Blood brain barrier shuttle

A blood-brain barrier and entity technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, for targeting specific cell fusion, etc., can solve problems such as limiting pharmacological effects

Inactive Publication Date: 2018-12-21
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low concentration of the antibody within the CNS, this would necessarily limit any pharmacological effect

Method used

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  • Blood brain barrier shuttle
  • Blood brain barrier shuttle
  • Blood brain barrier shuttle

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0157] Embodiment 1: Preparation of expression plasmid

[0158] Description of Basic / Standard Mammalian Expression Plasmids

[0159] The desired protein was expressed by transient transfection of human embryonic kidney cells (HEK 293). In order to express the desired gene / protein (e.g. antibody-Fab multimeric protein), a transcription unit comprising the following functional elements is used:

[0160]Immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

[0161] ●Human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0162] Mouse immunoglobulin heavy chain signal sequence (SS),

[0163] the gene / protein to be expressed (e.g. full-length antibody heavy chain), and

[0164] • Bovine growth hormone polyadenylation sequence (BGH pA).

[0165] In addition to the expression unit / cassette including the desired gene to be expressed, the basic / standard mammalian expression plasmid contains:

[0166] an origin of replication fr...

Embodiment 2

[0198] Example 2: Purification of single and double Mab31-Fab constructs

[0199] Antibody chains were prepared by transient transfection of HEK293 cells (derived from the human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). For transfection, "293-Fectin" transfection reagent (Invitrogen) was used. Expression of antibody chains from 2 (tetravalent Mab31-scFab(8D3)) or 3 (trivalent Mab31-scFab(8D3)) different plasmids encoding the tetravalent Mab31-scFab(8D3) heavy chain and the Mab31 counterpart Light chain, or overhang and hole trivalent Mab31-scFab(8D3) heavy chain and Mab31 corresponding light chain. After transfection, the 2 or 3 plasmids were used in equimolar plasmid ratios. Transfection was performed as described in the manufacturer's instructions. Seven days after transfection, cell culture supernatants containing antibody fusion proteins were harvested. Supernatants were stored frozen until purification.

[0200] Proteins were purified...

Embodiment 3

[0201] Example 3: ELISA binding data for single and double Fab constructs

[0202] Binding of mAb31-8D3 constructs to mouse transferrin receptor (mTfR) was assessed by indirect ELISA. For this purpose, recombinant mTfR (extracellular domain; Sino Biological) was coated onto Maxisorb microtiter plates (Nunc) at 1 μg / mL in PBS overnight at 4°C. After blocking in 1% Crotein-C / PBS (blocking buffer; Roche) for 1 h at room temperature and washing 4 times with 0.1% Tween-20 / PBS (washing buffer), the mAb31-8D3 construct was blocked in blocking buffer. Concentrations between 0.01-150 nM in ® were added to the wells and incubated for 1 h at room temperature. After 4 washing steps, the construct was detected by adding anti-human-IgG-HRP (Jackson Immunoresearch) at a 1:10,000 dilution in blocking buffer (1RT), followed by 6 washes and warming in TMB (Sigma) education. After stopping the color development with 1N HCl, the absorbance was read at 450 nm.

[0203] image 3 showed that th...

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Abstract

The present disclosure relates to blood brain barrier shuttles that bind receptors on the blood brain barrier (R / BBB) and methods of using the same.

Description

[0001] This application is a divisional application of the invention patent application 201380042265.1 submitted on August 26, 2013. technical field [0002] The present invention relates to blood-brain barrier shuttles that bind receptors (R / BBB) on the blood-brain barrier and methods of use thereof. Background technique [0003] Brain penetration of neurological disease drugs, such as large biotherapeutics or small molecules with low brain penetration, is restricted by the extensive and impermeable blood-brain barrier (BBB) ​​as well as other cell groups in the neurovascular unit (NVU) points are strictly limited. A number of strategies to overcome this barrier have been tried, and one strategy utilizes the endogenous receptor-mediated endocytic transport pathway expressed on the brain capillary endothelium. Recombinant proteins such as monoclonal antibodies or peptides have been designed against these receptors to achieve receptor-mediated delivery of biotherapeutics to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/395A61P25/28
CPCC07K16/18C07K16/2881A61K2039/505C07K2317/31C07K2317/34C07K2317/35C07K2317/55C07K2317/64C07K2317/732C07K2319/33C07K2317/21A61P25/00A61P25/28A61P43/00C07K16/28C07K2317/76A61K47/50A61K47/62A61K47/68A61K39/395C07K16/46C07K19/00C12N15/62C07K2317/622C07K16/00
Inventor 贝恩德·博尔曼佩尔-奥拉·弗雷斯克加德彼得·迈尔延斯·尼韦纳阿兰·蒂索-达盖特爱德华·乌里希
Owner F HOFFMANN LA ROCHE & CO AG
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