Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation
The invention provides antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming an antibody-like binding protein possesses dual variable domains having a cross-over orientation. The invention also provides methods for making such antigen-like binding proteins.
1 - 40 . (canceled)
41 : A method of making an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, comprising:
(a) identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ;
(b) assigning either the light chain or the heavy chain as template chain;
(c) assigning the V L of the first antibody variable domain or the second antibody variable domain as V L1 ;
(d) assigning a V L2 , a V H1 , and a V H2 according to formulas [I] and [II] below:
V L1 -L 1 -V L2 -L 2 -C L [I]
V H2 -L 3 -V H1 -L 4 -C H1 -Fc [II]
(e) determining maximum and minimum lengths for L 1 , L 2 , L 3 , and L 4 ;
(f) generating polypeptide structures of formulas I and II;
(g) selecting polypeptide structures of formulas I and II that bind the first target antigen and the second target antigen when combined to form the antibody-like binding protein; wherein:
V L1 is a first immunoglobulin light chain variable domain;
V L2 is a second immunoglobulin light chain variable domain;
V H1 is a first immunoglobulin heavy chain variable domain;
V H2 is a second immunoglobulin heavy chain variable domain;
C L is an immunoglobulin light chain constant domain;
C H1 is the immunoglobulin C H1 heavy chain constant domain;
Fc is the immunoglobulin hinge region and C H2 , C H3 immunoglobulin heavy chain constant domains; and
L 1 , L 2 , L 3 , and L 4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
42 : The method of claim 41 , wherein the first antibody variable domain and the second antibody variable domain are the same.
43 : A method of making an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, comprising:
(a) identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ;
(b) assigning either the light chain or the heavy chain as template chain;
(c) assigning the V L of the first antibody variable domain or the second antibody variable domain as V L1 ;
(d) assigning a V L2 , a V H1 , and a V H2 according to formulas [I] and [II] below:
V L1 -L 1 -V L2 -L 2 -C L [I]
V H2 -L 3 -V H1 -L 4 -C H1 [II]
(e) determining maximum and minimum lengths for L 1 , L 2 , L 3 , and L 4 ;
(f) generating polypeptide structures of formulas I and II;
(g) selecting polypeptide structures of formulas I and II that bind the first target antigen and the second target antigen when combined to form the antibody-like binding protein; wherein:
V L1 is a first immunoglobulin light chain variable domain;
V L2 is a second immunoglobulin light chain variable domain;
V H1 is a first immunoglobulin heavy chain variable domain;
V H2 is a second immunoglobulin heavy chain variable domain;
C L is an immunoglobulin light chain constant domain;
C H1 is the immunoglobulin C H1 heavy chain constant domain; and
L 1 , L 2 , L 3 , and L 4 are amino acid linkers;
and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
44 : The method of claim 43 , wherein the first antibody variable domain and the second antibody variable domain are the same.