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Soluble pd-1 variants, fusion constructs, and uses thereof

A PD-1 and soluble technology, which is applied in the direction of fusion with soluble cell surface receptors, fusion polypeptides, and targeting specific cell fusion, can solve the problems of not improving Th1CD8T cell responses

Active Publication Date: 2013-08-21
VERSITECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this HIV vaccine composition did not improve Th1 CD8 T cell responses

Method used

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  • Soluble pd-1 variants, fusion constructs, and uses thereof
  • Soluble pd-1 variants, fusion constructs, and uses thereof
  • Soluble pd-1 variants, fusion constructs, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1 - Construction of Mouse sPD-1 Vaccine Candidates

[0155] This example illustrates the construction of a mouse sPD-1-p24 fusion construct. To construct the mouse sPD-1-p24-Fc construct, the PVAX vector carrying the wild-type msPD-1 gene and p24 gene was fused to rabbit Fc DNA. The vector and p24-Fc DNA were ligated by a linker encoding GGGSGGG (SEQ ID NO: 29). Transcription is under the control of the promoter Pcmv.

[0156] The mouse sPD1 protein variant mspd1-IgVΔ was obtained by deleting amino acids 89-90 of the mouse PD-1 protein, which form the C''D loop of the IgV domain and for PD-1 and PD-L1 / L2 interactions are essential. The PVAX vector carrying mspd1-IgVA was ligated to p24-rabbit Fc DNA via a linker encoding the GGGSGGG (SEQ ID NO:29) linker sequence. In addition, a p24-Fc fusion construct was obtained by ligating a p24-carrying PVAX vector with rabbit Fc DNA. Transcription is under the control of the promoter Pcmv.

[0157] Figure 1A An alig...

Embodiment 2

[0158] Embodiment 2—the binding ability of mouse SPD-1 fusion protein and SPD-1 ligand

[0159] This example shows the binding ability of msPD-1 fusion protein to mouse sPD-1 ligand. Briefly, 293T cells were transfected with PD-L (PD-L1 and PD-L2). The binding of sPD-1 protein to PD-1 ligand was detected by FITC-anti-rabbit Fc antibody using flow cytometry, and the results were analyzed by flowJo.

[0160] Such as Figure 2A The results shown in -B reveal that mspd1-p24-Fc binds to mouse PD-1 ligands PD-L1 and PD-L2. In contrast, the variant mspd1-IgVΔ fusion protein did not bind the mouse PD-1 ligand. Furthermore, p24-Fc did not affect the interaction between PD-1 and PD-L1 / L2.

Embodiment 3

[0161] Example 3 - Wild-type mouse SPD1 vaccine induces humoral and cell-mediated immune responses

[0162] This example shows that wild-type msPDl-p24-Fc effectively induces both humoral and cell-mediated immune responses. Briefly, Balb / c mice were primed at week 0 and treated with 20 μg / mouse encoding msPd1-p24-Fc, mspd1-IgVA-p24-Fc, or p24-Fc at weeks 3 and 6. DNA vectors were enhanced via intramuscular electroporation. Mice receiving PBS served as controls.

[0163] Two weeks after the final immunization, mouse sera were collected and exposed to HIV-1 p24 viral protein. Levels of anti-p24 IgG1 and IgG2a antibodies were measured by ELISA. Anti-p24 antibody levels in control samples are not shown because absorbance readouts for these samples were below the cutoff value used to determine antibody titers. Anti-p24 antibody endpoint titers were defined as the reciprocal of the highest dilution of the test sample that produced a read at least twofold higher than that of the ...

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Abstract

Provided are soluble PD-1 (sPD-1) proteins, fusion proteins comprising said sPD-1 proteins, nucleic acids encoding said sPD-1 proteins and fusion proteins. The sPD-1 proteins have the amino acid sequences as shown in SEQ ID NO: 11, SEQ ID NO: 15 and SEQ ID NO: 25. The fusion proteins have the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23 and SEQ ID NO: 27. Also provided is pharmaceutical composition, especially a vaccine composition, comprising the sPD-1 proteins, fusion proteins or nucleic acids. The sPD-1 proteins, fusion proteins, nucleic acids and therapeutic composition of the present invention can provide protective immunity against pathogenic infection including HIV infection, and can be used in the prevention and / or treatment of tumor or cancer.

Description

[0001] manual [0002] Cross reference to related applications [0003] This application claims the benefit of US Provisional Application Serial No. 61 / 412,557, filed November 11, 2010, which is hereby incorporated by reference in its entirety. Background of the invention [0004] The programmed death 1 (PD-1) mainly expressed on T cells are B7-H1 molecules (also known as programmed death ligand 1 (PD-L1)) and B7-DC molecules (also known as programmed death ligand 1 (PD-L1) receptor for PD-L2). PD-L1 is expressed on many different cell types, whereas PD-L2 is only expressed on antigen-presenting cells such as B cells, dendritic cells and macrophages. [0005] The PD-1 / PD-L pathway, which transmits negative signals to immune cells, plays a key role in the regulation of immune responses during infection and cancer. Interaction of PD-1 with PD-L1 / L2 suppresses T cell function during HIV infection. Recent studies have shown that blockade of PD-1 by anti-PD-1 antibodies during ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K19/00C12N1/21C12N15/11C12N15/62A61K38/17A61P31/18A61P35/00
CPCA61K38/00A61K38/177A61K39/0011A61K39/002A61K39/02A61K39/12A61K48/005A61K2039/53A61K2039/54A61K2039/57C07K14/005C07K14/161C07K14/70521C07K2319/00C07K2319/30C07K2319/32C07K2319/33C12N2740/16034C12N2740/16222A61K2300/00A61K39/21A61P31/00A61P31/14A61P31/16A61P31/18A61P31/20A61P31/22A61P33/00A61P35/00A61P37/04
Inventor 陈志伟周京颖
Owner VERSITECH LTD
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