Engineered monomeric antibody fragments
View Patent ↗The present invention relates to monomeric polypeptides comprising an engineered monomeric antibody fragment (e.g., monomeric Fc-containing polypeptides) wherein the monomeric Fc comprises one or more engineered N-linked glycosylation sites in the CH3-CH3 dimerization interface. Methods for producing such engineered monomeric antibody fragments and their use in diagnostics and therapeutics are also provided.
1. A polypeptide comprising an IgG CH2 and an IgG CH3 domain,
(i) wherein the CH3 domain comprises one or more engineered N-linked glycosylation sites in a CH3-CH3 dimerization interface,
(ii) wherein the engineered N-linked glycosylation sites are selected from the group consisting of Q347N-X-Y349T, Q347N-X-Y349S, Y349N-X-L351T, Y349N-X-L351S, L351N-X-P353T, L351N-X-P353S, S354N-X-D356T, S354N-X-D356S, D356N-X-L358T, D356N-X-L358S, E357N-X-T359S, K360N-X-Q362T, K360N-X-Q362S, S364N-X-T366S, L368N-X-K370T, L368N-X-K370S, K370N-X-F372T, K370N-X-F372S, K392N-X-T394S, V397N-X-D399T, V397N-X-D399S, S400N-X-G402T, S400N-X-G402S, D401N-X-S403T, F405N-X-Y407T, F405N-X-Y407S, Y407N-X-K409T, Y407N-X-K409S, K409N-X-T411S, K439N-X-L441T, K439N-X-L441S, S444N-X-G446T, and S444N-X-G446S, wherein X is any amino acid except Pro, and
(iii) wherein said polypeptide forms a soluble monomer.
2. A fusion protein comprising at least two recombinantly linked polypeptides of claim 1 , wherein each polypeptide has the same or different engineered N-linked glycosylation sites in the CH3-CH3 dimerization interface.
3. The polypeptide of claim 1 , wherein the amino acid modifications in the CH3-CH3 dimerization interface are selected from the group consisting of S364N-X-T366S, L368N-X-K370T, L368N-X-K370S, F405N-X-Y407T, F405N-X-Y407S, Y407N-X-K409T, and Y407N-X-K409S.
4. A polypeptide comprising an IgG CH2 and an IgG CH3 domain,
(i) wherein the CH3 domain comprises two engineered N-linked glycosylation sites in a CH3-CH3 dimerization interface,
(ii) wherein the engineered N-linked glycosylation sites are selected from the group consisting of a) S364N and Y407N-X-K409T; b) S364N-X-T366S and Y407N-X-K409T; c) S364N and Y407N-X-K409S; and d) S364N-X-T366S and Y407N-X-K409S, wherein X is any amino acid except Pro; and
(iii) wherein said polypeptide forms a soluble monomer.
5. A fusion protein comprising at least two recombinantly linked polypeptides of claim 4 , wherein each polypeptide has the same or different engineered N-linked glycosylation sites in the CH3-CH3 dimerization interface.
6. The polypeptide of claim 1 , wherein the CH2 domain comprises one or more engineered N-linked glycosylation sites in a CH2-CH2 dimerization interface, and
wherein the N-linked glycosylation sites are selected from the group consisting of S239N-X-F241S, S239N-X-F241T, F241N-X-243T, F241N-X-243S, E258N, E258N-X-T260S, T260N-X-V262T, T260N-X-V262S, V262N-X-V264S, V262N-X-V264T, K288T, K288S, K288N-K290T, K288N-K290S, V305N, and V305-X-T307S.
7. A fusion protein comprising at least two recombinantly linked polypeptides, wherein each polypeptide comprises at least one engineered N-linked glycosylation site, wherein the engineered N-linked glycosylation site comprises at least one amino acid modification selected from the group consisting of E258N-X-T260S, T260N-X-V262T, T260N-X-V262S, V305N, V305N-X-T307S, Q347N-X-Y349T, Q347N-X-Y349S, S364N-X-T366S, T366N-X-L368T, T366N-X-L368S, L368N-X-K370T, L368N-X-K370S, D401N, D401N-X-S403T, F405N-X-Y407T, F405N-X-Y407S, Y407N-X-K409T, Y407N-X-K409S, and K409N-X-T411S, wherein X is any amino acid except Pro, and wherein each polypeptide has the same or different engineered N-linked glycosylation site.
8. The polypeptide of claim 1 , further comprising a Fab.
9. The fusion protein of claim 2 , further comprising a Fab.
10. The fusion protein of claim 2 , wherein each polypeptide is linked recombinantly via C-N terminus linkage or via a linker.
11. The polypeptide of claim 10 , wherein the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:89), wherein n=1-10.
12. The fusion protein of claim 2 , wherein the polypeptide comprises two recombinantly linked polypeptides, wherein each polypeptide has the same engineered N-linked glycosylation sites in each CH3-CH3 dimerization interface, and further wherein the engineered N-linked glycosylation sites are S364N-X-T366 and Y407N-X-K409T.
13. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1 .
14. A host cell comprising the polynucleotide of claim 13 .
15. A method for producing a polypeptide comprising the step of culturing the host cell of claim 14 and, optionally, recovering the polypeptide.
16. The polypeptide of claim 1 , wherein the amino acid modifications are F405N-X-Y407T.
17. The polypeptide of claim 1 , wherein the amino acid modifications are L368N-X-K370T.
18. The polypeptide of claim 1 , wherein a amino acid modifications are S364N-X-T366S.
19. The polypeptide of claim 1 , wherein the amino acid modifications are Y407N-X-K409T.
20. The polypeptide of claim 4 , wherein the amino acid modifications are S364N and Y407N-X-K409S.
21. The fusion protein of claim 5 , wherein the polypeptide comprises two recombinantly linked polypeptides, wherein each polypeptide comprises amino acid modifications of S364N and Y407N-X-K409T.