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

a recombinant antibody and ganglioside technology, applied in the direction of fused cells, drug compositions, immunological disorders, etc., can solve the problem that the therapeutic effectiveness of gangliosides has not been demonstrated in humans

Inactive Publication Date: 2004-12-16
CENT DE INMUNOLOGIA MOLECULAR CENT DE INMUNOLO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even when hybridoma technology has been developed during 15 years (Koehler y Milstein (1975): Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256: 495497) and when monoclonal antibodies are still very useful in diagnosis as well as research they have not demonstrated their therapeutic effectiveness in human.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

example 1

Obtaining of Chimeric MAb P3.

[0080] The cDNA synthesis was obtained by a reaction with reverse transcriptase enzyme, starting with RNA from the hybridoma producing Mab P3, as described previously. The sequence of the specific primers used in this reaction is shown:

[0081] For VH:

9 5' AGGTCTAGAA(CT)CTCCACACAC AGG(AG)(AG)CCAGTGGATAGAC 3'

[0082] For VK:

10 5' GCGTCTAGAACTGGATGGTGGGAAGATGG 3'

[0083] cDNA VHP3 and cDNA VKP3 were amplified by PCR using Taq Polymerase and specific primers. The restriction sites included in the primers were ECORV / NHEI, for VH and ECORV / SALI for VK. The primers sequences used were the following:

[0084] For VH:

[0085] Primer 1 (signal peptide):

11 5'GGGGATATCCACCATGG(AG)ATG(CG) AGCTG(TG)GT(CA)AT(CG)CTCTT 3'

[0086] Primer 2 (CH1):

12 5' GGGGCTAGCTGCAGAGACAGTGACCAGAGT 3'

[0087] For VK:

[0088] Primer 1 (signal peptide):

13 5' GGGGATATCCACCATGGAG(TA)CAC A(GT)(TA)CTCAGGTCTTT(GA)T 3'

[0089] Primer 2 (Ck):

14 5' AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT(GT)GTCCC 3'

[0090] PCR products w...

example 2

Obtaining Different Versions of the Humanized Antibody P3.

[0095] Murine VHP3 and VKP3 sequences (FIGS. 1 and 2) were compared with human sequences. FIGS. 3 and 4 show the most homologous human sequences. Helical amphipatic regions or potential T cell epitopes were searched on murine P3 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.

[0096] The analysis on VHP3 rendered (FIG. 3) 2 amphipatic segments, the first one embraces CDR1, FR2 and some residues of the CDR2, the second one embraces the end of FR3 and CDR3. The main differences of murine sequence in comparison with the most homologous human sequence were founded in CDRs or residues involved with the three dimensional structure of the binding site. For that reason it was decided do not replace any aminoacid in murine VHP3.

[0097] The analysis for VKP3 rendered also 2 amphipatic...

example 3

Biological Activity of Chimeric MAb P3.

[0100] The specific binding to antigen measured by ELISA tested the biological activity of the Mab P3 chimeric.

[0101] For recombinant MAb P3, microtiter plates were coated with GM3(NeuGc) ganglioside in methanol. After drying one hour at 37.degree. C., unspecific binding was blockade with bovine sera albumin (BSA) 1% in Tris-HCl buffer, incubated for one hour at 37.degree. C. The wells were washed with PBS and incubated for 1 hour at 37.degree. 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.degree. 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.

[0102] Mab Ti chimeric was used as negative control.

[0103] FIG. 5 shows the specific binding of Mab P3 chimeric to the antigen.

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Abstract

The present invention is related with the obtaining of modified antibodies by means of the DNA recombinant technology from the murine monoclonal antibody P3 (MAb P3) produced by the hybridoma cell line deposited under Budapest Treaty with accession number ECACC 94113026 and from its anti-idiotype murine monoclonal antibody 1E10(MAbai 1E10) produced by the hybridoma cell line with deposit number ECACC 97112901, with the objective of achieving monoclonal antibodies which preserve the biological function of specific binding to the antigen of the original antibodies, but being at the same time less immunogenic. The chimeric antibodies of the invention contain the variable domains of the murine immunoglobulin and the constant regions of the human immunoglobulin; and those humanized, besides containing the constant regions of the human immunoglobulins, they are modified in the region of the murine frameworks (FRs) and in particular in those zones that could be in an antigenic site for the T cells, so several positions of the FRS are human as well. These antibodies can be used in the diagnosis and therapy of different types of tumors. The present invention is also related with use of the antibodies for therapeutical and diagnostic purposes.

Description

[0001] 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).[0002] More specifically, 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. On the other hand, the anti-tumor effect of the MAbai 1E10 has also been demonstrated in experimental models.[0003] 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.PR...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/7032C12N15/09A61K31/739A61K39/00A61K39/395A61P35/00A61P35/04A61P37/00A61P37/04A61P43/00C07K16/00C07K16/18C07K16/30C07K16/42C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12N5/20C12P21/08
CPCA61K31/7032A61K31/739A61K39/0011A61K39/395A61K39/39558A61K39/39566A61K2039/505A61K2039/55505C07K16/00C07K16/18C07K16/30C07K16/3076C07K16/3084C07K16/4241C07K16/4266C07K2316/96C07K2317/24C07K2317/565C07K2317/567A61K2300/00G01N33/577G01N33/57415C12N5/163C12N5/0694G01N2800/7028C07K16/4208G01N2033/57403G01N33/686C07K2317/76A61P35/00A61P35/04A61P37/00A61P37/04A61P43/00A61K39/001171
Inventor DEL RIO, CRISTINA MATEO DE ACOSTAVALLADARES, JOSEFA LOMBARDEROROQUE NAVARRO, LOURDES TATIANALOPEZ REQUENA, ALEJANDRO
Owner CENT DE INMUNOLOGIA MOLECULAR CENT DE INMUNOLO
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