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Anti-CD4 protein monoclonal antibody and its active fragment and application

A monoclonal antibody and protein technology, applied in the fields of prevention, immunotherapy and diagnosis, can solve problems such as interference

Active Publication Date: 2014-10-08
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the recognition epitopes of these antibodies overlap with the epitopes recognized by the HIV envelope glycoprotein gp120, these antibodies cannot react with the CD4 protein molecule bound to the envelope glycoprotein gp120, so these antibodies are used as therapeutic drugs for HIV infection There are serious defects (Bates P.A. et al., Protein Eng. 1989, 3: 13-21; McDougal J.S. et al., J Immunol. 1986, 137: 2937-2944; Landau N.R. et al., Nature. 1988, 334: 159-162 ; Dalgleish A.G. et al., Lancet.1987, 2:1047-1050), and at the same time, there will be application defects in the body due to the immune suppression of the body (Samuele E.B. et al., J Transl Med.2009, 7:101)
[0013] According to reports, the monoclonal antibody OKT4B (T.Kieber-Emmons T. et al., Biochim Biophys Acta.1989,989: 281-300), which recognizes the D2 domain of the CD4 molecule, has the ability to bind the HIV envelope glycoprotein gp120 to the CD4 protein. Obvious interference (McDougal J.S. et al., J.Immunol., J Immunol.1986, 137:2937-2944; Lundin K. et al., J Immunol Methods.1987, 97:93-100; Lamarre D. et al., EMBO J., 1989, 8: 3271-3277), but its effect of preventing HIV-infected cells from forming syncytia is obviously weaker than that of OKT4A, and the weakness in treatment is more prominent (SattentauQ.J. et al., Science.1986, 234:1120-1123; Moore J.P. et al., Science. 1990, 250:1139-42)

Method used

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  • Anti-CD4 protein monoclonal antibody and its active fragment and application
  • Anti-CD4 protein monoclonal antibody and its active fragment and application
  • Anti-CD4 protein monoclonal antibody and its active fragment and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0190] Example 1. Can inhibit HIV infection CD4 + Cellular Anti-CD4 Monoclonal Antibody Preparation

[0191] Mice: 6-week-old female BALB / c mice were provided by the Experimental Animal Center of School of Life Sciences, Xiamen University.

[0192] Preparation of hybridomas: We use standard in vivo immunization methods and PEG fusion methods to obtain monoclonal antibodies. For detailed methods, see Ed Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory 1988. The brief process is as follows:

[0193] Mice immunization: mix and emulsify the recombinant sCD4 antigen solution dissolved in PBS and complete Freund's adjuvant (CFA) in equal volumes, inject at multiple points through the muscles of the limbs, and inject 5 μg of the purified recombinant sCD4 antigen prepared in Example 2 per mouse (total volume 50 μL). 15 days and 29 days after the first immunization, booster immunization was carried out with the same dose of recombinant antigen solution plu...

Embodiment 2

[0200] Example 2. Preparation of Recombinant Human CD4 Fragment Polypeptide Used as Antigen

[0201] Preparation of human CD4 ectoregion fragments used as template: human CD4 cDNA in TZM-bl cell line (Cat. -CGG CATATG AAG AAA GTG GTG CTG GGC-3′) is the forward primer, CD4(d4)R (5′-GCGAATTCTTACCATGTGGGCAGAACCTT-3′) is the reverse primer, and the PCR thermal cycler (Biometra T3) is carried out according to the following conditions PCR reaction: 94°C for 10 minutes; followed by 25 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 minutes. A specific 1.1 kb template DNA fragment was obtained and used as a template for preparing each truncated recombinant human CD4 polypeptide. The PCR product obtained above was ligated with a commercially available pMD 18-T vector (manufactured by TaKaRa Company), and identified by Nde I / EcoR I digestion to obtain a positive clone inserted into the sCD4 gene. Using M13(+) / (-) primers, the conserved se...

Embodiment 3

[0215] Example 3. Monoclonal antibody blocking HIV infection TZM-bl cell activity verification

[0216] Select 5 strains to belong to respectively B, C, D, D / C subtype HIV full-gene experimental strain virus (HIV NL4-3 , HIV 89.6 , HIV 94UG114 , HIV MJ4 , HIV WCML249 ), through the cell neutralization experiment to determine the monoclonal antibody 15A7, 14G7 to the above virus infection CD4 + Cellular blocking / neutralizing effect. TZM-bl cells according to 1.5 × 10 4 The concentration of cells / well was inoculated in 96-well cell culture plate and used for detection after 12 hours. as per image 3 Dilute monoclonal antibodies 15A7 and 14G7 at the dilution concentrations listed in the coordinates, add 100 μL of antibody diluent and 50 μL of each HIV virus suspension (diluted to 100 TCID50) in each well of a 96-well U-bottom plate, and incubate at 37°C for 1 hour. 150 μL of the mixture was added to the cultured TZM-bl cells on a 96-well cell culture plate, and cultured a...

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Abstract

Provided are an anti-CD4 protein monoclonal antibody, active fragments and uses thereof. The antibody of the present invention can block the binding of the human immunodeficiency virus and human CD4 protein for invasion into cells. The antibody of the present invention can be used for detecting, diagnosing, preventing and treating diseases of which target cells are CD4 cells, especially AIDS caused by the human immunodeficiency virus (HIV). Further provided are corresponding hybridoma cell lines, isolated nucleic acids molecule and oligopeptides, as well as pharmaceutical compositions and kits comprising the monoclonal antibody.

Description

field of invention [0001] The present invention relates to a monoclonal antibody that can specifically bind to a human surface antigen differentiation cluster 4 (Cluster of Differentiation 4, CD4) protein, a conservative variant or an active fragment thereof, and a related coding sequence of a polypeptide or a polypeptide analog thereof, to produce the Cell lines of monoclonal antibodies, and methods and uses of the antibodies or fragments for prevention, immunotherapy and diagnosis; prevention or treatment of primates, including humans, whose source of infection is CD4 + Diseases caused by the main target cells of cells: These diseases include acquired immunodeficiency syndrome (AIDS, AIDS), AIDS-related diseases, and human immunodeficiency virus (human immunodeficiency virus, HIV) infection. Background technique [0002] Since the discovery of HIV in the last century, many researchers have devoted themselves to research on HIV molecular biology, immunity, and vaccines. How...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/20G01N33/577A61K39/395A61P31/18
CPCC07K16/2812C07K2317/622C07K2317/54C07K2317/70C07K2317/55A61P31/18
Inventor 顾颖侯汪衡方楚高双全曹芳王海虹张军
Owner XIAMEN UNIV
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