Bifunctional fusion protein composed of IL-2 and antibody subunit

A fusion protein, IL-2 technology, applied in the field of biomedicine, can solve problems such as the increase in the number of effector T cells

Pending Publication Date: 2022-05-06
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the clinical trials of aOX40 antibody, no significant decrease in the number of Tregs was observed, but an increase in the number of effector T cells was observed[15]

Method used

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  • Bifunctional fusion protein composed of IL-2 and antibody subunit
  • Bifunctional fusion protein composed of IL-2 and antibody subunit
  • Bifunctional fusion protein composed of IL-2 and antibody subunit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209]Example 1, IL2 / aOX40-Fc bispecific antibody significantly improved the effect of treatment alone

[0210] 1. The treatment of IL2-Fc fusion protein can partially control the tumor

[0211] Free free IL2 alone has a short half-life and requires multiple administrations. In order to prolong the half-life of IL2, we designed an IL2-Fc fusion protein (see figure 1 A), and verify the function of fusion protein in MC38 tumor model. Compared with the untreated control group, IL2-Fc could effectively control the growth of tumor, but IL2-Fc treatment alone failed to completely clear the tumor when the tumor was large ( figure 1 B). C57BL / 6 mice were subcutaneously inoculated with 5×10 5 MC38 tumor cells, tumor-bearing mice were grouped and treated on D12 (n=5 / group): 9ug IL2-Fc fusion protein was injected intraperitoneally (ip), once every three days, and treated three times in total.

[0212] The results showed that treatment with IL2-Fc fusion protein alone did not sustai...

Embodiment 2

[0236] Example 2, IL2 / aOX40-Fc can activate CD8 T cells

[0237] 1. The therapeutic effect of IL2 / aOX40-Fc bispecific antibody does not depend on NK cells

[0238] Since CD25 (IL-2 receptor α) and OX40 are mainly expressed on activated effector T cells and NK cells, in order to determine which group of immune cells the treatment of IL2 / aOX40-Fc antibody mainly depends on, we carried out different cell populations respectively The deletion experiment.

[0239] C57BL6 mice were subcutaneously inoculated with 5×10 5 MC38 tumor cells were intraperitoneally injected with 25ug IL2 / aOX40wt Fc fusion protein on the 12th, 15th, and 18th days. 400ug NK cell-deleting antibody PK136 (prepared in our laboratory) was injected intraperitoneally one day before the start of treatment, once every 4 days, for a total of 3 injections.

[0240] Figure 6 A shows the change of peripheral blood NK cells 1 day after NK cell deletion. In the mouse tumor experiment, the IL2 / aOX40-Fc antibody still...

Embodiment 3

[0258] Example 3, IL2 / aOX40-Fc can delete intratumoral Treg and increase the ratio of CD8 / Treg cells

[0259] 1. Intratumoral Treg cells highly express CD25 and OX40 molecules

[0260] In order to further study the anti-tumor mechanism of IL2 / aOX40-Fc antibody, we analyzed the expression levels of CD25 and OX40 molecules on T cells of tumor-bearing mice to analyze which group of cells IL2 / aOX40-Fc antibody mainly binds to and functions. After MC38 tumor inoculation, we analyzed the expression of IL2 receptor CD25 and OX40 on T cells in the tumor, draining lymph nodes and spleen of the mice on the 12th day respectively: Specific scheme: Mice were inoculated with 5×10 5 For MC38 tumor cells, the tumor tissues, spleen and draining lymph nodes (dLN) of tumor-bearing mice were collected on the 12th day, and the expressions of CD25 and OX40 on different cells were detected by flow cytometry.

[0261] It was found that in tumor-bearing mice, both CD25 and OX40 were highly expressed ...

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Abstract

The invention relates to a bifunctional fusion protein composed of IL-2 and an antibody subunit. The bifunctional fusion protein comprises a heterodimer composed of the following first monomer and second monomer: (1) the first monomer formed by connecting interleukin 2 (IL-2) and an immune globulin Fc single chain; and (2) a second monomer obtained by linking an Fc single chain to Fab / ScFv of an antibody of a TNF family co-stimulatory or co-inhibitory molecule or a CTLA4 molecule. The bifunctional fusion protein also comprises a homodimer, and the homodimer monomer comprises: (1) an IL-2 functional block; and (2) a monomer of an antibody or a monomer formed by connecting Fab / ScFv of the antibody with an Fc single chain, wherein the antibody is a TNF family co-stimulatory or co-inhibitory molecule or a CTLA4 molecule antibody. The invention also relates to application of the bifunctional fusion protein in preparation of antitumor drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a bifunctional fusion protein composed of IL-2 and antibody subunits. Background technique [0002] Interleukin 2 (IL2), also known as T cell growth factor, was discovered in 1976 and stimulates T cell clonal expansion in vitro[1]. IL2 is a glycoprotein of four α-helices with a molecular weight of 15.5Kd[2], which is mainly secreted by activated CD4 T cells, and other cells such as activated CD8 cells, NK cells, NKT cells and ILCs cells can also produce a small amount of IL2[ 3,4]. IL2 binds to recipient cells through autocrine and paracrine pathways and plays an important role in regulating T and NK cells. [0003] The IL2 receptor is composed of three subunits, IL2Rα (CD25), IL2Rβ (CD122) and IL2Rγc (CD132), and there are differences in the affinity between IL2 and the three subunits[5,6]. IL2 binds with α subunit with low affinity (Kd~10-8M), binds with βγ di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/395A61K47/68A61K45/06A61P35/00A61K38/20
CPCC07K14/55C07K16/2878C07K16/2818A61K39/39566A61K39/39558A61K38/2013A61K47/6813A61K45/06A61P35/00C07K2319/30C07K2317/55C07K2317/622C07K2317/24A61K2300/00A61K38/20A61K39/395A61K47/68C07K19/00C12N15/62
Inventor 彭华曹帅帅其他发明人请求不公开姓名
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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