Anti-pd-1 antibodies, method for producing same and method for using same

A PD-1, antibody technology, applied in biochemical equipment and methods, antibodies, antibody medical components, etc., can solve problems such as no significant cross-reactivity

Pending Publication Date: 2019-07-16
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The prior art discloses monoclonal anti-PD-1 antibodies comprising a specific amino acid sequence according to WO2006 / 121168 (nivolumab, BMS), which exhibit some useful properties, such as high affinity binding to human PD-1 , but no significant cross-reactivity with human CD28, CTLA-4, or ICOS

Method used

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  • Anti-pd-1 antibodies, method for producing same and method for using same
  • Anti-pd-1 antibodies, method for producing same and method for using same
  • Anti-pd-1 antibodies, method for producing same and method for using same

Examples

Experimental program
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Effect test

Embodiment 1

[0207] Designing Natural Human Antibody Fab-Library MeganLibTM

[0208] Total RNA in B lymphocytes from blood samples of more than one thousand individual human donors was isolated using a kit (RNeasy Mini Kit) according to the suggested protocol (Qiagen, Germany). RNA concentration determination was performed using Nanovue kit (GE Healthcare); the quality of isolated RNA was detected by 1.5% agarose gel electrophoresis.

[0209] Reverse transcription reactions were performed using MMLV RT kit (Anolen) according to the recommended protocol with MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0210] Reverse transcription products were used as substrates in a two-stage polymerase chain reaction to obtain genes with variable domains flanked by restriction sites; using oligonucleotide kits and [Journal of Biological Chemistry, June 1999 , 25; 274(26):18218-30] for the reaction.

[0211] The obtained DNA preparation VL-CK-VH ( figure 1 ) was treated...

Embodiment 2

[0213] Selection of fab-libraries of phage antibodies

[0214] Specific anti-PD-1 phage Fab-antibodies were isolated from MeganLibTM's combinatorial library phage Fab-display library. Selection was performed using recombinant human PD-1, under conditions as previously described (Journal of Biochemistry, June 25, 1999, 274(26):18218-30; Nature Biotechnology, March 1996, 14(3) :309-14; Journal of Molecular Biology, 5 Dec. 1991, 222(3):581-97). For the selection process by the panning method, human PD-1 in 50 mM carbonate buffer (pH 9.5) was adsorbed on the surface of a high pipette (Nanken, Denmark) overnight at 4°C. In addition, the tubes were washed with PBS (pH 7.4) and then blocked with a solution containing PBS (pH 7.4)-non-fat milk (0.5% w / v) for 1 hour. Then, 2-4 ml of phage solution (10 13 phage particles / ml) - non-fat milk (0.5% w / vol) was transferred to a tube with antigen and the system was incubated for 1 hour with agitation. Unbound phage were removed by a serie...

Embodiment 3

[0216] Analysis of Fab specific binding to human PD-1

[0217] ELISA was used to measure the binding of the tested Fab fragments to human PD-1. A Fab with published Nivolumab sequence (Bristol-Myers Squibb) was used as a positive control. For the analysis of specific binding, ELISA plate wells (medium binding from Grena First Biochemical) were filled with 50 μl of PD1-H6F (0.5 μg / ml in 1× carbonate coating buffer, pH 9.5) Coat, seal and incubate overnight at 4°C. All further stages were carried out according to standard ELISA protocols utilizing high performance automation platforms based on robotic systems such as Genetix Qpix2xt (Megumolecular) and Tecan Friedham EVO 200 (Tecan). Non-specific binding was blocked by adding blocking buffer BB (200 [mu]l 0.5% non-fat milk in PBS). The plate was incubated on a shaker at room temperature for 1 hour. After washing with PBS-Tween, each cell was coated with 50 μl of the cell supernatant containing the test Fab mixed with an equa...

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Abstract

The present invention relates to biotechnology and comprises isolated monoclonal antibodies, in particular human monoclonal antibodies, which specifically bind to PD-1 with high affinity. The antibodies according to the invention may be chimeric, humanized or human antibodies, or antigen-binding fragments thereof, and may be used as a medicinal agent in oncology and immuno-oncology, for treating diseases related to various disorders of cell proliferation and development. The invention also relates to methods for producing the indicated antibodies, and to a method for treating human diseases using said antibodies.

Description

technical field [0001] The present invention relates to biotechnology and provides isolated monoclonal antibodies, particularly human monoclonal antibodies that specifically bind to high-affinity PD-1. Antibodies of the present invention may be chimeric, humanized or human antibodies, or antigen-binding fragments thereof, and may be used as pharmaceutical preparations in oncology and immuno-oncology, and for the treatment of various cell proliferation or developmental Disorder-related diseases. The invention also relates to methods of producing said antibodies and methods of using said antibodies to treat human diseases. Background technique [0002] The programmed death 1 (PD-1) protein is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al., supra; Okazaki et al., (2002) Current Perspectives in Immunology, 14:391779-82; Bennett et al., (2003) J...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63A61K39/395
CPCC12N15/63A61P31/00A61P35/00C07K16/2818C07K2317/21C07K2317/33C07K2317/76C07K2317/92C07K2317/94C07K2317/55C07K2317/90A61K2039/505C07K2317/732A61K39/39591C07K2317/24C07K2317/31C07K2317/52C07K2317/70C07K2317/734A61K39/395C07K16/2803C07K16/28C07K16/2896
Inventor 维多利亚·米哈伊洛芙娜·叶基莫娃德米特里·瓦列里耶维奇·科尔扎温尤利娅·谢尔盖耶芙娜·切尔内赫季莫费·亚历山大罗维奇·涅曼金瓦莱丽·弗拉基米罗维奇·索洛维约夫安娜·康斯坦丁诺芙娜·弗拉迪米罗娃伊琳娜·安德烈耶芙娜·布兰金娜谢尔盖·瓦西里耶维奇·迪杜克奥尔加·弗拉基米罗芙娜·贡恰罗娃安娜·弗拉基米罗芙娜·叶罗绍娃亚科夫·尤雷维奇·乌斯蒂乌戈夫玛丽娜·弗拉基米罗芙娜·阿尔秋霍娃安德烈·鲍里索维奇·乌利京罗曼·阿列克谢耶维奇·伊万诺夫德米特里·瓦连京诺维奇·莫罗佐夫
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