Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions.
The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.
Ill
C R
CR
Division of Imaging and Therapeutics Research and Development, Hybritech, Inc., San Diego, CA 92121, USA.
Gonzales
J N
JN
Houtz
E K
EK
Ludwig
J R
JR
Melcher
E D
ED
Hale
J E
JE
Pourmand
R
R
Keivens
V M
VM
Myers
L
L
Beidler
K
K
Stuart
P
P
Cheng
S
S
Radhakrishnan
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001667:Binding, Competitive; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D017421:Sequence Analysis; D014162:Transfection
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins