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A fusion protein of il-2 mutant and antibody and its application
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A fusion protein and mutant technology, applied in the field of biotechnology and antibody engineering, can solve the problem of low reaction rate
Active Publication Date: 2022-05-20
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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However, the response rate of single-drug patients is low
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Embodiment 1
[0142] Example 1: Treg cells inhibit WT IL-2 from exerting its biological function
[0143] Considering the toxic and side effects of clinical use of large doses of IL-2, we fused IL-2 with the Fc of human antibody IgG1, and expressed and purified it using a mammalian suspension cell expression system. Low doses of Free IL-2 could not control the growth of tumors. When B16F10 tumor-bearing mice were treated with the same dose of IL2-Fc, the tumor growth of mice was relatively controlled but still showed an upward trend ( figure 1 A). This result indicated that the wild-type IL-2 also played the function of suppressing the immune response in the process of functioning. Through detection, it was found that there were more Tregs in tumor tissue than in lymphoid organs ( figure 1 B). This suggests the necessity of reducing IL-2 binding and inducing activation of proliferating Tregs.
Embodiment 2
[0144] Example 2: sumIL-2 enhances the binding and expansion of activated CD8 T cells, reduces the binding and expansion of Treg cells
[0145] We introduced the above six mutation sites in IL-2. We expressed and purified the fusion Fc forms of WTIL-2-Fc and sumIL-2-Fc, and tested the ability of the two fusion proteins to bind to the receptor and bind to effector cells in vitro.
[0146] To characterize the ability of WT IL-2 and sumIL-2 to bind different T cell subsets, we compared the differences in T cell binding of IL-2 fusion proteins by in vitro cell binding assays. In the same spleen cells,
[0147] (1) The ability of WT IL-2 to bind Treg cells is higher than that of memory CD8+ T cells ( figure 1 C);
[0148] (2) On the contrary, sumIL-2 is more inclined to bind memory CD8 + T cells but not Treg cells ( figure 1 C).
[0149] These in vitro experiments demonstrate that sumIL-2 is designed to activate memory CD8+ T cells rather than Treg cells.
[0150] In order...
Embodiment 3
[0151] Example 3: SumIL-2 has stronger anti-tumor activity than WT IL-2
[0152] In order to compare the anti-tumor function of the constructed IL-2 mutant and wild-type IL-2 in vivo, we used the mouse melanoma model to evaluate. SumIL-2 showed a tendency to bind activated CD8+ T cells in in vitro cell binding experiments, so the changes in T cells in tumors after treatment with sumIL-2 or WT IL-2 were first detected.
[0153] The same dose (5 μg) of sumIL-2-Fc and WT IL-2-Fc fusion protein was injected into the tumor, and the change of the cell ratio in the mouse tumor was detected by flow cytometry 3 days later. CTL is a very important factor associated with tumor regression, we found that sumIL-2 can increase the ratio of CD8 / CD4 ( figure 1 F), and can increase the ratio of CD8 / Treg ( figure 1 G), which indicates that mutant IL-2 can indeed expand more CD8+ T cells in the tumor. With such strong evidence, we further evaluated the tumor therapeutic effects of sumIL-2 an...
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Abstract
The present invention relates to a fusion protein of an IL-2 mutant and an antibody and applications thereof, the fusion protein is a heterotrimeric protein composed of an IL-2 mutant (sumIL-2) and a tumor treatment antibody, and its structure Similar to antibodies, the structural schematic diagram is shown in Figure 2A, and the three monomers of the heterotrimer are: (1) Monomer 1: IL-2 mutant (sumIL-2) and immunoglobulin Fc The fusion protein formed by the fusion of fragments, including sumIL-2 and immunoglobulin Fc region from the N-terminal to the C-terminal sequence; (2) Monomer 2: a single heavy chain of the tumor treatment antibody, from the N-terminal to the C-terminal sequence Comprising the heavy chain variable region VH, heavy chain CH region and immunoglobulin Fc region of the antibody, the immunoglobulin Fc region contained in monomer two can pair with the immunoglobulin Fc fragment of monomer one to form a heterodimer (3) monomer three: a single light chain (including VL and CL regions) of the tumor treatment antibody, the CL region contained in monomer three can pair with the CH of monomer two to form a heterodimer of half-antibody structure; The tumor treatment antibody is an antibody targeting a highly expressed membrane protein on the surface of tumor cells.
Description
technical field [0001] The invention belongs to the technical fields of biotechnology and antibody engineering, and specifically relates to a fusion protein of an IL-2 mutant and an antibody and its application. Background technique [0002] Interleukin-2 (IL-2), a T-cell growth factor induced by antigen stimulation, is a pleiotropic cytokine that plays a key role in the immune response 1,2 . A potent inducer of cytotoxic T cells and NK cells, IL-2 was one of the first FDA-approved immunotherapies for metastatic melanoma and renal cell carcinoma 3,4 . Unfortunately, due to short in vivo half-life, therapeutic doses and severe toxicity 5 And other issues, IL-2 immunotherapy has not been widely used. [0003] IL2 induces the proliferation of regulatory T cells (Tregs) by binding to IL-2 receptor alpha (IL-2Ra), which is preferentially expressed on Tregs 6,7,8,9,10 . IL-2 depletion by Treg cells has been shown to reduce IL-2-induced antitumor immunity, suggesting that Tre...
Claims
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