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Method for preparing conjugate of gp96 protein and PD1 antibody, and application of conjugate

An antibody conjugate, gp96-pd1 technology, applied in the biological field, can solve problems such as inhibition of T cell killing effect, and achieve the effect of enhancing antiviral ability, enhancing activation effect, and enhancing immune activity

Active Publication Date: 2015-12-30
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been clinically found that in the tumor of tumor patients and in the liver of hepatitis B patients, the expression of PD1 molecules on the surface of activated T cells is up-regulated, and the interferon released by CTL simultaneously up-regulates the expression of PDL1 on the surface of target cell membranes, resulting in the killing of T cells The effect is significantly suppressed

Method used

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  • Method for preparing conjugate of gp96 protein and PD1 antibody, and application of conjugate
  • Method for preparing conjugate of gp96 protein and PD1 antibody, and application of conjugate
  • Method for preparing conjugate of gp96 protein and PD1 antibody, and application of conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1, Preparation of gp96-PD1 antibody conjugate

[0093] 1. Expression of gp96 protein in insect cells

[0094] 1. Recombinant plasmid pFastBac TM 1- Construction of gp96

[0095] (1) Design and synthesis of gp96 primers: Using the human gp96 gene sequence in GenBank as a template, an EcoRI restriction site was added to the 5' end of the gp96 gene, and an XbaI restriction site was added to the 3' end. The sequence of the forward primer is: 5'-GGAATTCATGGGCAGCAGCCATCAT-3'; the sequence of the reverse primer is: 5'-GCTCTAGACTATTACAATTCATCTTTTTC-3', which was synthesized by Shanghai Sangon Bioengineering Technology Service Company, and the sequence of the primer was verified by sequencing.

[0096] (2) The mRNA of human liver cancer cell HepG2 was extracted, and cDNA was synthesized by reverse transcription.

[0097] (3) Use the cDNA obtained in step (2) as a template, and use the primers designed in step (1) to carry out PCR amplification to obtain the PCR amplif...

Embodiment 2

[0156] Example 2, the application of gp96-PD1 antibody conjugates in inhibiting the growth of hepatitis B virus

[0157] 1. Preparation of Hepatitis B Vaccine

[0158] The hepatitis B vaccine of the present invention consists of hepatitis B surface antigen HBsAg (purchased from Beijing Tiantan Biological Products Co., Ltd., approval number: S10980007, amino acid sequence shown in sequence 3 in the sequence table), hepatitis B core protein HBc 149 (Hepatitis B core protein HBc 149 The amino acid sequence is shown in Sequence 4 in the Sequence Listing) and the gp96-PD1 antibody conjugate prepared in Example 1.

[0159] In this example, gp96 is the gp96 protein expressed by the insect cells prepared in Example 1.

[0160] The PD1 antibody in this example is a product of Bristol-Myers Squibb Pharmaceutical Co., Ltd., USA, with a product number of 0003-3772-11 and a trade name of OPDIVO.

[0161] The gp96-PD1 antibody conjugate in this example is the gp96-PD1 antibody conjugate ...

Embodiment 3

[0214] Example 3. Application of gp96-PD1 antibody conjugates in inhibiting the growth of melanoma

[0215] The gp96 protein in this example is the gp96 protein derived from melanoma B16 tumor tissue in step 2 of Example 1.

[0216] The gp96-PD1 antibody conjugate in this example is the gp96-PD1 antibody conjugate in Step 3 of Example 1 (a conjugate of gp96 protein and PD1 antibody derived from melanoma tumor tissue).

[0217] 1. Experimental grouping and processing

[0218] BALB / c mice were subcutaneously inoculated with melanoma cell line B16 (purchased from ATCC (American type culture collection), product number is CRL-6475) (5 × 10 4 cells), the mice after inoculation were divided into five groups, 10 in each group, and the mice were immunized on the second day after inoculation, and the treatment was as follows:

[0219] The first group (PBS): PBS solution: the abdomen was subcutaneously injected with 0.2ml of PBS solution, immunized once every three days, and immunized...

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Abstract

The invention discloses a method for preparing a conjugate of a gp96 protein and a PD1 antibody, and application of the conjugate. The combined application of the PD1 antibody and the gp96 protein is intended to reduce the inhibition of gp96 therapeutic vaccine activated T cells caused by PD1 / PDL1 to the greatest extent and improve the immune activity of the gp96 protein. An experiment proves that the PD1 antibody greatly improves the activation of a T cell immune response caused by the gp96 of a tumor tissue source, obviously inhibits the growth of tumor cells, remarkably improves the inhibition of hepatitis B vaccine (HBV) replication caused by a therapeutic HBV taking the gp96 as an adjuvant, and improves the anti-virus ability of the therapeutic HBV.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a conjugate of gp96 protein and PD1 antibody. Background technique [0002] Heat shock proteins (heatshock proteins, HSPs) have unique immunological functions, and their immunological mechanisms include two aspects: one is to participate in T cell antigen presentation; the other is to effectively stimulate natural immunity. Heat shock proteins exist in the cytoplasm and endoplasmic reticulum, and consist of multiple members, such as HSP60, HSP70, HSP90 and gp96. As a molecular chaperone, heat shock proteins play an important role in protein folding and transportation. Another important function is that they can bind to 5-25mer antigen polypeptides in cells, enter cells through CD91 molecules on the surface of antigen-presenting cells (APCs), and bind the antigens Epitopes are presented to MHC class I and II molecules to initiate sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61K39/39A61K39/29A61K47/48A61P35/00A61P37/04A61P31/20C07K19/00C07K1/107A61K47/68
Inventor 孟颂东侯军委刘炜炜邓蒙蒙陈立钊
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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