Method for producing a multivalent fab fragment with collagen-like peptide
AssigneeIndustrial Technology Research Institute
InventorsMin-Yuan Chou, Chuan-Chuan Huang, Hsiu-Chuan Li, Ya-Ping Lai
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Abstract
The present invention relates to a method for producing multivalent Fab fragments. In particular, the invention relates to a method for the generation of trimeric Fab fragments by co-expression of a gene construct comprising a heavy chain portion of a Fab fragment and an in-frame fused collagen-like peptide, and a gene construct consisting of a light chain portion of an IgG in mammalian cells. Uses of molecules generated using the method of the invention are also described.
Claims12
A method for producing a multivalent Fab fragment comprising the steps of
- (i) co-expressing in an isolated host cell:
- (1) a first nucleic acid coding for an amino acid sequence comprising, in an N-terminal to C-terminal direction, a signal peptide, a humanized OKT3 heavy chain variable region, a CH1 region of human IgG, a hinge region, SEQ ID NO:
- 32 and a collagen-like peptide,
- wherein the hinge region consists of SEQ ID NO:
- 19,
- wherein the nucleotides coding for the hinge region are directly linked to the nucleotides coding for SEQ ID NO:
- 32,
- wherein the nucleotides coding for SEQ ID NO:
- 32 are directly linked to the nucleotides coding for the collagen-like peptide;
- and
- (2) a second nucleic acid coding for an amino acid sequence comprising, in an N-terminal to C-terminal direction, a signal peptide, a humanized OKT3 light chain variable region and a kappa light chain constant domain of human IgG;
- and
- (ii) allowing polypeptides expressed in the isolated host cell from the first nucleic acid and the second nucleic acid to trimerize into one or more multivalent Fab fragments.
A trimeric, multivalent Fab fragment comprising (1) three identical heavy chains each comprising an amino acid sequence comprising, in an N-terminal to C-terminal direction, a humanized OKT3 heavy chain variable region, a CH1 region of human IgG, a hinge region, SEQ ID NO:
- 32, and a collagen-like peptide, wherein the hinge region consists of SEQ ID NO:
- 19, wherein the amino acid sequence of SEQ ID NO:
- 19 is directly linked to the amino acid sequence of SEQ ID NO:
- 32, wherein the amino acid sequence of SEQ ID NO:
- 32 is directly linked to the collagen-like peptide;
- and (2) three identical light chains each comprising an amino acid sequence comprising, in an N-terminal to C-terminal direction, a humanized OKT3 light chain variable region and a kappa light chain constant domain of human IgG.
A method for producing a multivalent Fab fragment comprising the steps of
- (i) co-expressing in an isolated host cell:
- (1) a first nucleic acid coding for the amino acid sequence set forth in SEQ ID NO:
- 5;
- and
- (2) a second nucleic acid coding for the amino acid sequence set forth in SEQ ID NO:
- 7;
- and
- (ii) allowing polypeptides expressed in the isolated host cell from the first nucleic acid and the second nucleic acid to trimerize into one or more multivalent Fab fragments.
Isolated nucleic acids, consisting of:
- (1) a first nucleic acid coding for the amino acid sequence set forth in SEQ ID NO:
- 5;
- and
- (2) a second nucleic acid coding for the amino acid sequence set forth in SEQ ID NO:
- 7.
Dependencies are inferred from the claim text.