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ADAMTS-8 proteases and uses thereof

A protein and protease technology, applied in the field of disease treatment, can solve problems such as unidentified protease activity

Inactive Publication Date: 2007-05-23
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No protease activity identified for ADAMTS-8

Method used

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  • ADAMTS-8 proteases and uses thereof
  • ADAMTS-8 proteases and uses thereof
  • ADAMTS-8 proteases and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1. Creating a Phylogenetic Diagram

[0114] Proteins from the following human ADAMTS family members were pooled to create a phylogenetic diagram: ADAMTS-1 / AB037767, ADAMTS-2 / AJ003125 (published sequences have the following changes from those used in the phylogenetic diagram: W643C, P1001L, and S1089C), ADAMTS-3 / AF247668, ADAMTS-4 / AF148213, ADAMTS-5 / AF142099, ADAMTS-6 / "SEQ ID NO: 2" in US Patent Application Publication 20020120113, ADAMTS-7 / AF140675, ADAMTS-8 / AF060153 (already The published sequence has the following changes compared to the sequence used in the phylogenetic map: L11P, F13L, L21P, P23Δ, L24Δ, and L129Q, where Δ represents a deletion), ADAMTS-9 / AF261918 (the published sequence and the sequence used in the phylogenetic map The sequence has the following changes compared to: G64S and S96T), "SEQ ID NO: 9" in ADAMTS-10 / PCT Publication No. WO 02 / 60942 (the published sequence has the following changes compared to the sequence used in the phylogenetic pl...

Embodiment 2

[0117] Example 2. Construction of ADAMTS-8 expression vector

[0118] The DNA sequence of ADAMTS-8 was deposited at GeneBank (Accession No. AF060153) by Vázquez et al., supra. To isolate the gene, 4 sets of oligonucleotide primer pairs spanning the ADAMTS-8 open reading frame were designed:

[0119] The first pair of primers included ATGTTCCCCGCCCCCGCCGCCCCCCGGTG (SEQ ID NO: 2) and GGATCCCCCGAGGCGCTCGATCTTGAACT (SEQ ID NO: 3). The second pair of primers included GGATCCGGCCGGGCGACCGGGGGC (SEQ ID NO: 4) and CTCTAGAAGCTCTGTGAGATACATGGCGCT (SEQ ID NO: 5). The third pair of primers included CTCTAGACGGCGGGCACGGAGACTGTCTCCTGGATGCCCCTGGTGCGGCCCTGCCCCTCCCCACA (SEQ ID NO: 6) and ACGTGTATTTGACTTTTGGGGGGAAGACCTCGCCAGGGACTGTCAGGAGCTGCACTGTCAGAGGCTC (SEQ ID NO: 7). The fourth pair of primers included CACACGTTCTTTGTTCCTAATGACGTGGACTTTAG (SEQ ID NO: 8) and GCGGCCGCTCACAGGGGGCACAGCTGGCTTTC (SEQ ID NO: 9).

[0120] The PCR amplification of adult lung cDNA library was carried out according to...

Embodiment 3

[0123] Example 3. Establishment of CHO cell lines expressing ADAMTS-8

[0124] CHO / A2 cells were used to establish a cell line stably expressing ADAMTS-8. The CHO / A2 cell line was derived from CHO DUKX B11 by stably integrating the transcriptional activator tTA, a fusion protein comprising the Tet repressor and the herpesvirus VP16 transcriptional domain. The ADAMTS-8 / pHTop expression vector contains six tet operator repeats upstream of the ADAMTS-8 sequence. In pHTop, tTA binds to the Tet operator to activate downstream gene transcription. A gene encoding dihydrofolate reductase can also be included in the pHTop expression vector, allowing selection of stable transfectants by viral methotrexate resistance. A CHO cell line expressing ADAMTS-8 extracellularly can also be established by transfecting pHTop / ADAMTS-8 DNA into CHO / A2 cells using the manufacturer's recommended lipofection (Lipofectin from InVitrogen) protocol. Clones were selected with 0.02 μM methotrexate. Cell ...

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Abstract

The present invention features methods of using ADAMTS-8 proteins or their functional derivatives to cleave aggrecan or other proteoglycan molecules. The present invention also features methods for identifying ADAMTS-8 modulators that are capable of inhibiting or enhancing ADAMTS-8 proteolytic activities. In addition, the present invention features pharmaceutical compositions comprising ADAMTS-8 proteins or their derivatives or modulators. These pharmaceutical compositions can be used to treat diseases that are characterized by deficiencies or abnormalities in proteoglycan cleavage or metabolism.

Description

[0001] This application claims the benefit of US Provisional Patent Application Serial No. 60 / 562,687, filed April 16, 2004, the entire disclosure of which is incorporated herein by reference. technical field [0002] The present invention relates to ADAMTS-8 proteins, derivatives and modulators thereof, and methods of using them to treat diseases characterized by defects or abnormalities in proteoglycan cleavage or metabolism. Background technique [0003] The ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondinmotifs, Disintegrin And Metalloprotease with ThromboSpondinmotifs) family contains at least 19 members that are related to each other based on their common domains. In contrast to ADAM family members, ADAMTS proteins lack a transmembrane domain but contain at least one thrombospondin type 1 motif. A typical ADAMTS protein contains, from N-terminus to C-terminus, a signal sequence, a prodomain, a metalloprotease catalytic domain, an disintegrin-like domain, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64A61K38/46A61K38/48C12N9/00C12Q1/37
CPCC12N9/6489G01N2333/96486G01N2500/00A61K38/4886C12Q1/37G01N2400/00A61P17/00A61P19/02A61P43/00
Inventor E·R·拉瓦利L·A·科林斯-拉奇C·J·科科伦M·J·阿戈斯蒂诺B·A·弗里曼M·阿赖C·R·弗兰纳里M·X·吉恩
Owner WYETH LLC
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