METHOD FOR ENGINEERING IMMUNOGLOBULINS

Abstract

The present invention relates to a method for engineering an immunoglobulin comprising a variable domain and at least one modification in at least two structural loops of said immunoglobulin and determining the binding of said immunoglobulin to an epitope of an antigen, wherein the unmodified immunoglobulin does not significantly bind to said epitope, comprising the steps of: providing a nucleic acid encoding an immunoglobulin comprising at least two structural loops, modifying at least one nucleotide residue of each of said structural loops, transferring said modified nucleic acid in an expression System, expressing said modified immunoglobulin, contacting the expressed modified immunoglobulin with an epitope, and determining whether said modified immunoglobulin binds to said epitope, immunoglobulins produced by such a method and libraries of immunoglobulins.

Claims

1. A polypeptide scaffold comprising an immunoglobulin fold of a human antibody variable domain, wherein structural loops A-B, C-C′, C″-D and E-F of said human antibody variable domain do not comprise a CDR loop, wherein at least two of said structural loops A-B, C-C′, C″-D and E-F (i) comprise at least one amino acid selected from the group consisting of tryptophan, tyrosine, phenylalanine, histidine, isoleucine, serine, methionine, alanine and asparagine, and (ii) form a solvent accessible surface.

2. The polypeptide scaffold of claim 1, wherein said human antibody variable domain is selected from the group consisting of a Variable Heavy (VH) domain, a Variable kappa (Vκ) domain, a Variable lambda (Vλ) domain.

3. The polypeptide scaffold of claim 1, wherein said at least two structural loops of said human antibody variable domain comprises at least one tyrosine in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at least one tryptophan in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at least one histidine in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at least one asparagine in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at least one methionine in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at least one serine in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, and/or at least one isoleucine in any one of the positions 6 to 20, amino acids 44 to 52, amino acids 67 to 76 and amino acids 92 to 101 and/or at least one phenylalanine in any one of the positions 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101, wherein the numbering is according to the ImMunoGeneTics numbering system (IMGT).

4. The polypeptide scaffold of claim 1, wherein said at least two structural loops of said human antibody variable domain form a solvent accessible surface within one or more of (i) amino acids 8 to 20, (ii) amino acids 44 to 50, (iii) amino acids 67 to 76 and (iv) amino acids 89 to 101, wherein the numbering is according to the ImMunoGeneTics numbering system (IMGT).sup.1.

5. The polypeptide scaffold of claim 1, wherein said polypeptide scaffold or fragment thereof, is further combined with one or more of said polypeptide scaffolds or fragment thereof, or with one or more polypeptides, or fragment thereof, to obtain a combination polypeptide.

6. The polypeptide scaffold of claim 5, wherein said combination polypeptide is a fusion protein further comprising one or more of the group consisting of an immunoglobulin, a ligand, an enzyme, and a toxin.

7. The polypeptide scaffold of claim 1, wherein each of said structural loops A-B, C-C′, C″-D and E-F comprises a solvent accessible surface.

8. A kit comprising the polypeptide scaffold of claim 1.

9. A polypeptide scaffold comprising an immunoglobulin fold of a human antibody Constant domain, wherein the structural loops of said human antibody constant domain are connected by beta strands, wherein at least two of said structural loops (i) comprise at least one amino acid selected from the group consisting of tryptophan, tyrosine, phenylalanine, histidine, isoleucine, serine, methionine, alanine and asparagine, and (ii) form a solvent accessible surface.

10. The polypeptide scaffold of claim 9, wherein said human antibody constant domain is selected from the group consisting of a CH1 domain, a CH2 domain and a CH3 domain.

11. The polypeptide scaffold of claim 9, wherein said at least two structural loops of said human antibody constant domain comprises at least one tyrosine in any one of the positions 12 to 17, 45 to 50, 69 to 75 and 93 to 98, and/or at least one tryptophan in any one of the positions 12 to 17, 45 to 50, 69, 71 to 75, 93 to 94 and 96 to 98, and/or at least one histidine in any one of the positions 12 to 17, 46, 47, 49, 50, 69 to 74 and 93 to 98, and/or at least one asparagine in any one of the positions 12 to 17, 45 to 47, 49, 50, 70 to 73, 75, 94 to 96 and 98, and/or at least one methionine in any one of the positions 12 to 17, 46 to 50, 69 to 71, 73 to 75, 93, 95, 96 and 98, and/or at least one serine in any one of the positions 13, 71, 75, 94, 95 and 98, and/or at least one isoleucine in any one of the positions 12, 14 to 17, 45 to 50, 69, 70, 72 to 75, 93 and 96 to 98, and/or at least one phenylalanine in any one of the positions 15, 46, 48, 70 to 73, 75, 93, 95 and 98, wherein the numbering is according to the ImMunoGeneTics numbering system (IMGT).

12. The polypeptide scaffold of claim 9, wherein said at least two structural loops of said human antibody constant domain comprise at least one solvent accessible surface within one or more of (i) amino acids 7 to 21, (ii) amino acids 25 to 39, (iii) amino acids 41 to 81, (iv) amino acids 83 to 85, (v) amino acids 89 to 103 and (vi) amino acids 106 to 117, wherein the numbering is according to the ImMunoGeneTics numbering system (IMGT).

13. The polypeptide scaffold of claim 9, wherein said polypeptide scaffold or fragment thereof, is further combined with one or more said polypeptide scaffold or fragments thereof, or with one or more polypeptides, or fragments thereof, to obtain a combination polypeptide.

14. The polypeptide scaffold of claim 13, wherein said combination polypeptide is a fusion protein further comprising one or more of the group consisting of another immunoglobulin, a ligand, an enzyme, and a toxin.

15. The polypeptide scaffold of claim 9, wherein three of said at least two structural loops of said human antibody constant domain form a solvent accessible surface.

16. A kit comprising the polypeptide scaffold of claim 9.

Description

[0361] FIG. 1: Sequence alignment of the amino acid sequences of SEQ ID No. 1 and SEQ ID No. 2.

[0362] FIG. 2: Nucleotide sequence encoding the synthetic gene coding for the anti-TNF-alpha camel VHH domain, as well as the translation in amino acid one-letter code. Restriction sites used for cloning are underlined in the nucleotide sequence. Amino acid residues to be randomized in the library are underlined in the amino acid sequence. (Inserted amino acids are not given in this sequence.)

[0363] FIG. 3 shows the schematic of the PCR reactions and ligation procedure. Horizontal arrows indicate the positions and directions of the PCR primers, vertical lines indicate the positions of the NcoI, BglII and NotI sites, respectively (from left to right).

[0364] FIG. 4: Schematic of the PCR reactions used to construct the library as described in example 6.

[0365] FIG. 5: scFv 2F5 wt synthetic gene library 1 (VH-Linker-VL) and translation, with two structural loops randomized

[0366] FIG. 6: scFv 2F5 wt synthetic gene library 2 (VH-Linker-VL) and translation, with two structural loops randomized

[0367] FIG. 7: scFv 2F5 wt synthetic gene library 3 (VH-Linker-VL) and translation, with three structural loops randomized

[0368] FIG. 8: scFv 2F5 wild-type synthetic gene with translation (VH-Linker-VL)