • murine variable region sequences were compared with the most homologous human sequence and different aminoacid residues at each position between the murine MAb and the most homologous human sequence were identified, only residues into FRs were taken into account (Kabat (1991), Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health), the previously defined residues were replaced by those residues present in the most homologous human sequence. Replacements were made by directed mutagenesis techniques.
  • the recombinant antibodies were purified by affinity chromatography using protein A (Pharmacia, Upssala, Sweden).
  • microtiter plates were coated with GM3(NeuGc) ganglioside in methanol. After drying one hour, unspecific binding was blockade with bovine sera albumin (BSA) 1% in Tris-HCl buffer, incubated for one hour at 37° C. The wells were washed with PBS and incubated for 1 hour at 37° C. with purified recombinant Mab P3. The wells were washed with tris-HCl and a goat anti-human antibody conjugated with alkaline phosphatase was added and incubated at 37° C. for one hour. Finally, the wells were washed and the substrate buffer containing p-nitrophenylphosphate was added. After half hour absorbance at 405 or 492 nm respectively, was measured.
  • BSA bovine sera albumin
  • VH 5′ AGGTCTAGAA(CT)CTCCACACAC AGG(AG)(AG)CCAGTGGATAGAC 3′
  • Primer 1 (signal peptide): 5′GGGGATATCCACCATGG(AG)ATG(CG) AGCTG(TG)GT(CA)AT(CG)CTCTT 3′
  • VKP3 rendered also 2 amphipatic segments (FIG. 4), the first segment embraces FR1, the second one embraces CDR2 and some residues of the FR3. It was decide to replace residues at positions 8,9,10,11 and 13 by residues at the same position in the most homologous human sequence.
  • the amino acids aminoacidos His, Lys, Phe, Met and Thr were replaced by Pro, Ser, Ser, Leu, and Ala, respectively.
  • the replacements were made by PCR overlapping (Kammann et al.
  • the point mutations were verified by sequencing.
  • the resultant construct was P3Vkhu and it was cloned in PAG 4622 expression vector.
  • the resultant construct was P3VKhu-PAG4622.
  • NS-0 cells were transfected with P3VH-PAH4604 and P3VKhu-PAG4622 P3hu antibody was transfected following the same procedure of electroporation and detection described previously for the chimeric antibodies.
  • microtiter plates were coated with GM3(NeuGc) ganglioside in methanol. After drying one hour at 37° C., unspecific binding was blockade with bovine sera albumin (BSA) 1% in Tris-HCl buffer, incubated for one hour at 37° C. The wells were washed with PBS and incubated for 1 hour at 37° C. with purified recombinant Mab P3. The wells were washed with tris-HCl and a goat anti-human antibody conjugated with alkaline phosphatase was added and incubated at 37° C. for one hour. Finally, the wells were washed with Tris-HCl and the substrate buffer containing p-nitrophenylphosphate was added. After half hour absorbance at 405 nm, was measured.
  • BSA bovine sera albumin
  • Mab Ti chimeric was used as negative control.
  • FIG. 5 shows the specific binding of Mab P3 chimeric to the antigen.
  • cDNA synthesis was obtained by a reaction with reverse transcriptase enzyme, starting with RNA from the hybridoma producing Mab 1E10, as described previously.
  • the sequence of the specific primers used in this reaction is shown following:
  • VK 5′GCGTCTAGAACTGGATGGTGGGAAGATGGA 3′
  • cDNA VH1E10 and cDNA VK1E10 were amplified by PCR using Taq Pol and specific primers.
  • Primer 1 (signal peptide): 5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG (TG)GT(CA)AT(CG)CTCTT 3′
  • Primer 2 (CH1): 5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′
  • VK For VK: Primer 1 (signal peptide): 5′GGGGTTAACCACCATGAGG(GT)CCCC(AT)GC TCAG(CT)T(CT)CT(TG)GG(GA)3′ Primer 2 (Ck): 5′AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)C TT(GT)GTCCC3′
  • PCR products were cloned into TA vector (TA cloning kit, Invitrogen). Twelve independent clones were sequenced (FIGS. 7 and 8) by the dideoxy method using T7 DNA Pol (Pharmacia). By homology search analysis it was determined the most homologous sequence group for VHIE10 and VK1E10. VH1E10 and VK1E10 sequences have high homology with groups miscellaneous and V respectively according to Kabat's classification.
  • NS-0 cells were transfected with 10 ⁇ g of 1E10VK-PAG4622, a clone expressing light chain was transfected with 10 ⁇ g 1E10VH-PAH4604, in both cases DNA is linearized with Pvul, ethanol precipitated and dissolved in 50 ⁇ l of PBS before transfection.
