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Novel hyaluronic acid-hydrolyzing enzyme mutant and pharmaceutical composition comprising same

A composition and variant technology, applied in the field of novel human hyaluronidase variants, can solve problems such as insufficient thermal stability or expression level

Pending Publication Date: 2020-11-20
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Meanwhile, recombinant PH20 still does not have sufficient thermostability or expression levels in recombinant cells

Method used

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  • Novel hyaluronic acid-hydrolyzing enzyme mutant and pharmaceutical composition comprising same
  • Novel hyaluronic acid-hydrolyzing enzyme mutant and pharmaceutical composition comprising same
  • Novel hyaluronic acid-hydrolyzing enzyme mutant and pharmaceutical composition comprising same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Example 1. Construction of PH20 variants

[0187]To construct PH20 variants, the cDNA of wild-type PH20 (clone ID: hMU002604) was purchased from Korean Human Gene Bank. Wild-type PH20 encodes amino acids from L36 to S490. The PH20 gene was amplified by polymerase chain reaction (hereinafter referred to as PCR), and inserted into the XhoI and NotI restriction enzyme sites of the pcDNA3.4-TOPO vector. For expression in ExpiCHO cells, the signal peptide of human growth hormone, human serum hormone, or human Hyal1 was used as the signal peptide instead of the original signal peptide of PH20. For protein purification using HisTrap columns, the DNA sequence of the 6xHis tag is located at the 3' end of the PH20 cDNA. Amino acid substitutions of the PH20 variants were made using the PCR method and confirmed by DNA sequencing.

[0188] The list of primers used to clone PH20 variants is summarized in Table 9 below and the specific sequences of the primers are summarized in Tab...

Embodiment 2

[0227] Example 2. Construction of PH20 variants HM1 and HM6

[0228] Such as figure 1 As shown in B, amino acid residues M345 to N363 of PH20 correspond to the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 in the protein tertiary structure model. Among the amino acids of α-helix 8, C351 forms a disulfide bond with C60 of α-helix 1; Y357 forms a hydrophobic interaction with F315 of α-helix 7; and N363 forms a hydrogen bond with D69 of α-helix 1, thereby making the The secondary structure is stable ( figure 1 C).

[0229] To construct variants with higher enzymatic activity and thermostability than WT by substituting amino acids located in the α-helix 8 region of PH20 and the linker region between α-helix 7 and α-helix 8, the following variants were constructed: variant HM1, in which 12 amino acids in the region M345 to N363 were substituted; variant HM2, in which 7 amino acids in the region Y365 to V379 were substituted; and variant HM3, in which 19 ...

Embodiment 3

[0233] Example 3. Construction of PH20 variants HM4, HM7, HM8, HM9, HM10, HM11 and HM12

[0234] It is believed that through the construction of variants HM1 and HM6 in Example 2, the amino acid substitutions in the M345 to N363 and M345 to I361 regions resulted in an increase in both enzymatic activity and thermostability of PH20, a great advance in protein engineering. Therefore, based on the variants HM1 and HM6, other amino acids in the N-terminal and C-terminal direction were additionally substituted.

[0235] First, variants HM4 and HM7 were constructed, comprising the substituted amino acids in variants HM1 and HM6, and wherein the amino acids between G340 and I344 were additionally substituted by G340V, T341S, L342W, S343E and I344N . The sequences of the substituted amino acid sequences in variants HM4 and HM7 are shown in Table 11 above. Variants HM4 and HM7 were expressed in ExpiCHO cells. The protein purification results of HM7 are in Figure 5 Shown in A. HM4...

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Abstract

The present invention relates to a technical field of protein engineering for increasing the enzyme activity and thermal stability of human hyaluronidase, which is a hyaluronic acid-hydrolyzing enzyme, and relates to a hyaluronidase PH20 mutant or a fragment thereof, comprising: the substitution of at least one amino acid residue, among the amino acid sequence of the wild-type PH20 having SEQ ID NO: 1, at an alpha helix site and / or a site corresponding to a connection site thereof; and selectively, the additional deletion of an N-terminal amino acid residue and / or a C-terminal amino acid residue. Specifically, the present invention relates to a PH20 mutant and a fragment thereof, the wild-type PH20, having a sequence of SEQ ID NO: 1, comprising: the substitution of at least one residue selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T; additionally, the substitution of an amino acid positioned at alpha helix site 8 and / or theconnection site of alpha helix site 7 and alpha helix site 8; and the deletion of some amino acids of an N-terminal site and a C-terminal site.

Description

technical field [0001] The present invention relates to novel human hyaluronidase variants having increased enzymatic activity and thermostability compared to human hyaluronidase as an enzyme that hydrolyzes hyaluronic acid; and More particularly to hyaluronidase PH20 variants or fragments thereof, in the region corresponding to wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, preferably consisting of amino acid residue L36 Comprising one or more amino acid residue substitutions in the region of the alpha-helix region and / or linker region of mature wild-type PH20 composed of S490, and in said hyaluronidase PH20 variant or fragment thereof, selectively one or more of the N-terminal or C-terminal amino acid residues; methods for producing said hyaluronidase PH20 variants or fragments thereof; and pharmaceutical combinations comprising said hyaluronidase PH20 variants or fragments thereof things. Background technique [0002] Human skin is composed of epidermis,...

Claims

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Application Information

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IPC IPC(8): C12N9/26A61K38/16A61K45/06A61P35/00
CPCA61P35/00C12N9/2474A61K38/47C12Y302/01035A61K38/16A61K45/06C07K2319/02C12N9/2402C07K14/00
Inventor 朴淳宰郑惠信李承柱柳善儿宋炯楠李昌禹
Owner ALTEOGEN
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