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Epitope-specific antibody screening method and screened antibody

A screening method and specific technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc., can solve the ineffective anti-PD-1 treatment, low antibody hit rate, affinity Advanced problems, to achieve the effect of reducing drug cost and price, high affinity, and improving hit rate

Pending Publication Date: 2019-06-25
TAIZHOU MABTECH PHARM CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The present invention provides a method for efficiently screening antibody libraries, which can solve the problem of low hit rate of the existing antibody library screening technology for functional antibodies specific to epitopes with potential to become drugs
The present invention also provides an anti-PD-1 monoclonal antibody obtained by this screening method, which has a different binding epitope from Nivolumab and Pembrolizumab, and has high affinity and good specificity, which can solve the problem of some patients with existing anti-PD -1 Healing doesn't work

Method used

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  • Epitope-specific antibody screening method and screened antibody

Examples

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Embodiment 1

[0038] Example 1. Synthetic PD-1 epitope peptide screening phage display antibody library

[0039] (1) Epitope peptide synthesis and magnetic bead coupling

[0040] According to the sequence of SEQ ID NO: 13, the human PD-1 cyclic epitope peptide was chemically synthesized (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.). The epitope peptide includes amino acids QTDKLAAF at positions 75-82 of human PD-1 , as well as the cysteine ​​M for magnetic bead coupling and the linking peptide consisting of 3 amino acids on both sides, with a molecular weight of 1475 Da. Dissolve the synthesized epitope peptide in PBS (phosphate buffer, pH7.2, containing 20mM sodium phosphate salt, 150mM sodium chloride) at 10mg / mL for later use;

[0041] Wash 2 mL of amino-derivatized magnetic beads (Thermofisher Scientific company, catalog number 21352, each mL of magnetic beads weighs 2.5 g and contains about 12 µmol amino groups) with 2 mL of PBS, wash three times, and magnetically separa...

Embodiment 2

[0047] Example 2. Secondary screening of functional antibodies by living cells with high expression of PD-1

[0048] (1) Construct a cell line that highly expresses PD-1 on the cell surface

[0049] Clone the full-length PD-1 coding gene (PDCD1 gene, containing the N-terminal signal peptide coding sequence, see NCBIReference Sequence: NM_005018.2 for the specific sequence), insert it into the pcDNA3.1 expression vector, and amplify it in Escherichia coli (E. coli) Transfect 293T cells (ATCC number CRL-3216), screen high expression cell lines, according to the literature "Flow cytometric detection and quantitation of the epidermal growth factor receptor incomparison to Scatchard analysis in human bladder carcinoma cell lines. Cytometry. 1994 Sep 1;17( 1):75-83" and "Antibody-dependent cellularcytotoxicity mediated by cetuximab against lung cancer cell lines. ClinCancer Res. 2007 Mar 1;13(5):1552-61" recorded flow cytometry method to measure the surface of each cell line The ex...

Embodiment 3

[0053] Example 3, Construction of a fully human anti-PD-1 monoclonal antibody expression system

[0054] (1) Identification of heavy and light chain variable regions

[0055] From the 22 phage-positive clones finally obtained in Example 2, 5 clones with high affinity identified by ELISA were selected for sequencing, and 1 clone numbered 5L12 was selected for further development. Sequencing results showed that the heavy chain variable region of 5L12 had the amino acid sequence of SEQ ID NO: 7, and the light chain variable region had the amino acid sequence of SEQ ID NO: 8. Further antibody sequence analysis showed that the complementarity determining regions of the heavy chain CDR1, CDR2 and CDR3 respectively have the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the complementary determining regions CDR1, CDR2, and CDR3 of the light chain have SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 5, respectively. Amino acid sequence of ID NO: 6.

[0056] (2)...

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Abstract

The invention discloses an epitope-specific antibody screening method, and is used for solving the problem of low screening efficiency of functional antibodies having treatment function in an antibodylibrary screening technology; the method includes the steps of screening of functional epitope peptides and screening of living cells. The invention also provides a fully human anti-PD-1 monoclonal antibody obtained by screening a phage display antibody library by the method; for specific functional epitopes, the fully human anti-PD-1 monoclonal antibody can effectively block the binding of PD-1to a ligand of PD-1, has the characteristics of high affinity and low EC50, and can produce treatment effect on animal models at low dose. The screening efficiency of the functional antibody is greatly improved, and a potential, safer and more economical immunotherapy drug is provided for patients with malignant tumors and immune diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an epitope-specific antibody screening method and the screened antibody. Background technique [0002] The "immune escape" of cancer cells is considered to be the main mechanism of tumorigenesis, development and drug resistance. Tumors can protect themselves from elimination by the immune system by directly or indirectly suppressing T cell signaling. Immune checkpoint therapy of tumors is a kind of therapeutic method that regulates T cell activity through a series of pathways such as co-inhibition or co-stimulation signals to improve anti-tumor immune response. [0003] PD-1 (programmed cell death protein 1, English full name Programmed Cell Death Protein 1, also known as CD279) is a cell surface receptor expressed on activated T cells and pre-B cells (pro-B cells), It is an immune checkpoint that negatively regulates the immune system and promotes self-tolerance by inhib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00A61P37/02
Inventor 钱卫珠郭怀祖徐进陶静
Owner TAIZHOU MABTECH PHARM CO LTD
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