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Tumor-associated antigen derivatives from MAGE family, and nucleic acid sequence encoding them

A tumor-related, nucleic acid sequence technology, used in tumor-specific antigens, tumor rejection antigen precursors, anti-tumor drugs, etc., can solve problems such as inability to detect antigens

Inactive Publication Date: 2005-11-16
GLAXOSMITHKLINE BIOLOGICALS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] On melanoma cells (including malignant melanoma) and some other cancer cells including NSCLC (non-small cell lung cancer), head and neck squamous cell carcinoma, bladder transitional cell carcinoma and esophageal cancer, the MAGE gene can be significantly expressed Family-encoded antigens, however, are not detectable on normal tissues other than testis and placenta (Gaugler, 1994; Weynants, 1994; Patard, 1995)

Method used

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  • Tumor-associated antigen derivatives from MAGE family, and nucleic acid sequence encoding them
  • Tumor-associated antigen derivatives from MAGE family, and nucleic acid sequence encoding them
  • Tumor-associated antigen derivatives from MAGE family, and nucleic acid sequence encoding them

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0087] A recombinant Escherichia coli strain expressing the fusion protein lipoprotein D-MAGE-3-His (LPD 1 / 3-MAGE-3-His or LpD MAGE-3-His) was prepared.

[0088] 1. Escherichia coli expression system:

[0089] To produce lipoprotein D, DNA encoding protein D has been cloned into the expression vector pMG81. This plasmid utilizes signals from λ-phage DNA to drive the transcription and translation of the inserted foreign gene. This vector contains the λPL promoter PL, the operator OL and two utilization sites (NutL and NutR) to depolarize transcription when N protein is provided (Gross et al., 1985, Molecular Cell Biology, 5:1015) . A vector containing the PL promoter was introduced into an E. coli lysogenic host in order to stabilize the plasmid DNA. Lysogenic host strains contain replication-defective lambda phage DNA integrated into the genome (Shatzman et al., 1983, in "Experimental Manipulation of Gene Expression", Inouya (Ed.), PP 1-14, Academic Press, NY). The λ-phage...

Embodiment II

[0107] Preparation of LPD 1 / 3-MAGE-3-His antigen:

[0108] 1. Cultivation and induction of strains - expression of LPD 1 / 3-MAGE-3-His:

[0109] AR58 cells transformed with plasmid pRIT 14477 were cultured in 2 liter shake flasks, each containing 400ml of LY 12 medium supplemented with yeast extract (6.4g / L) and kanamycin sulfate (50mg / L) . After incubation for 8±1 hours at 30°C on a shaker, a small sample was removed from each shaker flask for microscopic examination. The contents of the two shake flasks were combined to provide the inoculum to a 20 liter fermenter.

[0110] This inoculum (approximately 800 ml) was added to a pre-sterilized 20 liter (total volume) fermenter containing 7 liters of medium supplemented with 50 mg / L kanamycin sulfate. By timing the addition of NH 4 OH (25% v / v) calibrated and maintained pH at 6.8, and calibrated and maintained temperature at 30°C. The aeration rate was calibrated and maintained at 12 L / min using agitation rate feedback contro...

Embodiment III

[0119] Identification of fusion protein Lipo D-MAGE 3:

[0120] 1. Purification:

[0121] LPD-MAGE-3-His was purified from cell homogenate using the following sequence of steps:

[0122] a) - solubilization of the washed pellet fractions from the cell lysate,

[0123] b) - Chemical reduction of intramolecular and intermolecular disulfide bonds in proteins, with subsequent blocking of sulfhydryl groups to prevent oxidative recoupling,

[0124] c) - microfiltration of the reaction mixture in order to remove particulates and reduce endotoxins,

[0125] d) - Capture and preliminary purification of LPD-MAGE-3-His using the affinity interaction between polyhistidine tails and zinc-loaded chelating agarose,

[0126] e) - Removal of contaminating proteins by anion exchange chromatography.

[0127] This purified LPD-MAGE-3-His was subjected to several polishing steps:

[0128] f) - buffer exchange / urea scavenging by size exclusion chromatography using Superdex 75,

[0129] g) - f...

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Abstract

The present invention relates to novel proteins and to their production, from the MAGE family. In particular, to a MAGE protein fused to an immunological fusion partner, such as Lipoprotein D. Such antigens may be formulated to provide vaccines for the treatment of a range of tumours. Novel methods for purifying MAGE proteins are also provided.

Description

technical field [0001] The present invention relates to protein derivatives containing tumor-associated antigens which have been found to have therapeutic utility as cancer vaccines. Specifically, the derivatives of the present invention include fusion proteins containing an antigen encoded by the MAGE (melanoma antigen-encoding gene) family (eg MAGE-3, MAGE-1) linked to a T Immunological fusion partners for helper epitopes such as the lipidated form of protein D from Haemophilus influenzae B; also include chemically modified MAGE proteins in which the disulfide bonds of the antigen are reduced, the sulfhydryl groups formed are blocked, and also Including genetically modified MAGE proteins provided with an affinity tag and / or genetically modified to prevent disulfide bridge formation. Also described are methods of purifying the MAGE protein and formulating a vaccine for use in the treatment of a range of cancers including, but not limited to, melanoma, breast cancer, bladder ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09A61K38/00A61K38/17A61K39/00A61K39/385A61K39/39A61K48/00A61P35/00C07K1/00C07K1/16C07K14/11C07K14/245C07K14/285C07K14/315C07K14/47C07K19/00C12N1/21C12N15/12C12N15/62C12N15/70
CPCC07K2319/20C07K2319/00A61K2039/55572C07K14/4748A61K2039/55577C07K14/285A61K2039/6068A61K39/0011A61K48/00C07K14/3156C07K14/245A61K38/1709A61K2039/55566A61P35/00A61K39/001186C07K14/00
Inventor T·卡贝宗斯尔瓦J·科亨M·M·斯拉欧伊C·维纳尔斯巴索尔斯
Owner GLAXOSMITHKLINE BIOLOGICALS SA
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