FAB Molecules with a Rodent Hinge Region and a Non-Rodent CH1 Region

1 . A method of preparing a Fab comprising a modified heavy chain constant region, wherein the Fab comprises a CH1 domain and a hinge region in order from N- to C-terminus, comprising the steps of:

(a) providing a Fab comprising a hinge region that is not a rodent IgG hinge; and

(b) replacing the hinge with a rodent hinge.

2 . The method according to claim 1 , wherein the hinge region in step (a) is a human IgG hinge.

3 . The method according to claim 2 , wherein the human IgG1 hinge comprises the amino acid sequence EPKSC (SEQ ID NO: 94).

4 . The method according to claim 1 , wherein the rodent hinge is a wildtype rat IgG2a or rat IgG2b isotype.

5 . The method according to claim 1 , wherein the rodent hinge comprises the amino acid sequence of any one of ERRNGGIGHKC (SEQ ID NO: 32), EERNGGIGHKC (SEQ ID NO: 93), ERRQGGIGHKC (SEQ ID NO: 34), VPREC (SEQ ID NO: 26).

6 . The method according to claim 1 , wherein the rodent hinge contains not more than one cysteine.

7 . The method according to claim 1 , wherein the CH1 domain is a wildtype human IgG1 CH1 domain, any allotype of a wildtype human IgG1 CH1 domain or comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of a wildtype human IgG1 CH1 domain.

8 . The method according to claim 7 , wherein the CH1 domain comprises the amino acid sequence of an one of:

(SEQ ID NO: 1)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

V,

(SEQ ID NO: 2)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV,

(SEQ ID NO: 7)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

I,

(SEQ ID NO: 8)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVEKKV,

(SEQ ID NO: 9)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKGV

or

(SEQ ID NO: 10)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKE

V.

9 . The method according to claim 1 , wherein the modified heavy chain constant region inhibits or prevents recognition of the Fab by anti-Fab antibodies present in a host's serum.

10 . The method according to claim 1 , wherein replacing the hinge with a rodent hinge in step b) removes an epitope which is recognized by anti-Fab antibodies present in a host's serum.

11 . The method according to claim 1 , where the host's serum is human serum.

12 . The method according to claim 1 , wherein the modified heavy chain constant region consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 70-76.

13 . The method according to claim 1 , wherein the modified heavy chain constant region comprises a CH1 domain and a hinge region in order from N- to C-terminus, wherein

(a) the CH1 domain comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKEV (SEQ ID NO: 10) and

b) the hinge region comprises the amino acid sequence ERRQGGIGHKC (SEQ ID NO: 34).

14 . A Fab obtainable according to the method of claim 1 .

15 . A method of preventing or inhibiting recognition of a Fab by anti-Fab antibodies present in a host's serum comprising the steps of:

(a) providing a Fab comprising a hinge region that is not a rodent IgG hinge; and

(b) replacing the hinge with a rodent hinge.

16 . A method for treating or preventing a disease, comprising administering to a subject a Fab comprising a modified heavy chain constant region, wherein said modified heavy chain constant region comprises a CH1 domain and a hinge region, wherein the hinge region is of a rodent IgG isotype and the CH1 domain is not of a rodent IgG isotype, so that recognition of said Fab by anti-Fab antibodies present in a host's serum is inhibited.

17 . The method according claim 16 , wherein the rodent hinge region does not serve as an epitope for anti-Fab antibodies present in the host's serum.

18 . The method according to claim 16 , wherein the disease is associated with the undesired presence of a target molecule of interest specifically bound by said Fab comprising the modified heavy chain constant region.

19 . The method according to claim 16 , wherein the disease is an autoimmune disease, inflammatory disease, cancer, neovascular disease, infectious disease, thrombosis, myocardial infarction, and/or diabetes.