Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-03-22
2002-11-26
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S387100, C530S350000
Reexamination Certificate
active
06485943
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to methods for optimizing production of recombinant antibodies and nucleic acid sequences which code for novel light chain proteins, the later of which are used in conjunction with the inventive methods. More particularly, the novel light chains which demonstrate the efficacy of the inventive methods can also be utilized in conjunction with a panel for comparing the amino acid sequences of amyloid-associated unmunoglobulin light chains to sequences of non-pathogenic light chains. In such a manner protein regions responsible for self-association and fibril formation can be identified and, ultimately, provided a basis for rational drug design.
Detailed analyses of the structures and biophysical properties of unmunoglobulin molecules have, over the years, probed many aspects of immunoglobulin function, particularly antibody-antigen interactions and effector functions. See Padlan,
Anatomy of the Antibody Molecule,
Mol. Immunol. 31:169-217, 1994. Immunoglobulin genes have been cloned and altered by mutagenesis to investigate effects of the changes on biological activities, and synthetic immunoglobulin genes have been generated for the production of unique antibody reagents for medical and diagnostic purposes. Another important area of immunoglobulin biology and analysis is the structural characterization of pathological protein deposits formed in humans when plasma cell dyscrasias result in excess production of immunoglobulin protein chains.
Amyloidosis is a severe pathological condition in which deposits of extracellular protein form insoluble fibers in tissues. Amyloid fibers are non-branching fibrils of diameter 70-100 A. Birefringence of bound Congo Red dye demonstrates that proteins within an amyloid fibril are highly ordered. The fibrils are virtually insoluble, except under extremely denaturing conditions, suggesting a large number of molecular interactions contribute to amyloid stability. These tissue deposits impair organ function, and extensive amyloid deposition can lead to death due to organ failure. Many different types of proteins are known to form amyloids, but any particular amyloid deposit contains an essentially homogeneous protein core of primarily &bgr;-sheet structure. See Stone,
Amyloidosis: A Final Common Pathway for Protein Deposition in Tissues,
Blood 75:531-545, 1990. In light chain amyloidosis (AL-amyloidosis) a monoclonal immunoglobulin light chain forms the amyloid deposits. See Glenner et al.,
Amyloid Fibril Proteins: Proof of Homology with Immunoglobulin Light Chains by Sequence Analyses,
Science 172:1150-1151, 1971. Amyloid fibrils from patients suffering AL-amyloidosis occasionally contain only intact light chains, but more often they are formed by proteolytic fragments of the light chains which contain the VL domain and varying amounts of the constant domain, or by a mixture of fragments and fuil-length light chains. Not all light chains from plasma cell dyscrasias form protein deposits; some circulate throughout the body at high concentrations and are excreted with the patients urine without pathological deposition of the protein in vivo. See Solomon,
Clinical Implications of Monoclonal Light Chains,
Semin. Oncol. 13:341-349, 1986; Buxbaum,
Mechanisms of Disease: Monoclonal Immunoglobulin Deposition, Amyloidosis, Light Chain Deposition Disease, and Light and leavy Chain Deposition Disease,
Hematol/Oncol. Clinics of North America
6:323-346, 1992; and Eulitz,
Amyloid Formation from Immunoglobulin Chains, Biol. ChenL Hoppe-Seyler
373:629-633, 1992.
In some types of hereditary amyloidoses, single amino acid changes in normal human proteins are responsible for amyloid fibril fornation See Natvig et al.,
Amvloid and Amyloidosis
1990. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1991, and references cited therein. It is unlikely, however, that any single amino acid position or substitution will fully explain the many different immunoglobulin light chain sequences associated with AL-amyloidosis. Rather, several different regions of the light chain molecule may sustain one or more substitutions which affect a number of biophysical characteristics, such as dimer formation, exposure of hydrophobic residues, solubility, and stability.
Increased dimerization, for example, may promote amyloid deposition of a protein. It has been shown that an extremely high proportion of rREC occurs as dimers, even at very low concentrations of the recombinant protein. The calculated dimerization constant for rREC is ~10
7
, approximately two orders of magnitude higher than that of rLEN. The dimerization constant of rLEN, ~5×10
5
M
−1
, is in the range of self-association constants observed for other human immunoglobulin light chains. For KI protein AU, for example, a value of 6.6×10
4
M
−1
was experimentally determined (see Maeda et al., Kinetics of Dimerization of the Bence-Jones Protein AU, Biophys. Chem. 9:57-64, 1978); values from ~10
3
M
−1
to ~10
6
M
−1
were estimated for a large number of human immunoglobulin KI light chains (see Stevens et al.,
Self-association of Human Immunoglobulin &kgr;I Light Chains: Role of the Third Hypervariable Region,
Proc. Natl. Acad. Sci. USA 77:1144-1148, 1980); and values of ~2.5×10
5
M
−1
to ~5.0×10
6
M
−1
were calculated for variant REI VKI domains. Computer simulation of rREC dimerization, however, yield a dimerization constant of 5×10
7
M
−1
.
It has been suggested that unusual amino acids within the inner &bgr;-sheets which form the contact regions at the dimer interface may be responsible for increasing dimer stability of amyloidogenic light chains, thereby promoting fibril formation. See Dwulet et al.,
Amino Acid Sequence of a ⊖ VI Primary
(
AL
) Amyloid Protein. Scand. J. Immunol. 22:653-660, 1985; Liepnicks et al.,
Comparison of the Amino Acid Sequences of ten kappa I Amyloid Proteins for Amyloidogenic Sequences,
In: Natvig J B, et al. Amyloid and Amyloidosis 1990. Dordrecht, The Netherlands: Kluwer Academic Publishers, pp. 153—156, 1991; and Aucouturier et al.,
Complementary DNA Sequence of Human Amyloidogenic Immunoglobulin Light-Chain Precursors.
Biochem. J. 285:149-152, 1992. The positional effect of amino acids is illustrated by two unanticipated features in the crystallographic structures of naturally occuhing light chains obtained from human patients. In one structural investigation study, a glutamine residue at position
38
was observed to have been replaced by a histidine residue in the Bence-Jones protein Loc. The crystal structure of the protein crysted from ammonium sulfate differed from that of the protein crystallized from distilled water. The quaternary interactions exhibited by the protein in the two crystal forms were sufficiently different to suggest fimdalmentally different interpretations of the structural basis for the function of this protein. See Schiffer et al,
The Structure of a Second Crystal Form of Bence Jones Protein Loc: Strikingly Different Domain Associations in Two Crystal Forms of a Single Protein,
Biochemistry 28:4066-4072, 1989. In a second crystallographic analysis, a highly conserved tyrosine residue at position
36
was observed to have been replaced by a phenylalanine residue, the structural differences again suggesting an altered quaternary interaction. See Huang et al.,
Novel Immunoglobulin Variable Domain Interaction is Observed,
American Crystallographic Association Meeting, 1993, p 127. Notwithstanding findings of this sort, the stability of amyloidogenic dimers is not fully understood: The sequence of the amyloid protein REC differs from that of LEN primarily at CDR residues and not at residues comprising the &bgr;-sheet framework.
Nonetheless, there has been great interest in deteimining the sequences of amyloid-associated immunoglobulin light chains and comparing them to sequences of non-pathogenic light chains to identify regions of the proteins responsible for self-association and fibr
Raffen Rosemarie
Schiffer Marianne
Stevens Fred J.
Stevens Priscilla Wilkins
Foley & Lardner
Helms Larry R.
Huff Sheela
The University of Chicago
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