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Means and methods for oral protein delivery

A fusion protein, matrix technology, applied in peptide/protein components, chemical instruments and methods, animal/human proteins, etc., can solve problems such as expensive regulatory procedures, not edible vaccines, and lengthy

Pending Publication Date: 2019-12-13
VLAAMS INTERUNIVERSITAIR INST VOOR BIOTECHNOLOGIE VZW +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Plant production systems, although capable of producing large quantities of recombinant proteins, are not optimal for the production of edible vaccines due to lengthy and expensive regulatory procedures

Method used

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  • Means and methods for oral protein delivery
  • Means and methods for oral protein delivery
  • Means and methods for oral protein delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0183] Materials and methods of Example 1

[0184] Expression of VHH-IgAFc

[0185] Arabidopsis: A previously published Arabidopsis line expressing monomeric V2A-IgAFc and V3A-IgAFc fusion proteins (Virdi et al. (2013) PNAS 110, 29, 11809-11814), was scaled up in the greenhouse to produce approximately 100 g of V2A- IgAFc and V3A-IgAFc produced seeds and formulated antibody-containing feeds for Arabidopsis groups in challenge experiments.

[0186] soybean : Plasmids pEV2A and pEV3A (Virdi et al. (2013) PNAS 110, 29, 11809-11814) with the VHH-IgAFc fusion gene of antibodies V2A and V3A respectively, according to Cloning instruction manual (Invitrogen), recombined into the pGW43 multisite gateway cassette (Karimi et al. (2002) Trends Plant Sci 7, 193-195), said cassette has the gene conferring phosphinothricin resistance, for use herbicide selection of transformants. The resulting expression vectors were named pMXV2A and pMXV3A, and then the expression vectors were i...

Embodiment 2

[0220] Materials and methods of Example 2

[0221] Strains, media and reagents

[0222] Escherichia coli (E. coli) MC1061 or DH5α were used for standard molecular biology procedures. For plasmid propagation, E. coli were grown in LB broth (0.5% yeast extract, 1% tryptone and 0.5% NaCl) supplemented with the appropriate antibiotic: 50 μg / mL carbenicillin ( Duchefa Biochemie), 50 μg / mL Kanamycin (Sigma) Aldrich), 50 μg / mL Hygromycin B (Duchefa Biochemie) or 50 μg / mL (Life Technologies). All PCR reactions were performed using Phusion high-fidelity polymerase (NEB). PCR reagents (such as dNTPs and primers) were ordered from Promega and IDT, respectively.

[0223] Pichia pastoris NRRL-Y 11430 strain (same name Komagataella phaffi) was provided by A. Glieder (Graz University of Technology, Austria). This strain is called wild type. Yeast culture in liquid YPD (1% yeast extract, 2% peptone, 1% D-glucose) or on solid YPD-agar (1% yeast extract, 2% peptone, 1% D-glucose, 2% ...

Embodiment 3

[0245] Materials and methods of Example 3

[0246] Feed formulation based on VHH-IgA-Fc produced by Pichia pastoris

[0247] As used in the challenge experiment of Example 1, an effective dose of anti-ETEC VHH-IgAFc is about 5 mg VHH-IgAFc, or more suitably, each piglet is fed with the dried product from a 0.5 L shake flask growth culture per day. Feed preparations. This dose consisted of two aliquots of anti-F4-ETEC VHH-IgAFcs, V2A and V3A (see Virdi et al. (2013) 110, 29, 11809-11814). To prepare similar doses for 18 piglets (three groups of six animals each) receiving Pichia-produced VHH-IgAFcs; 45 L each of Pichia cultures expressing V2A and V3A were grown in shake flasks (total 90 L ). As summarized in Table 5 below, six production batches were performed (in a five-day process, as in Example 1, growth in BMGY medium for 48 hours followed by induction in BMMY medium for 48 hours). Therefore, 15L culture batches were produced weekly. At the end of each run, the mediu...

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Abstract

The present invention relates to the field of recombinant protein production in a host cell. More specifically the invention relates to the field of oral protein delivery. Specifically, the inventionprovides oral pharmaceutical formulations comprising the culture medium of a recombinant host secreting a recombinant protein. The resulting oral pharmaceutical formulations are useful for the treatment of gastro-intestinal and / or buccal disorders. Additionally the oral pharmaceutical formulations are useful for prophylactic and vaccine purposes.

Description

[0001] field of invention [0002] The present invention relates to the field of production of recombinant proteins in host cells, such as yeast cells. More specifically, the present invention relates to the field of oral protein delivery. Specifically, the present invention provides an oral pharmaceutical formulation comprising a culture medium of a recombinant host secreting a recombinant polypeptide. Background technique [0003] Peptides or proteins (including hormones, enzymes, ligands or inhibitors), including antibodies, regulate various cellular functions. Therefore, they can treat or prevent human diseases clinically by regulating physiological or pathological processes. Compared with small-molecule drugs, the high selectivity of peptides or proteins for their targets can reduce side effects and toxicity to host cells. The use of proteins or peptides for therapeutic purposes is expected to continue to increase in the treatment of cancer, metabolic disorders, gastro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16A61K47/46A61K9/19A61K38/00A61K39/00A23L33/195A23L33/18A23K20/10C12P21/02C07K14/54C07K16/18
CPCA61K38/00A61K38/20A61K9/19A61K36/06C07K14/54C12P21/02C12N1/16A61K2121/00A61K2300/00C07K2319/30A23K20/10A23L33/17A23L33/18A61K47/68C07K16/1232A61K2039/505C07K2317/22A61P31/04A61K9/0095A61K47/36A61K36/064A61K47/6813A61P1/00A61P1/02A23K20/147A23V2002/00
Inventor N·卡勒维尔特R·万卢申B·劳肯斯A·德皮克V·维蒂
Owner VLAAMS INTERUNIVERSITAIR INST VOOR BIOTECHNOLOGIE VZW
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