Claims
- 1. A method for improved recombinant antibody subunit dimerization, comprising:
providing nucleic acid sequences which code for a plurality of antibody subunits having heavy polypeptides and light polypeptides, each said polypeptide further including at least one of constant and variable regions with interacting interface segments; modifying at least one codon of said nucleic acid sequence to replace an amino acid occurring naturally in said antibody with a charged amino acid at a position in each said interface segment of said light polypeptide variable region, said charged amino acid having a first polarity; modifying at least one codon of said nucleic acid sequence to replace an amino acid occurring naturally in said antibody with a charged amino acid at a position in each said interface segment of said heavy polypeptide variable region corresponding to said position in said light polypeptide variable region, said charged amino acid having a second polarity opposite said first polarity; expressing said modified nucleic acid sequence; and interacting said modified heavy and light polypeptide variable regions of said expressed sequence.
- 2. The method as defined in claim 1, wherein said modified nucleic acid sequences are cloned and expressed by bacteria.
- 3. The method as defined in claim 2, wherein said modified heavy polypeptide variable region is expressed in a first recombinant bacteria, and said modified light polypeptide variable region is expressed in a second recombinant bacteria.
- 4. The method as defined in claim 3, wherein said expressed modified variable regions are assembled into heterologous aimers.
- 5. The method as defined in claim 1, wherein said charged amino acids are selected from the group consisting of aspartic acid, lysine, arginine, and glutamic acid.
- 6. The method as defined in claim 5, wherein said light polypeptide variable region is modified by replacement of glutamine with aspartic acid in said light polypeptide interface segment; and said heavy polypeptide variable region is modified by replacement of glutamine with lysine in said heavy polypeptide interface segment.
- 7. The method as defined in claim 5, wherein said polypeptide variable region is modified by replacement of glutamine with aspartic acid in said light polypeptide interface segment; and said heavy polypeptide variable region is modified by replacement of glutamine with arginine in said heavy polypeptide interface segment.
- 8. The method as defined in claim 1, wherein said nucleic acid sequences code for a light polypeptide interacting with said heavy polypeptide through at least one of positions 36, 38, 87, 89, 96, and 98 of said interface segment of said light polypeptide variable region.
- 9. The method as defined in claim 8 wherein said light polypeptide is selected from the group consisting of kappa and lambda constructs.
- 10. The method as defined in claim 9, wherein said charged amino acids are selected from the group consisting of aspartic acid, lysine, arginine, and glutamic acid.
- 11. The method as defined in claim 10, wherein:
said light polypeptide variable region is modified by replacement of glutamine with one of aspartic acid and glutamic acid at interfacial position 38; and said heavy polypeptide variable region is modified by replacement with one of lysine and arginine at a corresponding interfacial position of said heavy polypeptide variable region.
- 12. The method as defined in claim 10, wherein:
said light polypeptide variable region is modified by replacement of glutamine with one of lysine and arginine at interfacial position 38; and said heavy polypeptide variable region is modified by replacement with one of aspartic acid and glutamic acid at a corresponding interfacial position of said heavy polypeptide variable region.
- 13. The method as defined in claim 1, further including modifying said nucleic acid sequences to remove hydrophobic amino acids naturally occurring in said antibody from said interface segment of said heavy polypeptide variable region.
- 14. A method for increasing heterologous immunoglobulin domain affinity, comprising:
providing a light chain having a variable domain, said domain modified by substitution of at least one interfacial position with a first charged amino acid, said first charged amino acid having a first polarity; providing a heavy chain having a variable domain, said domain modified by substitution of at least one interfacial position with a second charged amino acid, said heavy chain interfacial position corresponding to said light chain interfacial position, said second charged amino acid having a second polarity opposite said first polarity; and interacting said modified heavy and light chains to induce heterologous dimerization, such that production of functional antigen binding and variable domain fragments is enhanced.
- 15. The method as defined in claim 14, wherein said first and second charged amino acids are selected from the group consisting of aspartic acid, lysine, arginine, and glutamic acid.
- 16. The method as defined in claim 15, wherein:
said light chain variable domain is modified by substitution with one of aspartic acid and glutamic acid at said interfacial position; and said heavy chain variable domain is modified by substitution with one of lysine and arginine at said interfacial position.
- 17. The method as defined in claim 15, wherein:
said light chain variable domain is modified by substitution with one of lysine and arginine at said interfacial position; and said heavy chain variable domain is modified by substitution with one of aspartic acid and glutamic acid at said interfacial position.
- 18. The method as defined in claim 14, wherein said immunoglobulin is selected from the group consisting of IgG, IgA, IgM, IgD and IgE globulins.
- 19. The method as defined in claim 18, wherein said light chain is a kappa IV protein.
- 20. The method as defined in claim 19, wherein said light chain protein variable domain is modified in at least one of interfacial positions 36, 38, 87, 89, 96, and 98, and said heavy chain protein variable domain is modified at a corresponding subset of interfacial positions.
- 21. The method as defined in claim 20, wherein said first and second charged amino acids are selected from the group consisting of aspartic acid, lysine, arginine, and glutamic acid.
- 22. The method as defined in claim 21, wherein:
said light chain variable domain is modified by substitution with one of aspartic acid and glutamic acid at interfacial position 38; and said heavy chain variable domain is modified by substitution with one of lysine and arginine at interfacial position 39.
- 23. The method as defined in claim 22, wherein said light chain variable domain is modified by aspartic acid substitution, and said heavy chain variable domain is modified by arginine substitution.
- 24. A variant kappa IV light chain protein having an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
- 25. A nucleic acid sequence comprising a coding region for a protein having an amino acid sequence selected from the group consisting of sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
- 26. The nucleic acid sequence as defined in claim 25, wherein the sequence of said coding region is selected from the group consisting of:
(a) the codons of SEQ ID NO. 1; (b) the codons sequence of SEQ ID NO. 2; (c) the codons sequence of SEQ ID NO.3; and (d) nucleic acid sequences complementary to the codons of (a), (b), or (c).
- 27. The nucleic acid sequence as defined in claim 25, further including vector DNA, such that said coding region can be introduced into a host cell.
BACKGROUND OF THE INVENTION
[0001] The United States Government has rights in this invention pursuant to Contract No. W-31-109-ENG-38 between the U.S. Department of Energy (DOE) and the University of Chicago representing Argonne National Laboratory.
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
08373380 |
Jan 1995 |
US |
Child |
09274163 |
Mar 1999 |
US |