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Anti-tnf-α/pd-1 bispecific antibody and its application

A bispecific antibody, PD-1 technology, applied in the direction of antibodies, applications, specific peptides, etc., can solve the problems of low tissue penetration rate, long half-life, etc., and achieve the effect of good antigen binding activity

Active Publication Date: 2022-02-11
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IgG-like BsAbs retain the corresponding functions of the constant region. In addition, their molecular weight is relatively large, so they have high stability and long half-life, and are more conducive to the purification of antibody products, but their tissue permeability is relatively low.

Method used

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  • Anti-tnf-α/pd-1 bispecific antibody and its application
  • Anti-tnf-α/pd-1 bispecific antibody and its application
  • Anti-tnf-α/pd-1 bispecific antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Anti-TNF-α plasmid construction of bispecific antibody

[0075] (1) Anti-TNF-αVL region gene and Anti-TNF-αKappa region gene amplification and connection

[0076] Use P1, P2 to amplify Anti-TNF-α VL region gene, use P3, P4 to amplify Anti-TNF-α Kappa region gene, P1, P2, P3, P4 primer sequences are as follows:

[0077] P1: 5'-GACCCAAGCTTGCATTCCTGCGCCATGGCTCCCGTGCAGCT-3' (SEQ ID NO: 17)

[0078] P2: 5'-GAGCGGCCACGGTCCGTTTGATTTCCACCTTGGTCCC-3' (SEQ ID NO: 18)

[0079] P3: 5'-ACCAAGG TGGAAATCAA ACGGACCGTGGCCGCTC-3' (SEQ ID NO: 19)

[0080] P4: 5'-AAGTACTGCTTAAGATCGATGTCGATCAGCACTCGCCCCTGTT-3' (SEQ ID NO: 20)

[0081] 1) Anti-TNF-α VL region gene amplification. The reaction system is shown in Table 1; the amplification reaction conditions are shown in Table 2.

[0082] Table 1 Anti-TNF-α VL region gene amplification reaction system

[0083]

[0084] Table 2 Anti-TNF-α VL region gene amplification reaction conditions

[0085]

[0086] 2) Anti-TNF-α Ka...

Embodiment 2

[0155] Example 2: Anti-PD-1 plasmid construction of bispecific antibody

[0156] (1) Anti-PD-1 light chain vector construction

[0157]Use P13 and P14 to amplify the Anti-PD-1 light chain gene, and use P5 and P6 to amplify the pBudCE 4.1 vector fragment. The primer sequences of P13 and P14 are as follows:

[0158] P13: 5'-GACCCAAGCTTGCATTCCTGCGCCACCATGGCTCCCGTG-3' (SEQ ID NO: 29)

[0159] P14: 5'-AAGTACTGCTTAAGATCGATGTCGACTCGAGTCAGCAGGACTTGGGC-3' (SEQ ID NO: 30)

[0160] 1) Anti-PD-1 light chain gene amplification. The reaction system is shown in Table 15; the amplification reaction conditions are shown in Table 2.

[0161] Table 15 Anti-TNF-α VL region gene amplification reaction system

[0162]

[0163] 2) PCR amplification of the pBudCE 4.1 vector fragment. The reaction system is shown in Table 5; the amplification reaction conditions are shown in Table 6.

[0164] The amplified products were subjected to agarose gel electrophoresis and gel recovery, the results we...

Embodiment 3

[0199] Example 3: Expression and purification of bispecific antibodies

[0200] (1) Suspension cell culture

[0201] Resuscitate 293F suspension cells at 37°C, 8% CO 2 The shaker was cultured at a speed of 120rpm, and OPM-293 CD05 Medium was used to culture for five generations until the cell state was stable. After the cell survival rate was greater than 90% by trypan blue staining, the cells were cultured at 1×10 6 Subculture at a cell / ml density, after 12-16 hours of subculture, wait until the cell density reaches 1.5×10 6 Transfection was performed at 1 cell / ml.

[0202] (2) Cell transfection

[0203] The amount of Anti-PD-1 plasmid and Anti-TNF-α plasmid was 2 μg / ml, and the amount of PEI transfection reagent was 5 μg / ml. The amount of plasmid and transfection reagent was calculated according to the volume of suspended cells. Add Anti-PD-1 plasmid and Anti-TNF-α plasmid to 5ml OPM-293 CD05 Medium, mix gently, and let stand for 20min. Add PEI transfection reagent to 5...

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Abstract

The invention discloses an anti-TNF-α / PD-1 bispecific antibody and its use. The present invention uses anti-human PD-1 antibody and anti-human TNF-α antibody to construct a bispecific antibody, which can neutralize TNF-α that promotes tumor cell proliferation, and at the same time bind to PD-1 to block The PD-1 / PD-L1 signal between tumor cells and T cells can effectively kill tumor cells, and has good biological activity and application prospects.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to an anti-TNF-α / PD-1 bispecific antibody and its application. Background technique [0002] Bispecific antibodies (BsAbs) can simultaneously recognize two different epitopes on the same or different antigens, overcome the shortcomings of traditional monoclonal antibodies and improve their efficacy, and can be used in multiple therapeutic areas, such as: cancer, chronic inflammation diseases and autoimmune diseases. BsAbs have become a hotspot in the field of antibody engineering and have broad application prospects in tumor immunotherapy. BsAbs have the following advantages: (1) Mediate the killing of tumors by immune cells. BsAbs have two antigen-binding arms, one of which binds to the target antigen, and the other binds to the labeled antigen on the effector cells, which can activate the effector cells. Make it target and kill tumor cells; (2) Dual-target signal blocking, exert uniqu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCC07K16/241C07K16/2818A61P35/00C07K2317/31C07K2317/56C07K2317/76C07K2317/92C07K2317/73
Inventor 刘煜纪雪梅毛颖清陈岳张祎帆
Owner CHINA PHARM UNIV
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