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Polypeptide-antibody immune conjugate and preparation method thereof

A technology of immunoconjugates and antibodies, which is applied in the direction of antineoplastic drugs, drug combinations, pharmaceutical formulations, etc., to achieve the effect of simple synthesis methods

Inactive Publication Date: 2017-06-13
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to construct an antibody or antibody fragment that can be widely used in various solid tumors. The antibody or antibody fragment can not only target tumor tissue, but also mediate antibody-dependent cell-mediated cytotoxicity and kill tumor cells. , to make up for the limitation that the antibody that triggers ADCC is limited by the enlargement of tumor cell surface antigens, and to expand the scope of use of immunotherapy with ADCC as the killing mechanism

Method used

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  • Polypeptide-antibody immune conjugate and preparation method thereof
  • Polypeptide-antibody immune conjugate and preparation method thereof
  • Polypeptide-antibody immune conjugate and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of membrane-penetrating peptide-antibody Fc fragment conjugate (pHLIP-Fc)

[0036]1. Covalent cross-linking of mouse IgG2a Fc protein with sulfo-SMCC

[0037] 200 μg of mouse IgG2a Fc protein and 55.2 μg of sulfo-SMCC were dissolved in 0.5 mL of 10 mM phosphate buffer solution with pH=7.4 and stirred at room temperature for 1 h. Equilibrate the NAP-5 SephadexG25 column with 10mL of 10mM phosphate buffer (pH=7.4), add 0.5mL of the reaction product to the column, remove unreacted SMCC, and concentrate and centrifuge the product to 200 μL through an Amiconultra ultrafiltration tube with a molecular weight cut-off of 3KDa. A maleimide-modified Fc protein was obtained.

[0038] 2. The product obtained in step 1 and 0.2 mg pHLIP were dissolved in 0.3 mL of 10 mM phosphate buffer solution with pH = 7.4 and stirred at room temperature for 4 hours to generate pHLIP-Fc. The unreacted pHLIP was removed by centrifugation with an Amicon ultrafiltration tube w...

Embodiment 2

[0039] Example 2 Cell Membrane Anchoring of pHLIP-Fc Immunoconjugates under Acidic pH and Physiological pH Conditions

[0040] Seed B16 cells in confocal dishes at 37 °C 5% CO 2 Cultivate for a while. The DMEM medium (containing 10% fetal bovine serum) was adjusted with a pH meter to obtain complete medium with pH 6.8 and pH 7.4. The pH of the phosphate buffer was adjusted with a pH meter to obtain phosphate buffers of pH 6.8 and pH 7.4.

[0041] (1) Observation of the cell membrane anchoring effect of pHLIP-Fc under slightly acidic conditions (pH=6.8). When the cells were confluent to 60%, they were washed three times with pH=6.8 phosphate buffer, and pHLIP-Fc / a and pHLIP-Fc / b were diluted in complete medium with pH=6.8, and added to the confocal dish. 37°C 5% CO 2 After culturing the cells for 2 hours, discard the medium, wash three times with pH=6.8 phosphate buffer, and fix with 4% paraformaldehyde for 15 minutes. After blocking with blocking solution for 60 min at ro...

Embodiment 3

[0044] Example 3 pHLIP-Fc conjugates cause antibody-dependent cell-mediated cytotoxicity ADCC ability identification

[0045] The whole splenocytes of C57 / BL-6 mice were extracted as ADCC effector cells, and the splenocytes were counted. The target cells were mouse B16 melanoma cells and breast cancer 4T1 cells.

[0046] (1) ADCC capability identification under slightly acidic conditions (pH 6.8)

[0047] The target cells were treated with different drug groups, each drug was incubated in the medium with pH 6.8 for 1 h, and the pH value of the buffer used in the subsequent experiments was adjusted to 6.8. Target cells were trypsinized and counted. The effector cells and target cells were mixed into a 96-well plate at a ratio of 25:1, and the number of target cells was 20,000 per well. The effector cells and target cells interacted for 4 hours in a medium with pH 6.8.

[0048] (2) Identification of ADCC ability under physiological conditions (pH 7.4)

[0049] The target cel...

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Abstract

The invention provides a polypeptide-antibody immune conjugate and a preparation method thereof. The immune conjugate is formed by connecting a polypeptide and an antibody or an antibody FC segment through a covalent bond, wherein the polypeptide is pH-sensitive cell-penetrating peptide. According to the cell-penetrating peptide-antibody immune conjugate, the characteristic of pH response cell penetration of the cell-penetrating peptide is reserved, and the function of causing antibody dependent cell-mediated cytotoxin (ADCC) of the antibody is reserved. An in vitro experiment proves that acid response cell penetration can be achieved by the polypeptide-antibody immune conjugate and the protein of the antibody or the antibody Fc segment is anchored on a cell membrane to induce ADCC. An in vivo experiment proves that the polypeptide-antibody immune conjugate is capable of obviously inhibiting growth of murine melanoma and breast cancer and can be applied to immunotherapy of a clinical solid tumor.

Description

technical field [0001] The invention relates to the technical field of pharmaceutical engineering, in particular to a polypeptide-antibody immunoconjugate and a preparation method thereof. Background technique [0002] Malignant tumors are the second largest killer of humans after cardiovascular and cerebrovascular diseases. In recent years, with the gradual increase in the clinical application of therapeutic monoclonal antibodies, antibody-dependent cell-mediated cytotoxicity (antibody-dependent cellmediated cytotoxicity, ADCC) The antitumor effect has been paid more and more attention. Antibody-dependent cell-mediated cytotoxicity refers to that cells with killing activity (such as NK cells, macrophages, and neutrophils) recognize the surface-expressed Fc receptors (FcR) coated on the target antigen ( Such as the Fc segment on bacteria or tumor cells), directly kills target cells. Natural killer cells (NK cells) are the main effector cells that mediate ADCC's specific an...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K47/68A61P35/00
CPCA61K38/16
Inventor 赵颖聂广军郎佳妍季天骄齐菲菲
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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