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Anti-human CCL1 antibody and application thereof

An antibody and antibody conjugate technology, applied in the field of biomedicine, can solve the problems of inability to effectively cause CDC and incapable of inhibiting the biological activity of CCL1, and achieve the effect of high affinity and low immunogenicity

Pending Publication Date: 2022-07-29
BEIJING WEIFENG YIMIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In therapeutic applications, fully human antibodies can overcome many shortcomings of mouse monoclonal antibodies in clinical applications: such as inducing anti-mouse antibody (HAMA) reactions in the human body, inability to effectively induce CDC and ADCC, etc.
[0019] Antibodies against CCL1 have been reported. For example, R&D Company has produced mouse anti-human CCL1 monoclonal antibody MAB272, but it is only used as a reagent in the field of detection, but cannot inhibit CCL1-related biological activities and is used for the treatment of CCL1-related diseases

Method used

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  • Anti-human CCL1 antibody and application thereof
  • Anti-human CCL1 antibody and application thereof
  • Anti-human CCL1 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0077] Preparation Example 1 Construction of large-capacity natural phage antibody library

[0078] 1.1 Preparation of phagemid pHLS

[0079] 1) On the basis of the phagemid pUC119 vector (purchased from TAKARA company), design the gene sequence, such as figure 1 , the phagemid pHLS was constructed by using whole gene synthesis technology.

[0080] 2) Transfer the plasmid into XL1-blue (invitrogen) competent cells, select a single colony, and insert 5mL of 2YT-A + In the nutrient solution (16g peptone, 10g yeast powder, 5g sodium chloride, add water to 1000mL, pH7.0, contain 100 μg / mL ampicillin), transfer overnight as 200mL 2YT-A+ nutrient solution the next day.

[0081] 3) Use Qiagen plasmid extraction kit to extract plasmids according to the kit instructions. Finally, the DNA concentration was detected in a UV spectrophotometer, and the final concentration was about 0.3 μg / mL.

[0082] 1.2 Extraction of total RNA from lymphocytes

[0083] 1) A total of 65 peripheral bl...

preparation example 2

[0157] Preparation Example 2 Screening of fully human anti-CCL1 single chain antibody

[0158] 2.1 Preparation of helper phage

[0159] 1) Pick a single XL1-BLUE strain and inoculate it into 40 mL of SB (containing 10 μg / mL tetracycline), and culture with shaking at 37°C overnight.

[0160] 2) The next day, it was diluted 1:500 into 10 mL of the same SB medium, and cultured with shaking at 37°C for 1 h.

[0161] 3) Pick a single M13K07 phage plaque, inoculate it into the above-mentioned 10 mL bacterial solution, and culture with shaking at 37°C for 2 hours.

[0162] 4) Add to SB medium containing 10 μg / mL tetracycline and 70 μg / mL kanamycin, to 500 mL, and shake culture at 37°C overnight.

[0163] 5) When cultured to an OD600 of about 1, centrifuge at 12000rpm at 4°C for 15min. The supernatant was taken, aseptically divided into test tubes, 50 ml per tube, and stored at 4°C.

[0164] 2.2 Titration of phage virus species

[0165] 1) Prepare 6 LB culture plates without any ...

preparation example 3

[0187] Preparation Example 3 Screening and Identification

[0188] 3.1 Selection of random clones

[0189] 1) Prepare 2YT medium, 100 μg / mL ampicillin and 10 μg / mL tetracycline, and add them to a 96-well deep-well culture plate, with about 600 μL per empty space.

[0190] 2) In the third and fourth rounds of screening output culture plates, toothpicks randomly select colonies and insert into 96-well deep-well culture plates. Incubate overnight at 37°C with shaking.

[0191] 3) The next day, transfer at 1:10 into a new 96-well deep-well culture plate containing 600 μL of medium, and culture with shaking at 37° C. for 3 hours. Add helper phage, incubate at 37°C for 20 minutes, and then incubate at 30°C for 8 hours with shaking.

[0192] 4) Centrifuge at 3000 rpm for 10 minutes. The supernatant was used as the scFv phage solution to be tested.

[0193] 3.2 Polyclonal Phage ELISA

[0194] 1) Human CCL1 recombinant protein and bovine serum albumin (BSA) were diluted with PBS ...

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Abstract

The invention discloses an antibody capable of being combined with human CCL1, the antibody comprises a VH structural domain and a VL structural domain, the VH structural domain comprises HCDR1-3 as shown in SEQ ID NO: 5-7, and the VL structural domain comprises LCDR1-3 as shown in SEQ ID NO: 8-10. The invention also discloses a method for preparing the antibody and application of the antibody. The brand-new antibody combined with the human CCL1 has the effect of inhibiting CCL1 related biological activity, and can be used for treating CCL1 related diseases or detecting CCL1.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-human CCL1 antibody and an application thereof. Background technique [0002] Chemokines refer to small molecular cytokines that enable cells to undergo chemotactic movement. They transmit signals into cells by binding to receptors and play an important role in inflammation, cell migration, and angiogenesis. These factors have similar structures and functions, and can be divided into four categories: CXC, CC, C and CX3C according to the relative positions of the two cysteine ​​residues at the amino terminus. Similar to chemokines, the nine human chemokine receptors are divided into two subclasses, CXC chemokine receptors (CXCR) and CC chemokine receptors, based on their selectivity for CXC or CC (CCR). [0003] Chemokine C-C motif ligand 1 (CCL1) is one of the genes in the CC subfamily chemokine gene cluster located on human chromosome 17q11.2-q12. CCL1 is known to be produc...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13A61K39/395A61K47/68A61P43/00A61P1/16A61P11/00A61P11/06A61P35/00
CPCC07K16/24A61K47/6803A61K47/6845A61P43/00A61P1/16A61P11/00A61P11/06A61P35/00C07K2317/565C07K2317/567C07K2317/56C07K2317/92C07K2317/76C07K2317/622C07K2317/73
Inventor 胡卓伟刘姗姗严君崔冰
Owner BEIJING WEIFENG YIMIN TECH
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