Materials and methods for multidirectional biological transport

A single-domain antibody, molecular technology, applied in the field of materials and methods for multidirectional biological transport, which can solve problems such as restricted transport

Pending Publication Date: 2022-03-11
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their relatively large size limits their transepithelial transport, including across the mucosal epithelial barrier, and presents transport challenges for biologics reaching and passing the mucosa
Therefore, the most common mode of administration is invasive, often requiring the services of a health professional in an expensive healthcare setting

Method used

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  • Materials and methods for multidirectional biological transport
  • Materials and methods for multidirectional biological transport
  • Materials and methods for multidirectional biological transport

Examples

Experimental program
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preparation example Construction

[0568] 5.2.6. Preparation of single domain antibody

[0569] Single domain antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding a single domain antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody producing cells such as hybridoma cells or B cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a host cell. Many vectors available and known in the art can be used for the purposes of this disclosure. Selection of an appropriate vector will depend primarily on the size of the nucleic acid ...

Embodiment 1

[0700] 6.1. Example 1: Immunization, Recovery and Screening of pIgR Conjugates

[0701] To generate a panel of single domain antibodies that bind to pIgR, llamas were immunized with recombinant human pIgR (hpIgR) and / or mouse pIgR (mpIgR) for approximately 90 days. Whole blood and PBMC were isolated from llamas and RNA was prepared. After first-strand eDNA synthesis, llama-specific primers annealing to (i) VH (heavy chain variable region), (ii) VHH leader sequence gene, and (iii) CH2 gene were used to PCR amplify the VH and VHH gene pools.

[0702] The VHH pool was separated from the VH pool by running the PCR fragments on a gel and excising the smaller bands. The VHH gene pool was reamplified and cloned into CMV-based mammalian vectors. VHH-genes are formatted as Ig-fusions. The library was transformed into E. coli. Single colonies were picked in a 96-well format for Sanger sequencing. From approximately 300 unique sequences, a selected number of VHH sequences were sel...

Embodiment 2

[0713] 6.2. Example 2: Biophysical characterization of hpIgR-specific binders

[0714] Ten plgR binders from Example 1 (8 hpIgR specific and 2 human / mouse cross-reactive) were selected for further biophysical and functional assays. Ten pIgR binders were expressed and purified from CHO cells using protein-A affinity chromatography. Size-exclusion chromatography combined with multi-angle light scattering revealed 10 VHH-mono-Fc conjugate species (VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11, and VHH12) in the molecular weight range of 41.3 kDa to 48.7kDa.

[0715] The thermal stability of the samples was determined by differential scanning fluorimetry, specifically the NanoDSF method, using an automated Prometheus instrument. Measurements were performed by loading samples from a 384-well sample plate into a 24-well capillary. Duplicate runs were performed for each sample. The Prometheus NanoDSF user interface (Fusion Scan tab) was used to set the experimental par...

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Abstract

The present invention describes a method for delivering a single domain antibody or a therapeutic molecule from an apical surface of a polymeric immunoglobulin receptor (pIgR)-expressing cell to a basal lateral surface of the pIgR-expressing cell, the method comprising contacting the pIgR-expressing cell with the single domain antibody or the therapeutic molecule, wherein the single domain antibody binds to pIgR, and the therapeutic molecule comprises an agent and the single domain antibody. Also described is a method for transporting such a therapeutic molecule to the systemic circulation or the intrinsic layer or the gastrointestinal tract of a subject comprising administering the therapeutic molecule to the subject via oral delivery, buccal delivery, nasal delivery, or inhalation delivery.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application No. 62 / 940,232, filed November 25, 2019, U.S. Provisional Patent Application No. 62 / 940,228, filed November 25, 2019, U.S. Provisional Patent Application No. 62 / 940,228, filed November 25, 2019 62 / 940,220, U.S. Provisional Patent Application No. 62 / 940,208, filed November 25, 2019, U.S. Provisional Patent Application No. 62 / 940,206, filed November 25, 2019, U.S. Provisional Patent Application No. 62 / 940,206, filed November 25, 2019 62 / 940,200, U.S. Provisional Patent Application No. 62 / 940,196, filed November 25, 2019, U.S. Provisional Patent Application No. 62 / 882,387, filed August 2, 2019, U.S. Provisional Patent Application No. 62 / 882,387, filed August 2, 2019 Application No. 62 / 882,361, U.S. Provisional Patent Application No. 62 / 882,346, filed August 2, 2019, and U.S. Provisional Patent Application No. 62 / 882,291, filed August 2, 2019, each of these Provision...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K45/00A61P1/00A61P1/14
CPCA61P1/00A61P1/14C07K2317/569C07K2317/22C07K16/283C07K2317/52C07K2319/00C07K2317/24C07K2317/33C07K2317/94C07K2317/92C07K2317/70C07K2317/30A61K47/6849A61K45/06A61K38/28A61K38/26Y02A50/30A61K47/65A61P35/00A61P29/00A61K2039/505A61P11/00A61K2039/544C07K2317/565C07K2317/567C07K2317/76
Inventor R·贾内桑B·V·马鲁塔查拉姆A·兹沃拉克B·盖斯特X·林-施米德特S·文卡塔拉马尼S·辛赫
Owner JANSSEN BIOTECH INC
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