Anti-cd73 antibodies and uses thereof

An antibody and antigen technology, applied in the field of immunology, can solve serious clinical symptoms and life-threatening problems, and achieve the effect of reducing the production of adenosine

Pending Publication Date: 2022-02-22
AKESO BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Infection with the above viruses can lead to severe clinical symptoms and even life-threatening

Method used

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  • Anti-cd73 antibodies and uses thereof
  • Anti-cd73 antibodies and uses thereof
  • Anti-cd73 antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1: Preparation of anti-CD73 antibody 19F3

[0103] 1. Preparation of hybridoma cell line LT014

[0104] The antigen used to prepare the anti-CD73 antibody is human NT5E-His (NT5E is GenbankID: NP_002517.1, position: 1-552). Splenocytes from immunized mice were fused with mouse myeloma cells to produce hybridoma cells. Using human NT5E (NT5E is GenbankID: NP_002517.1, position: 1-552)-Biotin as an antigen, hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that can secrete antibodies that specifically bind to CD73. For the hybridoma cells obtained by screening, a stable hybridoma cell line was obtained by the limiting dilution method. The above hybridoma cell lines were respectively named hybridoma cell line LT014, and the monoclonal antibodies secreted by them were named 19F3 respectively.

[0105] Hybridoma cell line LT014 (also known as CD73-19F3), which was deposited in the China Center for Type Culture Collection (CCTCC) on June 21,...

Embodiment 2

[0108] Example 2: Sequence Analysis of Anti-CD73 Antibody 19F3

[0109] The mRNA was extracted from the LT014 cell line cultured in Example 1 according to the method of the Total RNA Extraction Kit from Cultured Bacteria (Tiangen, Cat. No. DP430).

[0110] According to Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions Synthesize cDNA and perform PCR amplification.

[0111] The PCR amplification product was directly cloned by TA, and the specific operation was performed according to the instructions of the pEASY-T1 Cloning Kit (TransgenCT101) kit.

[0112] The products of TA clones were directly sequenced, and the sequencing results were as follows:

[0113] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, with a length of 363bp.

[0114] The amino acid sequence encoded by it is shown in SEQ ID NO: 2, with a length of 121aa;

[0115] The sequence of the heavy chain CDR1 is shown in SEQ ID NO:15, the sequence of t...

Embodiment 3

[0119] Example 3: Design and preparation of light chain and heavy chain of humanized antibody against human CD73

[0120] 1. Light chain and heavy chain design of anti-human CD73 humanized antibodies 19F3H1L1, 19F3H2L2 and 19F3H2L3

[0121] According to the three-dimensional crystal structure of human CD73 protein (Hage T, Reinemer P, Sebald W. Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain. Eur J Biochem.1998; 258 (2): 831 -6.) and the sequence of the antibody 19F3 obtained in Example 2, through computer simulation of the antibody model, design mutations according to the model, to obtain the variable region sequences of the antibodies 19F3H1L1, 19F3H2L2, and 19F3H2L3 (antibody constant region sequences, from the NCBI database, heavy The chain constant region is Iggamma-1 chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834).

[0122] The designed variab...

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Abstract

The invention relates to an anti-CD73 antibody and application thereof in treating diseases caused by coronavirus infection. Specifically, a heavy chain variable region of the antibody contains HCDR1-HCDR3 of which the amino acid sequence is shown as SEQ ID NOs: 15-17; and the light chain variable region of the antibody contains LCDR1-LCDR3 of which the amino acid sequence is shown as SEQ ID NOs: 18-20.

Description

technical field [0001] The present invention relates to the field of immunology. In particular, it relates to anti-CD73 antibodies and uses thereof. Background technique [0002] Extracellular-5'-nucleotidase (Ecto-5'-nucleotidase), that is, CD73 protein, is a multifunctional glycoprotein with a protein molecular weight of 70KD encoded by the NT5E gene. phosphatidylinositol (GPI) is anchored to the cell membrane (Zimmermann H. Biochem J. 1992; 285:345-365). [0003] CD73 is widely distributed on the surface of human tissue cells, and is also widely expressed on the surface of immune cells, such as dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), and myeloid-derived suppressor cells (MDSC). [0004] CD73 has both hydrolase activity and non-hydrolase activity. An immunosuppressive mechanism of the enzymatic and non-enzymatic functions of CD73 is mediated by the CD73-adenosine (Adenosine) metabolic signaling pathway. CD39 upstream of CD73 can cata...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/40C12N15/13A61K39/395A61P31/14
CPCC07K16/2896C07K16/40A61P31/14C07K2317/565C07K2317/76C07K2317/56C07K2317/24C07K2317/77C07K2317/74A61K2039/505C07K2317/92
Inventor 王忠民金小平李百勇夏瑜
Owner AKESO BIOPHARMA
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