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Novel monoclonal antibody against PD-1

A PD-1 and antibody technology, applied in the direction of antibodies, carriers, anti-tumor drugs, etc., can solve problems such as limiting the application of antibody drugs and reducing the therapeutic effect of antibody drugs

Active Publication Date: 2018-03-27
CSTONE PHARM (SUZHOU) CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most PD-1 antibody drugs do not bind to the mouse PD-1 protein, which limits the application of antibody drugs in preclinical animal experiments, and because most of their antibody sequences are derived from immunity to mice, serious Immunogenic responses reduce therapeutic efficacy of antibody drugs in humans

Method used

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  • Novel monoclonal antibody against PD-1
  • Novel monoclonal antibody against PD-1
  • Novel monoclonal antibody against PD-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0244] Example 1 Preparation of experimental materials

[0245] 1. Antigen preparation

[0246] Synthesize the DNA encoding the full length or extracellular region of PD-1 and PD-L1, and insert them into the expression vector pcDNA3.3. After extracting a large amount of plasmid DNA, sequencing to verify the sequence of the inserted DNA fragment. The fusion protein PD-1 extracellular region and PD-L1 extracellular region contain different tags, including human Fc, murine Fc and His tags, etc., by transfecting PD-1 extracellular region gene sequence into CHO-S or HEK293 Expressed in cells. Five days after the transient transfection of the cells, the cell culture supernatant was collected, and the fusion protein was purified and quantified for immunization and screening.

[0247] 2. Establishment of stable cell line

[0248] In order to obtain antibody screening verification tools, PD-1 and PD-L1 transfected cell lines were prepared. Briefly, the pcDNA3.3 vector expression plasmid ...

Embodiment 2

[0249] Example 2 Production of antibody hybridomas

[0250] 1. Immunity

[0251] Female SD rats aged 6 to 8 weeks were sensitized with 10 μg of human PD-1 extracellular domain protein and 10 μg of mouse PD-1 extracellular domain protein (in TiterMax) via plantar injection, and then used in each week. The human PD-1 extracellular domain protein or mouse PD-1 extracellular domain protein in the aluminum phosphate gel adjuvant is immunized once through the sole of the foot until it is suitable for fusion. During the immunization period, the serum titer of anti-PD-1 antibodies was detected by ELISA or FACS every two weeks.

[0252] 2. Cell Fusion

[0253] When the antibody titer reaches a sufficiently high level, the rats are given the final adjuvant-free immunogen (human PD-1 extracellular domain protein and mouse PD-1 extracellular domain protein) to stimulate (with an equal volume of phosphate buffer Solution (PBS) instead of adjuvant). The SP2 / 0 cells were resuscitated one week b...

Embodiment 3

[0266] Example 3 Sequencing of antibody hybridoma cells, humanized construction of antibodies and affinity maturation

[0267] 1. Hybridoma antibody sequencing

[0268] Trizol reagent was used to isolate RNA from monoclonal hybridoma cells. The VH and VL segments of the PD-1 chimeric antibody are amplified by the following method: First, reverse transcription of RNA into cDNA by reverse transcriptase.

[0269] Reaction system (20μL)

[0270]

[0271] Reaction conditions

[0272]

first step

Second step

third step

the fourth step

Temperature(℃)

25

37

85

4

time

10 minutes

120 minutes

5

∞

[0273] The obtained cDNA was used as a template, and the following PCR amplification was performed using specific primers for the gene of interest. The PCR reaction operation is as follows:

[0274]

[0275] Reaction conditions:

[0276]

[0277]

[0278] The resulting PCR reaction product (10 μL) was ligated to the pMD18-T vector. 10μL ligation product was transformed into Top10 competent cel...

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Abstract

The invention provides a monoclonal antibody against PD-1, especially a human monoclonal antibody against PD-1. The antibody specifically binds to PD-1 in virtue of high affinity and comprises a heavychain and a light chain. The invention also provides a nucleic acid sequence coding the antibody, a cloning or expression vector, a host cell, a method for expressing or isolating the antibody, and an immunoconjugate and therapeutic composition containing the antibody. The invention further provides application of the anti-PD-1 antibody to treatment of various cancers.

Description

Technical field [0001] The present invention mainly relates to PD-1 monoclonal antibodies and their compositions, and the immunotherapy of human diseases using anti-PD-1 antibodies. Background technique [0002] More and more evidence of preclinical and clinical results shows that targeted immune checkpoints are becoming the most promising method for treating cancer patients. Programmed cell death molecule 1 (PD-1) is an inhibitory member of the immunoglobulin superfamily that has homology to CD28 and is expressed in activated B cells, T cells and bone marrow cells (Agata et al, supra; Okazaki et al (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) JImmunol 170: 711-8) and play an important role in regulating activation and suppression signals in the immune system (Okazaki, Taku et al. 2007 International Immunology 19:813-824). In fact, PD-1 was found in the differential expression screening of apoptotic cells (Ishida et al (1992) EMBO J 11:3887-95). [0003] The ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A01K67/027C12N5/10A61K39/395A61P35/00A61P37/02
CPCA61K2039/505C07K2317/24C07K2317/92C07K2317/565C07K16/2818C12N15/85C12N5/0686A61P35/00A61P37/02C12N2800/107C12N2510/00C07K2317/732C07K2317/734C07K2317/56C07K2317/76
Inventor 郑勇李竞根纳迪·戈洛洛波夫李栋徐建清王卓智
Owner CSTONE PHARM (SUZHOU) CO LTD
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