Modified il-2 molecules and uses thereof

An IL-2, molecular technology, applied in the field of modified IL-2 molecules, which can solve problems such as side effects, chemotherapeutic drug resistance, and cancer cells cannot be completely removed from the patient's body.

Pending Publication Date: 2022-04-22
LETO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, surgery may not be feasible due to patient health or advanced disease conditions, and cancer cells are often not completely removed from the patient after surgery; radiotherapy is only indicated when tumor tissue is more sensitive to radiation than normal tissue and high doses of radiation often cause serious side effects; hormone therapy is rarely used as a single agent, and while it may be effective, it is usually used to prevent or delay cancer recurrence after other treatments have removed most of the cancer cells The biggest problem with chemotherapy is that patients may quickly develop resistance to chemotherapy agents
[0004] In addition, immunoregulatory factors (such as interleukins) can also be adapted to exert anti-tumor effects on animal models and cancer patients, and avoid the above-mentioned defects in various traditional therapies. Systemic toxic and side effects limit its use to a large extent

Method used

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  • Modified il-2 molecules and uses thereof
  • Modified il-2 molecules and uses thereof
  • Modified il-2 molecules and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Preparation of IL-2 / 15 chimera from mammalian cells

[0096] 1.1 Synthesis and construction of expression plasmids

[0097] Entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize IL-2 / 15 chimera 1 (SEQ ID NO.14), sushi-hIgG1Fc (SEQ ID NO.19), sushi-hIgG4Fc (SEQ ID NO.21), sushi- The target gene fragments of the 4 proteins of mIgG2aa.1Fc (SEQID NO.22) were cloned between the EcoRI and HindIII sites of the pZD vector to obtain 4 expression plasmids, numbered respectively PM619, PM432, PM657, and PM599. On the basis of PM619, a point mutation was carried out according to the method described in "Molecular Cloning", and the 115th position (corresponding to the 121st position of the natural IL-2 molecular amino acid sequence) phenylalanine (F) was mutated into tryptophan (W), Further, the plasmid of IL-2 / 15 chimera 2 (SEQ ID NO.23) was obtained, and the plasmid number was PM824. Then the synthesized or constructed plasmids were transformed into DH10B, seq...

Embodiment 2

[0121] Embodiment 2 prepares IL-2 / 15 chimera from escherichia coli

[0122] 2.1 Synthesis and construction of expression plasmids

[0123] Entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the IL-2 / 15 chimera 1 gene fragment (SEQ ID NO.26), and clone it into the pET41a vector between the NdeI and XhoI sites to obtain the expression plasmid, numbered 1187.

[0124] 2.2 Expression and purification of IL-2 / 15 chimera in Escherichia coli

[0125] According to the operation method mentioned in "Molecular Cloning", the plasmid was extracted, and the expression strain BL21(DE3) was transformed. The expression of IL-2 / 15 chimera 1 was carried out according to the routine method of protein prokaryotic expression. Afterwards, through techniques such as classical denaturation techniques and chromatography (see for example: Yunier Rodríguez- et al, Preparative Biochemistry and Biotechnology, 47:9, 889-900) were purified to obtain a relatively pure chimeric protein.

Embodiment 3I

[0126] Example 3 Determination of the Binding Ability of IL-2 / 15 Chimera to IL2Rα and IL2 / 15Rβ

[0127] Biolayer interferometry (biolayer interferometry, BLI) was used to measure the affinity between the target protein and the receptor. For the method, see (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013.5(2): p.270-8) to proceed. The receptor proteins IL-15Rα-his, IL2Rα-his and IL2 / 15Rβ-Fc / Fc used in the experiment are all produced by our company, and no IL2Rα-binding IL-2 derivatives and no IL2Rα-binding IL-2 complexes are based on the patent publication The record preparation of CN111018961A. The IL-15(N72D) / sushi-hIgG1Fc complex was prepared according to the literature (K.-p.Han et al. / Cytokine 56(2011)804-810). The buffer formulation was 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.1% BSA and 0.05% tween20; the receptor proteins were all immobilized on the corresponding sensors in advance, and then according...

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Abstract

The present application discloses a modified IL-2 molecule wherein the modification comprises replacing a region in which the IL-2 molecule mediates the binding of the IL-2 molecule to IL2R [alpha] with a region in which the IL-15 molecule mediates the binding of the IL-15 molecule to the IL15R [alpha], the region in which the IL-15 molecule mediates the binding of the IL-15 molecule to the IL15R [alpha] comprising nine or more amino acid residues. The invention also discloses a protein heterodimer, which comprises the modified IL-2 molecule, and IL15R alpha (sushi structural domain) or a variant of IL15R alpha (sushi structural domain). According to the modified IL-2 molecule and / or the protein heterodimer containing the modified IL-2 molecule, the affinity with IL2R alpha is remarkably reduced, so that the side effects of the modified IL-2 molecule and / or the protein heterodimer in clinical treatment are greatly reduced, and the modified IL-2 molecule and / or the protein heterodimer have a good patent medicine prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a modified IL-2 molecule and its application. Background technique [0002] According to public information, data released by the World Health Organization in February 2017 showed that there are more than 14 million new cancer cases worldwide every year, and this number is expected to increase to more than 21 million by 2030; 8.8 million people die of cancer every year , the global cancer death rate accounts for nearly one-sixth of the total death toll, and the prevention and treatment of cancer is a worldwide problem that needs to be solved urgently in the medical field. [0003] Currently, the traditional methods used to treat cancer include surgery, chemotherapy, hormone therapy, and radiotherapy (see for example: Stockdale, 1998, Principles of Cancer Patient Management, Scientific American: Medicine American: Medicine), Vol. 3, eds. Rubenstein and Federman, Chapter 12, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K38/20A61K38/17A61K47/68A61P35/00
CPCC07K14/55C07K14/5443C07K14/7155C12N15/85C12N5/0686A61K47/68A61K47/6813A61P35/00C07K2319/00C07K2319/30C12N2800/107C12N2510/02A61K38/00A61P35/04A61P37/04C12R2001/19A61K38/17A61K38/20C07K19/00C12N5/10C12N15/70C12N15/74
Inventor 赵耀张玉刘慧杰朴锦华张建军张秋磊张维王国永张天赋
Owner LETO LAB CO LTD
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