  • the wells were washed with PBS-T and peroxidase of spicy radish-conjugated goat anti-human kappa light chain or alkaline phosphatase-conjugated goat anti-human IgG (gamma chain specific), were added and incubated at room temperature one hour.
  • the wells were washed with PBS-T and substrate buffer containing o-phenylendiamine or p-nitrophenylphosphate, respectively, was added. After half hour absorbance at 492 or 405 nm respectively, was measured.
  • Murine VH1E10 VK1E10 sequences (FIGS. 6 and 7) were compared with human sequences, FIGS. 8 and 9 shown the most homologous human sequences.
  • Helical amphipatic regions or potential T cell epitopes were searched on murine 1E10 variable region sequences and according with the method a judiciously strategy for aminoacid replacements was established in order to break or humanize potential T cell epitopes into the murine sequences
  • Primers for mutation at position 5 of the heavy chain were 1 and 2 and 3 and 4 whose sequences are the following: Primer 1: 5′ CAGGTTCAGCTGGTGCAGTCTGGAGCT 3′ Primer 2: 5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′ Primer 3: 5′ AGCTCCAGACTGCACCAGCTGAACCTG 3′ Primer 4: 5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG (TG)GT(CA)AT(CG)CTCTT 3′
  • Primer for mutations at positions 40 and 42 of the heavy chain Primer 1: 5′ TGGGTGAGGCAGGCGCCTGGGCAGGGACTTGAG 3′
  • Primer 2 5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′
  • Primer 3 5′ CTCAAGTCCCTGCCCAGGCGCCTGCCTCACCCA 3′
  • Primer 4 5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG) GT(CA)AT(CG)CTCTT 3′
  • Primer for mutations at position 87 (83 according to Kabat's numbering) of the heavy chain Primer 1: 5′ CTCAGCAGGCTGCGGTCTGAGGACTCT 3′
  • Primer 2 5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′
  • Primer 3 5′ AGAGTCCTCAGACCGCAGCCTGCTGAG 3′
  • Primer 4 5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG (TG)GT(CA)AT(CG)CTCTT 3′
  • the point mutations were verified by sequencing.
  • the resultant construct was 1E10VHhu and it was cloned in PAH4604 expression vector.
  • the resultant construct was 1E10 VH-PAH4604.
  • VKlEl0 rendered also 3 amphipatic segments (FIG. 9), the first segment embraces FR1, the second one embraces CDR1 and the thirst one embraces FR3. It was decide to replace residues at positions 7,8 and 15 by residues at the same position in the most homologous human sequence.
  • the amino acids Thr, Thr and Leu were replaced by Ser, Pro and Val, respectively.
  • the replacements were made by PCR overlapping (Kammann et al. (1989) Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR), Nucleic Acids Res., 17: 5404) using primers 1 and 2 and 3 and 4 whose sequences are the following:
  • the point mutations were verified by sequencing.
  • the resultant construct was 1 El OVkhu and it was cloned in PAG 4622 expression vector.
  • the resultant construct was 1E10 VKhu-PAG4622.
  • NS-0 cells were transfected with 1E10 VHhu-PAH4604 and 1 El OVKhu-PAG4622
  • FIG. 10 shows the specific binding of Mab 1E10 chimeric to Mab P3.
  • FIG. 2 VKP3 DNA and deduced amino acid sequences. Sequences are aligned according Kabat's numbering (Kabat and collaborators (1991), Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health), CDRs appeared marked with dotted lines.
  • FIG. 3 VHP3 was aligned with the most homologous human sequence. Amphipatic segments are underlined and CDRs in bold.
  • FIG. 4 VKP3 was aligned with the most homologous human sequence. Amphipatic segments are underlined and CDRs in bold.
  • FIG. 5 Specific binding to GM3(NeuGc) by chimeric Mab P3. Different concentrations of Mab P3 and MAb TI (negative control) were tested by ELISA. Microtiter plates were coated with GM3(NeuGc) and GM3(NeuAc) (negative control) ganglioside in methanol and specific binding was measured.
  • FIG. 6 VH1E10 DNA and deduced amino acid sequences. Sequences are aligned according Kabat's numbering (Kabat and collaborators (1991), Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health), CDRs appeared marked with dotted lines.
  • FIG. 7 VK1E10 DNA and deduced amino acid sequences. Sequences are aligned according Kabat's numbering (Kabat et al. (1991), Sequences of proteins of immunological interest, Fifth Edition, National Institute of Health), CDRs appeared marked with dotted lines.
  • FIG. 9 VK1E10 was aligned with the most homologous human sequence. Amphipatic segments are underlined and CDRs in bold.
  • FIG. 10 Specific binding murine Mab P3 by chimeric Mab 1E10. Different concentrations of Mab 1E10 and MAb C5 (negative control) were tested by ELISA. Microtiter plates were coated with Mab P3 and Mab A3 (negative control) and specific binding was measured.

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