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Peptide immunogens targeting interleukin 6 (il-6) and formulations thereof for immunotherapy of diseases impacted by il-6 dysregulation

A technology of IL-6 and peptide immunogen, applied in blood diseases, bone diseases, immunoglobulin, etc., can solve problems such as unclear mechanism of action and weak immunogenicity

Pending Publication Date: 2021-08-06
UNITED BIOMEDICAL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] As mentioned above, there are many limitations and problems in current IL-6 vaccine design (e.g. complex chemical coupling procedure for immunogen preparation, using KLH as carrier protein but most of the antibodies elicited are against carrier protein, using clinically Permissive Freund's complete adjuvant to enhance the immunogenicity of vaccination, weak immunogenicity against target IL-6 in vaccinated animals despite using the most aggressive immunization regimen, unclear mechanism of action, etc.)

Method used

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  • Peptide immunogens targeting interleukin 6 (il-6) and formulations thereof for immunotherapy of diseases impacted by il-6 dysregulation
  • Peptide immunogens targeting interleukin 6 (il-6) and formulations thereof for immunotherapy of diseases impacted by il-6 dysregulation
  • Peptide immunogens targeting interleukin 6 (il-6) and formulations thereof for immunotherapy of diseases impacted by il-6 dysregulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0298] Example 1. Synthesis of IL-6-related peptides and preparation of preparations thereof

[0299] a. Synthesis of IL-6-related peptides

[0300] The methods involved in the development of IL-6 peptide immunogen constructs to synthesize IL-6 related peptides are described. Peptides synthesized in small scale quantities were used in serological assays, laboratory tests and field trials, and in large scale (kilogram) quantities for industrial / commercial production of pharmaceutical compositions. For epitope mapping, and for screening and selection of optimal peptide immunogen constructs for effective targeting of IL-6 in pharmaceutical compositions, a large number of IL-6 sequences with a length of about 7 to 70 amino acids were designed. 6 related antigenic peptides.

[0301] Representative full-length IL-6 (SEQ ID NOs: 1-4), IL-6 peptide fragments, and 10 for epitope mapping in various serological assays from human, mouse, rat, and macaque species -mer peptides (SEQ ID N...

Embodiment 2

[0313] Example 2. Serological tests and reagents

[0314] Serological assays and reagents used to assess the functional immunogenicity of IL-6 peptide immunogen constructs and formulations thereof are described in detail below.

[0315] a. ELISA assay based on IL-6 or IL-6 peptide fragments for antibody specificity analysis

[0316] The ELISA assay described in the Examples below to evaluate immune serum samples was developed and described below. IL-6 or IL-6 fragment peptides (SEQ ID NOs: 1 to 20, 72 to 75), which were applied in a volume of 100 [mu]L at 37[deg.] C. for 1 hour to coat the wells of a 96-well plate individually.

[0317] React wells coated with IL-6 or IL-6 fragment peptides with 250 μL of gelatin prepared in PBS at a concentration of 3% by weight at 37°C for 1 hour to block non-specific protein binding sites, and then use 0.05 volume percentage Wash the wells three times with 20 liters of PBS and dry. Utilize containing 20 volume percent normal goat serum,...

Embodiment 3

[0326] Example 3. Evaluation in Animals of the Functional Properties of Antibodies Induced by IL-6 Peptide Immunogen Constructs and Their Preparations

[0327] Further testing the ability of immune serum or purified anti-IL-6 antibodies in immunized animals: (1) Block the interaction between IL-6 and its receptor IL-6R (IL-6α and IL-6Rβ / gp130) and (2) inhibit IL-6-induced STAT3 phosphorylation in RPMI 8226 cells and (3) inhibit IL-6-dependent proliferation of TF-1 cells, and (4) inhibit monocyte chemoattractant proteins in U937 cell line -1 (MCP-1) generated.

[0328] a. cells

[0329] (1) The RPMI 8226 cell line was purchased from the American Type Culture Collection (Manassas, VA), and placed at 37°C with 5% CO 2 maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 4.5 g / L L-glutamine, sodium pyruvate and 1% penicillin / streptomycin in a humidified incubator.

[0330] (2) Place the TF-1 cell line at 37°C containing 5% CO 2 Maintained in a humidif...

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Abstract

The present disclosure is directed to individual peptide immunogen constructs targeting portions of the Interleukin-6 (IL-6) protein, compositions containing the constructs, antibodies elicited by the constructs, and methods for making and using the constructs and compositions thereof. The disclosed IL-6 peptide immunogen constructs contain a B cell epitope from IL-6 linked to a heterologous T helper cell (Th) epitope directly or through an optional heterologous spacer. The IL-6 peptide immunogen constructs stimulate the generation of highly specific antibodies directed to the IL-6 receptor (IL-6R) binding site for the prevention and / or treatment of diseases impacted by IL-6 dysregulation.

Description

technical field [0001] The present invention relates to peptide immunogen constructs targeting interleukin 6 (IL-6) and formulations thereof for immunotherapy of diseases affected by IL-6 dysregulation. Background technique [0002] IL-6 is a small (-25 kD) secreted glycoprotein consisting of 184 amino acids (Table 1), characterized by a four-helix bundle structure. It is produced by several cell types, including leukocytes (T and B lymphocytes, monocytes, macrophages), fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells (mesangial cells), adipocytes, skeletal muscle cells, cardiomyocytes, brain cells (astrocytes, microglia, neurons), and in response to various stimuli (such as lipopolysaccharide and other bacterial products, viruses, cytokines (TNF-α , IL-1, transforming growth factor (TGF)-β), adenosine triphosphate, parathyroxine, vitamin D3, homocysteine, and type II angiotensin (Sebba, 2008)) in response to some tumor cells. [0003] Circulatin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K39/00A61K39/385A61K39/39
CPCA61K38/00A61K2039/55505A61K39/00114A61K2039/55566A61K2039/545A61K2039/6031A61K2039/627A61K2039/55561A61K39/0008A61K2039/804A61K2039/884A61K39/0005C07K16/248C07K2317/76C07K2317/73C07K2317/34C07K2317/33A61K2039/6037A61K2039/6075A61P29/00A61P35/00A61P19/02A61P7/06A61K2039/505
Inventor 王长怡林峰陈君柏丁双
Owner UNITED BIOMEDICAL INC
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