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Lnterleukin-2 receptor (IL2R) and interleukin-2 (IL2) variants for specific activation of immune effector cells

A technology of interleukin and IL2, applied in the direction of blood/immune system cells, animal cells, albumin peptides, etc., can solve problems such as limiting the efficacy of cytokines and the influence of resident immune cells

Pending Publication Date: 2021-12-31
BIONTECH CELL & GENE THERAPIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only limits the efficacy of cytokines to ACT, but also requires concomitant lymphoablative treatment (Gattinoni, L. et al. J. Exp. Med. 202, 907-12 (2005))
[0012] (3) Cytokine administration may have undesired effects on resident immune cells

Method used

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  • Lnterleukin-2 receptor (IL2R) and interleukin-2 (IL2) variants for specific activation of immune effector cells
  • Lnterleukin-2 receptor (IL2R) and interleukin-2 (IL2) variants for specific activation of immune effector cells
  • Lnterleukin-2 receptor (IL2R) and interleukin-2 (IL2) variants for specific activation of immune effector cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0495] Example 1: Construct Design

[0496] To design an interactive pair of human IL2 (hIL2) and interleukin 2 receptor subunit alpha (hIL2RA, CD25), various amino acid substitutions were made at the hIL2:hIL2RA binding interface. In more detail, three pairs of basic and acidic amino acid residues were identified as part of the ionic interactions driving hIL2:hIL2RA binding. The respective residues are swapped in a reciprocal fashion, mutating basic amino acids to acidic amino acids and vice versa. For both hIL2 and the corresponding hIL2RA, up to three amino acid positions were mutated, resulting in four hIL2 variants with predicted altered hIL2RA binding and four hIL2RA mutants with predicted altered hIL2 binding.

[0497] Four different hIL2 variants contain the following amino acid substitutions:

[0498] -hIL2_A3: K35E, K43E and E61K

[0499] -hIL2_A4: K43E and E61K

[0500] -hIL2_A5:E61K

[0501] -hIL2_A8:K43E

[0502] Four different hIL2RA mutants contained the ...

Embodiment 2

[0510] Example 2: mRNA Production

[0511]Cytokines encoding mRNA for in vitro transcription are based on the pST1-T7-AGA-dEarI-hAg-MCS-FI-A30LA70 plasmid-backbone and derived DNA-constructs. These plasmid constructs contain a 5'UTR (untranslated region, a derivative of the 5'-UTR of the Homosapiens hemoglobin subunit α1 (hAg)), a 3'FI element (where F is the amino-terminal enhancer of the split mRNA I is a 136 nucleotide long 3'-UTR segment of the 12S RNA, and I is a 142 nucleotide long segment of the mitochondrial encoded 12S RNA, both identified in Homo sapiens; WO 2017 / 060314) and 100 nucleotides Acid poly(A) tail with a linker after 70 nucleotides.

[0512] Cytokine and serum albumin (hAlb) coding sequences were derived from Homo sapiens and no changes were introduced in the resulting amino acid sequences except for the expected mutations in the hIL2 variants described above (hIL2: NP_000577.2; NCBI Protein Resource [AM1] ). For the cytokine constructs, the hIL2 varian...

Embodiment 3

[0520] Example 3: In vitro expression of RNA-encoded hAlb-hIL2 variants

[0521] The resulting hAlb-hIL2-encoding variants were analyzed by transfecting mRNA liposomes into HEK293T / 17 cells and subsequently analyzing CD25-independent activation of reporter cells expressing IL2Rβγ by supernatants containing hAlb-hIL2 variants. In vitro expression of body mRNA ( figure 1 A, B). One day before liposome transfection, 1.2×10 6 HEK293T / 17 cells were seeded in 3 mL of DMEM (Life Technologies GmbH, Cat. No. 31966-021 ) + 10% Fetal Bovine Serum (FBS, Biochrom GmbH, Cat. No. S0115 ) in 6-well plates. For liposome transfection, under sterile and RNase-free conditions, 3 μg of IVT-mRNA was prepared using 400 ng mRNA / μL Lipofectamine MessengerMax (Thermo Fisher Scientific, cat. 2 The dish should be applied to approximately 80% confluent HEK293T / 17 cells. After 20 hours of expression, supernatants were collected under sterile conditions and stored at -20°C until further use. The CD25-i...

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Abstract

The invention relates to variants of the alpha subunit of interleukin-2 receptor (IL2R) and interleukin-2 (IL2). In one embodiment, the IL2 variants described herein have amino acid substitutions at the region of IL2 that contacts the alpha (alpha) subunit of the heterotrimeric IL2 receptor complex, IL2Ralpha Beta Gamma, reducing its ability to bind and activate the heterotrimeric receptor complex. Conversely, the corresponding IL2R Alpha variants described herein have amino acid substitutions compensating for such reduced ability of IL2 variants to bind to and activate IL2R Alpha Beta Gamma, preferably at amino acid residues contacted by IL2 amino acid residues that are substituted in the IL2 variants described herein.

Description

technical field [0001] The present invention relates to the alpha subunit of interleukin-2 receptor (IL2R) and variants of interleukin-2 (interleukin-2, IL2). In one embodiment, the IL2 variants described herein have amino acid substitutions in the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex IL2Rαβγ, thereby reducing its binding and activation of the heterotrimeric IL2 receptor complex. Trimeric receptor complex capacity. In contrast, the corresponding IL2Rα variants described herein have such reduced amino acid substitutions that compensate for the IL2 variant's ability to bind to and activate IL2Rαβγ, preferably in contact with the substituted IL2 amino acid residues in the IL2 variants described herein. of amino acid residues. IL2 variants show impaired binding and / or activation of wild-type IL2R, ie IL2R comprising the alpha subunit of (wild-type) IL2R. However, variation of the alpha subunit of IL2R at least partially re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55A61P35/00
CPCC07K14/55A61P35/00A61K39/4631A61K39/4611A61K39/464402C12N5/0636A61K2239/59C07K14/7155C07K14/76C07K2319/31C07K2319/30C12N2510/00A61K38/2013
Inventor 乌尔·沙欣西纳·费勒梅尔-科普夫亚历山大·穆伊克马蒂亚斯·比特尔
Owner BIONTECH CELL & GENE THERAPIES
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