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Anti-gprc5d antibody and molecule containing same

An antibody, human antibody technology, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problem of undeveloped pharmaceutical products and so on

Inactive Publication Date: 2019-11-15
DAIICHI SANKYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no medicinal product targeting GPRC5D has been developed

Method used

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  • Anti-gprc5d antibody and molecule containing same
  • Anti-gprc5d antibody and molecule containing same
  • Anti-gprc5d antibody and molecule containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1132] Example 1. Preparation of rat anti-human GPRC5D antibody

[1133] 1)-1 Immunization using human GPRC5D expression vector

[1134] 1)-1-1 Construction of human GPRC5D expression vector (pcDNA3.1-DEST-hGPRC5D)

[1135] pcDNA3.1-DEST engineered as a destination vector was prepared from pcDNA3.1(+) using Gateway Vector Convention System (Thermo Fisher Scientific Inc.). The cDNA encoding human GPRC5D protein (NP_061124.1) was cloned into the pcDNA3.1-DEST vector using Gateway LR ClonaseEnzyme mixture (Life Technologies Corp.) to construct the human GPRC5D expression vector pcDNA3.1-DEST-hGPRC5D. For large-scale preparation of human GPRC5D expression vector, Endofree Plasmid Giga Kit (Qiagen N.V.) was used.

[1136] 1)-1-2 Rat Immunization

[1137]For immunization, WKY / Izm female rats (Japan SLC, Inc.) were used. First, both calves of each rat were pretreated with hyaluronidase (Sigma-Aldrich Corp.). Then, pcDNA3.1-DEST-hGPRC5D was intramuscularly injected into these sit...

Embodiment 2

[1171] Example 2. In Vitro Evaluation of Rat Anti-GPRC5D Antibodies (2A4, 2B1 and 7B4)

[1172] 2)-1 Binding activity of rat anti-GPRC5D antibodies (2A4, 2B1 and 7B4) obtained by flow cytometry to human GPRC5D

[1173] The human multiple myeloma cell line KHM-1B cells (JCRB Cell Bank) expressing GPRC5D was adjusted to 5 × 10 with PBS containing 5% FBS 6 cells / mL, inoculate 100 μL / well into a 96-well U-bottom microplate, and centrifuge to remove the supernatant. Each of the rat anti-GPRC5D antibodies (2A4, 2B1 and 7B4) adjusted to 0.32 ng / mL to 10 μg / mL in Example 1)-7 was added thereto in an amount of 100 μL / well, and the plate was incubated at 4 ℃ for 1 hour. Cells were washed twice with PBS containing 5% FBS. Then, PE goat anti-rat Ab (Becton, Dickinson and Company) diluted 100-fold with PBS containing 5% FBS was added thereto at a concentration of 100 μL / well, and the plate was left to stand at 4° C. for 1 hour. Cells were washed twice with PBS containing 5% FBS and the...

Embodiment 3

[1186] Example 3. Sequencing of cDNAs encoding the variable regions of rat anti-GPRC5D antibodies (2A4, 2B1 and 7B4)

[1187] 3)-1 Sequencing of cDNA encoding the variable region of 2A4

[1188] 3)-1-1 Preparation of total RNA from hybridoma producing 2A4

[1189] To amplify the cDNA encoding the variable region of 2A4, total RNA was prepared from 2A4-producing hybridomas using TRIzol reagent (Ambion / Thermo Fisher Scientific Inc.).

[1190] 3) Synthesis of -1-2 cDNA (5'-RACE-Ready cDNA)

[1191] cDNA (5'-RACE-Ready cDNA) was synthesized using approximately 1 µg of the total RNA prepared in Example 3)-1-1 and the SMARTer RACE cDNA Amplification Kit (Clontech Laboratories, Inc.).

[1192] 3)-1-3 The cDNA encoding the heavy chain variable region of 2A4 was amplified and sequenced by 5'-RACE PCR

[1193] The primers used for PCR amplification of the cDNA encoding the variable region of the heavy chain gene of 2A4 were UPM (Universal Primer A Mixture; attached to the SMARTer RAC...

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PUM

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Abstract

The present invention provides a novel antibody binding to human GPRC5D and a molecule which contains the antibody and is capable of binding to an antigen. Provided are: a novel antibody binding to human GPRC5D; a molecule which contains the antibody and is capable of binding to an antigen; an antitumor medicinal composition which contains the antibody or the molecule as an active ingredient, etc.

Description

[technical field] [0001] The present invention relates to novel anti-GPRC5D antibodies and molecules comprising said antibodies. [Background technique] [0002] G protein-coupled receptor family C group 5 member D (GPRC5D) is one of the G protein-coupled receptors discovered by homology search of EST database using amino acid sequences of a series of human GPCRs (Non-Patent Document 1). GPCR family C group 5 receptors (GPRC5 receptors) have 4 subtypes (GPRC5A, GPRC5B, GPRC5C, and GPRC5D) and are also known as retinoic acid-induced orphan G protein-coupled receptors (RAIGs) because of their Expression is induced by retinoic acid stimulation (Non-Patent Document 2). However, the biological functions or biological ligands of GPRC5D, the isoforms of G proteins coupled to it, etc. have not been revealed yet. [0003] Regarding the association of the GPRC5D gene with cancer, it is known that GPRC5D is highly expressed in multiple myeloma. Specifically, it is known that, for exa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395A61P35/00A61P35/02C07K16/28C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08
CPCA61P35/00C07K16/28C07K16/46C12N5/10C12N15/09C07K16/2809C07K2317/565C07K2317/24C07K2317/31C07K2317/33C07K2317/76C07K2317/21C07K2317/732A61K2039/505C07K2317/52C07K2317/73C07K2317/54C07K2317/55C07K2317/569C07K2317/622
Inventor 茶圆贵志大塚敏明饭田谦二中村健介油谷隆秀市川淳也工藤翔太奇恩·李·皮斯契特里马里欧·桑奇斯
Owner DAIICHI SANKYO CO LTD
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