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Novel cldn18.2-binding molecules

A technology for combining fragments and nucleic acid molecules, applied in the field of antibodies

Active Publication Date: 2022-06-28
SANYOU BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although there are currently clinically researched monoclonal antibody drugs targeting CLDN18.2, as therapeutic agents, there is still an urgent need to continue to develop antibodies targeting CLDN18.2

Method used

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  • Novel cldn18.2-binding molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0254] Construction and identification of overexpressed cell lines and tumor cell lines

[0255] In this example, different types of overexpression cell lines and tumor cell lines were constructed and identified by flow cytometry.

[0256] 1. Construction and identification of overexpression cell lines

[0257] The nucleic acid sequences of full-length human CLDN18.1 (SEQ ID NO: 16), murine CLDN18.2 (SEQ ID NO: 17), murine CLDN18.1 (SEQ ID NO: 18) were constructed into the pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electroporated into HEK293 cells ( CRL-1573 TM )middle. Through screening, an overexpression cell line expressing full-length human CLDN18.1 (human CLDN18.1-HEK293), an overexpression cell line expressing murine CLDN18.2 (murine CLDN18.2-HEK293) and a cell line expressing murine CLDN18.1 were obtained. Overexpressing cell line (murine CLDN18.1-HEK293). Afterwards, through the IMAB362 antibody (expressed and purified according to...

Embodiment 2

[0278] Animal immunity and serum immune titer detection

[0279] 1. Immunization

[0280] In this example, alpaca immunization was used. The specific operations are as follows: the immunogen uses the cell line human CLDN18.2-HEK293T (Kangyuan Borchuang, KC-0986) and hCLDN18.2-pLVX-puro plasmid containing human CLDN18.2 ECD1 (SEQ ID NO: 19). Use 2×10 respectively 7 Individual CLDN18.2-HEK293T cells (subcutaneous multi-point injection) and 2 mg plasmid (muscular multi-point injection) were alternately immunized with alpacas (Nanchang Dajia Biological Breeding) on ​​a weekly basis. Finally, with 2×10 7 Individual CLDN18.2-HEK293T cells were boosted.

[0281] 2. Serum immune titer determination

[0282] The immune titer was determined by ELISA method according to the signal of the immune serum on the antigen recombinant protein CLDN18.2 (GenScript, CP0007). The specific method is as follows.

[0283] One day before the immunotiter assay, the antigen recombinant protein CLDN...

Embodiment 3

[0287] Construction and Screening of Alpaca Immune Library

[0288] After the animals were immunized, 80 mL of blood was collected from alpaca, and PBMCs were separated by Ficoll-Paque density gradient separation solution (GE, 17144003S) for the construction of alpaca immune library. The specific method is as follows:

[0289] Take 15mL of Ficoll-Paque density gradient separation solution and slowly add it to a 50mL centrifuge tube, and then slowly add 15mL of the collected alpaca blood, so that the two liquids maintain a clear separation interface. Centrifugation was performed at about 15°C under the following conditions: 400 g, 20 min, acceleration of 3, and deceleration of 0. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. cell layer. First, the upper plasma mixture was carefully aspirated with a sterile Pasteur...

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Abstract

The present invention provides novel CLDN18.2 binding molecules. The present invention also provides nucleic acid molecules encoding the CLDN18.2 binding molecules, expression vectors and host cells for expressing the CLDN18.2 binding molecules. The present invention further provides methods for producing the CLDN18.2 binding molecules and uses thereof.

Description

[0001] sequence listing [0002] This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. technical field [0003] This application relates generally to antibodies. More specifically, the present application relates to single domain antibodies that specifically recognize CLDN18.2, methods for their preparation and uses thereof. Background technique [0004] Cell junction is the connection structure between cells, which is an important basis for the mutual connection and synergy between adjacent cells in multicellular organisms. In general, animal cells have four types of junctions: tight junctions, adhesive junctions, gap junctions, and desmosomes / hemidesmosomes. [0005] Tight junctions, also known as occlusive junctions, are structures formed between endothelial or epithelial cells that prevent interstitial substances from diffusing from the intercellular space and can only enter cells by active transport. Tight ju...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/70C12N15/81C12N15/85C12N1/21C12N1/19C12N5/10G01N33/68G01N33/574A61K39/395A61P35/00
CPCC07K16/28C12N15/70C12N15/81C12N15/85G01N33/68G01N33/574A61P35/00C07K2317/565A61K2039/505C07K2317/22C07K2319/30C07K2319/60C07K2317/24
Inventor 谭永聪郎国竣孔超刘婵娟邓敏吴琪张静张文海范宝国
Owner SANYOU BIOPHARMACEUTICALS CO LTD
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