Multispecific antigens binding fragments and multispecific antibodies derived therefrom comprising mutant ch1 and cl-kappa domains

A multispecific antigen-binding fragment comprising at least two Fab fragments with different CH1 and CL domains, wherein said Fab fragments comprise:

• a. a mutated Fab fragment consisting of:
• i. the VH and VL domains of an antibody recognizing an epitope of interest;
• ii. a CH1 domain of a human immunoglobulin comprising substitution of the leucine residue at position 143 of said CHI domain with a glutamine residue and substitution of the serine residue at position 188 of said CH1 domain with a valine residue;
• and
• iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the valine residue at position 133 of said CL domain with a threonine residue and substitution of the serine residue at position 176 of said CL domain with an valine residue;
• and
• b. at least another Fab fragment which is selected from the group consisting of
• b1) a mutated Fab fragment consisting of:
• i. the VH and VL domains of an antibody recognizing an epitope of interest;
• ii. a CH1 domain of a human immunoglobulin comprising substitution of the threonine residue at position 192 of said CH1 domain with a glutamic acid residue;
• and
• iii. a CL-kappa domain of a human immunoglobulin by substitution of the asparagine residue at position 137 of said CL domain with a lysine residue and substitution of the serine residue at position 114 of said CL domain with an alanine residue;
• b2) a mutated Fab fragment consisting of:
• i. the VH and VL domains of an antibody recognizing an epitope of interest;
• ii. a CH1 domain of a human immunoglobulin comprising substitution of the leucine residue at position 124 of said CH1 domain with an alanine residue and substitution of the leucine residue at position 143 of said CH1 domain with a glutamic acid residue;
• and
• iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the valine residue at position 133 of said CL domain with a tryptophane residue;
• b3) a mutated Fab fragment consisting of:
• i. the VH and VL domains of an antibody recognizing an epitope of interest;
• ii. a CH1 domain of a human immunoglobulin comprising substitution of the valine residue at position 190 of said CHI domain with an alanine residue;
• and
• iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the leucine residue at position 135 of said CL domain with a tryptophane residue, and substitution of the asparagine residue at position 137 of said CL domain with an alanine residue;
• and
• b4) a wild-type Fab fragment consisting of wild consisting of wild-type CH1 and wild-type CL domains of an immunoglobulin, and the VH and VL domains of an antibody recognizing an epitope of interest;
• wherein the position numbers used for the CH1 and CL domains refer to Kabat numbering and each Fab fragment recognizes a different epitope of interest, and said Fab fragments are tandemly arranged in any order, the C-terminal end of the CH1 domain of the first Fab fragment being linked to the N-terminal end of the VH domain of the following Fab fragment through a polypeptide linker.