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Artificial antibody polypeptides

a technology of artificial antibodies and polypeptides, which is applied in the direction of library member identification, directed macromolecular evolution, fungi, etc., can solve the problems of limiting the level of uptake, the size of antibodies and the complexity of six loops is a major design hurdle, and the antibody fragments are not suitable for structural analysis using nmr spectroscopy, etc., to achieve the effect of increasing the melting point and increasing the melting poin

Inactive Publication Date: 2013-10-10
RES CORP TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a fibronectin type III (Fn3) molecule with a stabilizing mutation that increases its melting point by at least 0.1°C compared to a molecule without the mutation. The Fn3 molecule contains a mutation that increases its stability, such as a substitution of an amino acid residue or the deletion of an amino acid residue. The Fn3 molecule also contains a plurality of β-strand domain sequences linked to a plurality of loop region sequences. The loop region sequences contain a unique restriction enzyme site, and the DNA molecule encoding the Fn3 molecule is cleaved at this site to produce a DNA molecule that encodes a polypeptide monobody with the stabilizing mutation. The Fn3 polypeptide monobody has improved stability and can be used for various applications such as drug development and research.

Problems solved by technology

However, the size of the antibodies and the complexity of six loops represents a major design hurdle if the end result is to be a relatively small peptide ligand.
These antibody fragments are not suitable for structural analysis using NMR spectroscopy due to their size, low solubility or low conformational stability.
However, the short persistence of scFvs in the circulation limits the exposure of tumor cells to the scFvs, placing limits on the level of uptake.
Since X-ray crystallography is not suited for characterizing flexible parts of molecules, structural studies in the solution state have not been possible to provide dynamic pictures of the conformation of antigen-binding sites.
However, as expected, they are too small and too flexible to maintain full affinity and specificity.
Mouse CDRs have been grafted onto the human Ig framework without the loss of affinity (Jones et al., 1986; Riechmann et al., 1988), though this “humanization” does not solve the above-mentioned problems specific to solution studies.
Thus, while Tendamistat may be antigenic in humans, its small size may reduce or inhibit its antigenicity.
Also, Tendamistat's stability is uncertain.
Disulfide bonds, however, are a significant disadvantage to such molecules in that they can be broken under reducing conditions and must be properly formed in order to have a useful protein structure.
Further, the size of the loops in Tendamistat are relatively small, thus limiting the size of the inserts that can be accommodated in the scaffold.
Moreover, it is well known that forming correct disulfide bonds in newly synthesized peptides is not straightforward.
When a protein is expressed in the cytoplasmic space of E. coli, the most common host bacterium for protein overexpression, disulfide bonds are usually not formed, potentially making it difficult to prepare large quantities of engineered molecules.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example i

Construction of the Fn3 Gene

[0145]A synthetic gene for tenth Fn3 of fibronectin (FIG. 1) was designed on the basis of amino acid residue 1416-1509 of human fibronectin (Kornblihtt, et al., 1985) and its three dimensional structure (Main, et al., 1992). The gene was engineered to include convenient restriction sites for mutagenesis and the so-called “preferred codons” for high level protein expression (Gribskov, et al., 1984) were used. In addition, a glutamine residue was inserted after the N-terminal methionine in order to avoid partial processing of the N-terminal methionine which often degrades NMR spectra (Smith, et al., 1994). Chemical reagents were of the analytical grade or better and purchased from Sigma Chemical Company and J. T. Baker, unless otherwise noted. Recombinant DNA procedures were performed as described in “Molecular Cloning” (Sambrook, et al., 1989), unless otherwise stated. Custom oligonucleotides were purchased from Operon Technologies. Restriction and modific...

example ii

Modifications to Include Restriction Sites in the Fn3 Gene

[0151]The restriction sites were incorporated in the synthetic Fn3 gene without changing the amino acid sequence Fn3. The positions of the restriction sites were chosen so that the gene construction could be completed without synthesizing long (>60 bases) oligonucleotides and so that two loop regions could be mutated (including by randomization) by the cassette mutagenesis method (i.e., swapping a fragment with another synthetic fragment containing mutations). In addition, the restriction sites were chosen so that most sites were unique in the vector for phage display. Unique restriction sites allow one to recombine monobody clones which have been already selected in order to supply a larger sequence space.

example iii

Construction of M13 Phage Display Libraries

[0152]A vector for phage display, pAS38 (for its map, see FIG. 8) was constructed as follows. The XbaI-BamHI fragment of pET12a encoding the signal peptide of OmpT was cloned at the 5′ end of the Fn3 gene. The C-terminal region (from the FN5F and FN5R oligonucleotides, see Table 2) of the Fn3 gene was replaced with a new fragment consisting of the FN5F and FN5R′ oligonucleotides (Table 2) which introduced a MluI site and a linker sequence for making a fusion protein with the pIII protein of bacteriophage M13. A gene fragment coding the C-terminal domain of M13 pIII was prepared from the wild-type gene III of M13 mp18 using PCR (Corey, et al., 1993) and the fragment was inserted at the 3′ end of the OmpT-Fn3 fusion gene using the MluI and HindIII sites.

[0153]Phages were produced and purified using a helper phage, M13K07, according to a standard method (Sambrook, et al., 1989) except that phage particles were purified by a second polyethylene...

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Abstract

The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

Description

[0001]Portions of the present invention were made with support of the United States Government via a grant from the National Institutes of Health under grant number GM 55042 The U.S. Government therefore may have certain rights in the invention.FIELD OF THE INVENTION[0002]The present invention relates generally to the field of the production and selection of binding and catalytic polypeptides by the methods of molecular biology. The invention specifically relates to the generation of both nucleic acid and polypeptide libraries encoding the molecular scaffolding of a modified Fibronectin Type III (Fn3) molecule. The invention also relates to “artificial mini-antibodies” or “monobodies,” i.e., polypeptides containing an Fn3 scaffold onto which loop regions capable of binding to a variety of different molecular structures (such as antibody binding sites) have been grafted.BACKGROUND OF THE INVENTIONAntibody Structure[0003]A standard antibody (Ab) is a tetrameric structure consisting of...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/78C12N15/10C12N15/09C07K16/00C07K16/18C12N1/15C12N1/19C12N1/21C12N5/10C12N15/12C12Q1/68C40B30/04G01N33/68
CPCC07K14/78C07K16/00C07K2317/565C12N15/1037C40B30/04G01N33/6845C12N15/1044
Inventor KOIDE, SHOHEI
Owner RES CORP TECH INC
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