Humanized CTLA-4 single chain antibody and human perforin path formed peptide P34 recombinant immunotoxin

A CTLA-4 and single-chain antibody technology, applied in the field of fusion proteins, can solve the problems of strong immunogenicity, low internalization steps, and low efficiency of action, and achieve the effects of weak antigenicity, high efficiency, and low efficiency

Inactive Publication Date: 2007-01-24
WEST CHINA HOSPITAL SICHUAN UNIV
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AI Technical Summary

Problems solved by technology

[0007] 1. High toxicity and side effects: These toxins are harmful to normal cells and often cause non-specific damage. Even after molecular modification, the toxicity still exists (Onda M. et al., The Journal of Immunology, 2000, 165: 7150-6);
[0008] 2. Low efficiency: These toxins must be internalized by molecules, that is, penetrate the cell membrane and be transported into the cell to initiate a series of enzymatic reactions leading to cell apoptosis, and the internalization steps are often not high, which greatly affects the immune system. Toxin effect play (Panchal RG., Biochemical Phamacology, 1998, 55:247-52);
[0009] 3. Strong immunogenicity: the molecular weight of these toxins is still very large, they have strong antigenicity, and are easy to produce antibodies
Moreover, there are often natural antibodies to pathogenic bacterial toxins in the human body, which have a certain neutralizing effect on both natural toxins and modified toxins, which will inevitably reduce the killing efficiency of targeted molecules (Hall PD. et al., Clinical Immunology , 2001, 100:191-7)
[0010] 4. Easy to produce drug resistance: At present, killer molecules mainly kill target cells through the mechanism of inhibiting intracellular protein synthesis, which is easy to produce drug resistance
[0011] In addition to the above-mentioned toxins, there is another class of membrane-active toxins that do not require internalization and kill cells by forming channels on the cell membrane, but the lack of structural understanding has restricted their application
[0013] Currently, there is no relevant literature on recombinant immunotoxins based on CTLA-4 scFv and small channel-forming peptides

Method used

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  • Humanized CTLA-4 single chain antibody and human perforin path formed peptide P34 recombinant immunotoxin
  • Humanized CTLA-4 single chain antibody and human perforin path formed peptide P34 recombinant immunotoxin
  • Humanized CTLA-4 single chain antibody and human perforin path formed peptide P34 recombinant immunotoxin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Cloning of guide fragment gene

[0054] The amplified template is the pCANTAB 5E plasmid (GE Company) loaded with the Anti-CTLA-4-hS83 gene, and the amplified template can also be prepared according to the method described by Pistillo et al. (Pistillo MP et al, Molecular characterization and applications of recombinant scFv Abs to CTLA-4 co-stimulatory molecule. Tissue Antigens, 2000, 55: 229-38).

[0055] Primers were designed according to the gene sequence of Anti-CTLA-4-hS83:

[0056] P1 5'-CAGTGAGCTCATGGCCGAGGTGCAGCTGG-3'

[0057] P2 5'-AGTCAAGCTTACCTAGGACGGTCAGCTTGG-3'

[0058] High-fidelity Platimum Pfx DNA polymerase (Invitrogen, USA) was used for PCR amplification. The PCR amplification conditions were as follows: 94°C for 10 min, 94°C for 45 sec, 50°C for 1 min, 68°C for 2 min, a total of 30 cycles. A guide fragment gene of about 750bp was amplified by PCR, and the corresponding restriction site was introduced.

[0059] The PCR product was purified by a ...

Embodiment 2

[0078] 1. Cloning of hS83 gene

[0079] Using the Anti-CTLA-4-hS83 plasmid as a template, design primers based on its gene sequence:

[0080] P5 5'-CAGTGAGCTCATGGCCGAGGTGCAGCTGG-3'

[0081] P6 5'-CAGTGTCGACTCAACCTAGGACGGTCAGCTTGG-3'

[0082] PCR was performed with high-fidelity Platimum Pfx DNA polymerase, and the PCR conditions were as follows: 10 min at 94°C, 45 sec at 94°C, 1 min at 50°C, 2 min at 68°C, a total of 30 cycles. A guide fragment gene of about 750bp was amplified by PCR, and the corresponding restriction site was introduced.

[0083] After the PCR product was purified by the Mini Gel Recovery Kit, it was digested with SacI / SalI, and then cloned into pBlueselect. SacI / SalI double enzyme digestion was used to identify, and the identified positive clones were re-sequenced for verification, and the plasmid with correct sequencing was named pBS-hS83. Restriction map see Figure 4 .

[0084] 2. Construction and induced expression of hS83 expression plasmid

[0...

Embodiment 3

[0094] The cell lines used in the following experiments were all purchased from the Cell Bank of the Chinese Academy of Sciences, and their product numbers and full names are as follows:

[0095] Raji: number TcHu44, tumor cell line of human Burkitt's lymphoma;

[0096] 6T-CEM: number TcHu2, human T cell leukemia cell line;

[0097] (The above two cell lines both constitutively express CTLA-4.)

[0098] ECV-304: numbered GNHu3, normal human umbilical vein endothelial cells.

[0099] 1. Cell killing specificity analysis

[0100] 1. Comparison of the killing effect of activated and non-activated T cells

[0101] Collect activated T cells (Ta) and non-activated T cells (To) by 1.5×10 5 Each well was inoculated in a 96-well plate, and then hS83-P34 was added to a final concentration of 1.5 μmol / L. After 24 hours, the amount of surviving cells was measured by MTS method at 490 nm, and hS83 was used as a control. Taking the cell survival rate of the PBS blank control group as 1...

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Abstract

The present invention relates to new recombinant humanized immunotoxin hS83-P34, which contains fusion protein comprising the antigen associated molecule of humanized cytotoxic T cell antigen 4 and human porforin channel forming active molecule. The immunotoxin of the present invention has strict targeting, less tendency of damaging health cell, less toxic side effect, no need of internalization and intracellular transport, high acting efficiency, less likelihood of generating corresponding antibody, weak immunogenicity and less tendency of generating drug resistance. It may be used in treating some tumors and autoimmune diseases and suppressing immunologic rejection reaction in organ transplantation.

Description

technical field [0001] The present invention relates to a new type of recombinant immunotoxin, in particular to an active small molecular fragment based on the antigen binding molecule of cytotoxic T cell antigen 4 (Cytotoxic T Lymphocyte Antigen 4, CTLA-4) and the channel of membrane active toxin. Recombinantly constructed fusion protein hS83-P34. [0002] The present invention also relates to the nucleotide sequence and amino acid sequence of the immunotoxin. [0003] The present invention also relates to a pharmaceutical composition comprising the recombinant immunotoxin. Background technique [0004] According to the principle of mutual recognition between antigens and antibodies, cytokines, growth factors, hormones and receptors, molecules that can kill cells are connected with guiding molecules such as antibodies to construct immunotoxins, which can specifically kill cells and realize drug delivery. targeted therapy. [0005] Humanized cytotoxic T cell antigen 4 (Cy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/395A61P37/06A61P35/00
Inventor 卢晓风程惊秋曾令宇万琳丘小庆陆燕蓉李胜富陈利弘李幼平步宏
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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