RANKL ANTIBODY-PTH/PTHrP CHIMERIC MOLECULES

a technology of pthrp and pthrp, which is applied in the field of pthrp/pthrp chimeric molecules, and can solve problems such as bone density increas

Inactive Publication Date: 2014-04-10
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Paradoxically, intermittent administration of PTH or PTH-related protein (PTHrP) can actually cause an increase in bone density.

Method used

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  • RANKL ANTIBODY-PTH/PTHrP CHIMERIC MOLECULES
  • RANKL ANTIBODY-PTH/PTHrP CHIMERIC MOLECULES
  • RANKL ANTIBODY-PTH/PTHrP CHIMERIC MOLECULES

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparing synPTH-αRANKL-1 Light Chain Expression Plasmid

[0450]A synthetic oligonucleotide having the sequence shown in FIG. 7 (SEQ ID NO: 5) was obtained from Picoscript (Houston, Tex.). That oligonucleotide sequence contains a 5′ XbaI restriction site (TCTAGA) followed by a Kozak sequence (CCACC), which are shown in bold in FIG. 7. The oligonucleotide sequence also contains a synthetic coding sequence for the first 65 amino acids of human preproparathyroid protein (prepropTH). See, e.g., Genbank accession no. CAA23843. The first 65 amino acids of human prepropTH contains a prepro domain and amino acids 1-34 of the human PTH modulating domain. The oligonucleotide sequence also contains a coding sequence for a helical linker sequence, GGGAP. That linker coding sequence also contains a BSSHII restriction site (GCGCGC). The synthetic oligonucleotide was cloned into plasmid pCR4.0-TOPO by Picoscript prior to delivery (oligo-pCR4.0 TOPO). Oligo-pCR4.0 TOPO was digested with XbaI and BssH...

example 2

Preparing synPTH-αRANKL-1 Heavy Chain Expression Plasmid

[0455]The synPTH coding sequence was prepared as described above in Example 1.

[0456]The αRANKL-1 (also called αOPGL-1) heavy chain was amplified from αRANKL-1-IgG2 / pDSRα19 plasmid (also called αOPGL-1-IgG2 / pDSRα19, described in PCT Publication No. WO 03 / 002713) as follows. Ten ng of αRANKL-1-IgG2 / pDSRα19 plasmid DNA was used in a PCR reaction using Pfu polymerase (Stratagene). The following primers were included in the reaction:

5′αRANKL-1 IgG2 BssHII primer (SEQ ID NO: 216):            BssRII           G   A   P   E   V   Q   L   L   E5′-AA CTT GGC GCG CCC GAG GTG CAG CTG TTG GAG-3′3′ human IgG2 constant region primer(SEQ ID NO: 217):5′-G CAT GTC GAC TCA TTT ACC CGG AGA CAG GGA           SalI   *   K   G   P   S   L   S GAG-3′ L

[0457]The PCR reaction generated a 1372 base pair PCR product, which encodes the amino acid sequence of αRANKL-1 heavy chain with 3 amino acids (GAP) of the linker sequence on the N-terminus. The 1372 ba...

example 3

Expression of synPTH-αRANKL-1

Expression in Chinese Hamster Ovary (CHO) Cells

[0460]For expression of synPTH-αRANKL-1 heavy chain fusion, dihydrofolate reductase deficient (DHFR-) serum-free adapted CHO AM-1 / D (described in U.S. Pat. No. 6,210,924) cells were co-transfected with synPTH-αRANKL-1-IgG2 pDSRα20 and αRANKL-1-kappa / pDSRα19 (also called αOPGL-1-kappa / pDSRα19; see PCT Publication No. WO 03 / 002713) using the calcium phosphate method. For expression of synPTH-αRANKL-1 light chain fusion, dihydrofolate reductase deficient (DHFR-) serum-free adapted CHO AM-1 / D cells were co-transfected with synPTH-αRANKL-1-kappa pDSRα20 and αRANKL-1-IgG2 / pDSRα19 (also called αOPGL-1-IgG2 / pDSRα19; see PCT Publication No. WO 03 / 002713) using the calcium phosphate method.

[0461]Expression of each of the synPTH-αRANKL-1 heavy chain fusion and the synPTH-αRANKL-1 light chain fusion was carried out as follows. Transfected cells were plated in 10 cm plates and selected in DMEM media supplemented with 1× ...

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Abstract

Chimeric molecules comprising receptor activator of NF-κB ligand (RANKL) antibodies and parathyroid hormone / parathyroid hormone-related protein (PTH / PTHrP) peptides are described. Compositions and methods for the treatment of bone diseases are also described.

Description

[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 736,664, filed Nov. 14, 2005. U.S. Provisional Application No. 60 / 736,664 is incorporated by reference herein in its entirety for any purpose.FIELD[0002]Chimeric molecules comprising antibodies that bind receptor activator of NF-κB ligand (RANKL) and parathyroid hormone / parathyroid hormone-related protein (PTH / PTHrP) peptides (RANKL antibody-PTH / PTHrP chimeric molecules) are provided. Compositions and methods for the treatment of bone diseases are also described.BACKGROUND[0003]Bone tissue provides support for the body and includes mineral (including calcium and phosphorous), a matrix of collagenous and noncollagenous proteins, and cells. Living bone tissue exhibits a dynamic equilibrium between formation of bone, which is called deposition, and break-down of bone, which is called resorption. Three types of cells found in bone, osteocytes, osteoblasts and osteoclasts, are involved in this equilibrium. O...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/24
CPCC07K16/241A01K67/0278A01K2207/15A01K2217/00A01K2227/105A01K2267/03A61K2039/505C07K14/635C07K14/70575C07K16/2875C07K2317/21C07K2317/76C07K2319/00A61P19/00A61P19/02A61P19/08A61P19/10A61P29/00A61P35/00A61P37/00A61P37/06
Inventor KOSTENUIK, PAULSHEN, WENYANBOONE, THOMAS C.
Owner AMGEN INC
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