Methods and compositions for targeting polyubiquitin

Patent metadata

Field	Value
Publication number	US-10808028-B2
Application number	US-201816021248-A
Country	US
Kind code	B2
Filing date	Jun 28, 2018
Priority date	Jan 18, 2008
Publication date	Oct 20, 2020
Grant date	Oct 20, 2020

Abstract

Anti-K63-linked polyubiquitin monoclonal antibodies, and methods for using the antibodies, are provided.

First claim

We claim:

1. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to K63-linked polyubiquitin, comprising:
the HVR-H1 sequence of SEQ ID NO: 5;
an HVR-H2 sequence;
the HVR-H3 sequence of SEQ ID NO: 6;
the HVR-L1 sequence of SEQ ID NO: 3;
an HVR-L2 sequence;
and
the HVR-L3 sequence of SEQ ID NO: 4; wherein
(i) the HVR-H2 sequence is SEQ ID NO: 60 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a T at position 4;
(ii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with a D at position 1 and an S at position 3, and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A at position 2 and an A at position 4;
(iii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with a D at position 1 and an S at position 3, and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a T, V, A, or S at position 4;
(iv) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an S at position 4;
(v) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A, L, or N at position 4;
(vi) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A at position 2 and an A at position 4;
(vii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a V at position 4;
(viii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and the HVR-L2 sequence is unsubstituted SEQ ID NO: 8; or
(ix) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and an L at position 6 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a V at position 4.

2. The nucleic acid molecule of claim 1 , wherein the antibody or antigen-binding fragment does not specifically bind to K48-linked polyubiquitin or monoubiquitin.

3. The nucleic acid molecule of claim 1 , wherein the antibody or antigen-binding fragment does not specifically bind to monoubiquitin.

4. The nucleic acid molecule of claim 1 , wherein the antibody or antigen-binding fragment specifically binds to a K63-linked polyubiquitinated protein.

5. The nucleic acid molecule of claim 4 , wherein the antibody or antigen-binding fragment, has at least one property selected from inhibiting degradation of the K63-linked polyubiquitinated protein, modulating at least one polyubiquitin-mediated signaling pathway, inhibiting at least one polyubiquitin-mediated signaling pathway, and stimulating at least one polyubiquitin-mediated signaling pathway.

6. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a T at position 4.

7. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with a D at position 1 and an S at position 3, and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A at position 2 and an A at position 4.

8. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with a D at position 1 and an S at position 3, and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a T, V, A, or S at position 4.

9. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an S at position 4.

10. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A, L, or N at position 4.

11. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A at position 2 and an A at position 4.

12. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a V at position 4.

13. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and the HVR-L2 sequence is unsubstituted SEQ ID NO: 8.

14. The nucleic acid molecule of claim 1 , wherein the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and an L at position 6 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a V at position 4.

15. The nucleic acid molecule of claim 1 , wherein the antibody has a Kd for K63-linked polyubiquitin of about 6 nM to about 90 nM.

16. A method of producing the antibody or antigen-binding fragment encoded by the nucleic acid molecule of claim 1 , wherein:
(i) the HVR-H2 sequence is SEQ ID NO: 60 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a T at position 4;
(ii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with a D at position 1 and an S at position 3, and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A at position 2 and an A at position 4;
(iii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an S at position 4;
(iv) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A, L, or N at position 4;
(v) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an A at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with an A at position 2 and an A at position 4;
(vi) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a V at position 4;
(vii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and the HVR-L2 sequence is unsubstituted SEQ ID NO: 8; or
(viii) the HVR-H2 sequence is SEQ ID NO: 60 substituted with an S at position 3 and an L at position 6 and the HVR-L2 sequence is SEQ ID NO: 8 substituted with a V at position 4; and
the method comprises culturing a host cell comprising the nucleic acid molecule under conditions wherein the antibody or antigen-binding fragment is produced.

17. A vector that comprises the nucleic acid of claim 1 .

18. A host cell comprising the vector of claim 17 .

Assignees

• Genentech Inc

Inventors

• Kelley Robert F
• Matsumoto Marissa L

Classifications

• C07K2317/51

  Complete heavy chain or Fd fragment, i.e. VH + CH1 · CPC title

• C07K2317/565

  Complementarity determining region [CDR] · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• G01N2333/47

  Assays involving proteins of known structure or function as defined in the subgroups · CPC title