Antibodies comprising C-terminal light chain polypeptide extensions and conjugates and methods of use thereof

Patent metadata

Field	Value
Publication number	US-11208497-B2
Application number	US-201415107884-A
Country	US
Kind code	B2
Filing date	Dec 23, 2014
Priority date	Dec 23, 2013
Publication date	Dec 28, 2021
Grant date	Dec 28, 2021

Abstract

The present disclosure provides light chain polypeptides that include a C-terminal extension, as well as antibodies and antibody conjugates containing such modified light chain polypeptides, where the C-terminal extension includes one or more cysteine residues. Conjugates that include an antibody of the present disclosure conjugated to an agent via the cysteine residue of the C-terminal amino acid extension are also provided. The present disclosure further provides nucleic acids encoding an antibody light chain polypeptide that includes a C-terminal amino acid extension including a cysteine residue. Pharmaceutical compositions including the antibodies or conjugates of the present disclosure are also provided, as are methods of making and use of the modified anti-bodies and conjugates of the present disclosure.

First claim

What is claimed is:

1. An antibody, or an antigen-binding fragment thereof, comprising two heavy chains and two light chains, wherein each of the light chain polypeptides comprises a C-terminal amino acid extension comprising one or more cysteine residues,
wherein the C-terminal amino acid extension is from 6 to 50 amino acids or less in length, and comprises the amino acid sequence that is selected from the group consisting of ESSCDVKLVEKSFET (SEQ ID NO: 32), DCGFTS (SEQ ID NO: 33), DVITMDPKDNCSKDAN (SEQ ID NO: 34), DHVKPKETENTKQPSKSCHKPK (SEQ ID NO: 35), DVITMDPKDNC (SEQ ID NO: 98), ESSCDVKLV (SEQ ID NO: 101), and DHVKPKETENTKQPSKSC (SEQ ID NO: 102);
wherein the antibody or antigen-binding fragment is an IgG or antigen-binding fragment thereof, and
wherein the C-terminal amino acid extension does not specifically bind antigen.

2. The antibody or antigen-binding fragment of claim 1 , wherein the C-terminal amino acid extension comprises one or more amino acid spacers that do not comprise a cysteine residue.

3. The antibody or antigen-binding fragment of claim 2 , wherein the spacer comprises from 1 to 30 amino acids, or from 3 to 20 amino acids, or from 4 to 17 amino acids.

4. The antibody or antigen-binding fragment of claim 2 , wherein the spacer comprises one or more glycine (G) residues and one or more serine (S) residues.

5. The antibody or antigen-binding fragment of claim 4 , wherein the spacer comprises the sequence GGGS (SEQ ID NO: 1).

6. The antibody or antigen-binding fragment of claim 2 , wherein the C-terminal amino acid extension comprises from 2 to 10 spacers and wherein (a) the spacers have the same amino acid sequence, or (b) have different amino acid sequences.

7. The antibody or antigen-binding fragment of claim 1 , wherein the cysteine residue of the C-terminal amino acid is conjugated to a therapeutic agent or a labeling agent.

8. The antibody or antigen-binding fragment of claim 7 , wherein the C- terminal amino acid extension comprises two or more cysteine residues, and wherein at least two of the two or more cysteine residues are conjugated to an agent independently selected from the group consisting of a therapeutic agent and a labeling agent.

9. The antibody or antigen-binding fragment of claim 1 , wherein the antigen-binding fragment is selected from the group consisting of: a Fab, a F(ab′) 2 , a Fab′, an Fv, and an scFv.

10. A conjugate, comprising:
the antibody or antigen-binding fragment of claim 1 ; and
an agent conjugated to the antibody or antigen-binding fragment via a cysteine residue of the C-terminal amino acid extension.

11. The conjugate of claim 10 , wherein the agent is conjugated to the cysteine residue via a linker.

12. The conjugate of claim 10 , wherein the C-terminal amino acid extension comprises one or more amino acid spacers that do not comprise a cysteine residue.

13. The conjugate of claim 12 , wherein the spacer comprises from 1 to 30 amino acids, or from 3 to 20 amino acids, or from 4 to 17 amino acids.

14. The conjugate of claim 12 , wherein the spacer comprises one or more glycine (G) residues and one or more serine (S) residues.

15. The conjugate of claim 14 , wherein the spacer comprises the sequence GGGS (SEQ ID NO: 1).

16. The conjugate of claim 12 , wherein the C-terminal amino acid extension comprises from 2 to 10 spacers and wherein (a) the spacers have the same amino acid sequence, or (b) at least two of the spacers have different amino acid sequences.

17. The conjugate of claim 10 , wherein the agent is a therapeutic agent or a labeling agent.

18. The conjugate of claim 10 , wherein the agent is a cytotoxic agent.

19. The conjugate of claim 10 , wherein the agent is an in vivo imaging agent.

20. The conjugate of claim 10 , wherein the agent is conjugated to a cysteine residue of the C-terminal amino acid extension.

21. The conjugate of claim 10 , wherein the C-terminal amino acid extension comprises two or more cysteine residues, and wherein at least two of the two or more cysteine residues are conjugated to an agent independently selected from a therapeutic agent and a labeling agent.

22. The conjugate of claim 10 , wherein the antigen-binding fragment is selected from the group consisting of: a Fab, a F(ab′) 2 , a Fab′, an Fv, and an scFv.

23. A pharmaceutical composition comprising: the conjugate of claim 10 ; and
a pharmaceutically acceptable excipient.

24. A method comprising administering to a patient in need thereof a therapeutically effective amount of the conjugate of claim 10 .

25. A method of making an antibody conjugate, comprising: conjugating an agent to the antibody or antigen-binding fragment of claim 1 , wherein the agent is conjugated to a cysteine residue of the C-terminal amino acid extension.

26. The method of claim 25 , wherein the method further comprises reducing the sulfhydryl group of the cysteine residue in the C-terminal amino acid extension prior to conjugating the agent to the antibody or antigen-binding fragment.

27. The method of claim 26 , wherein the reducing comprises (a) reducing one or more cysteine residues in the C-terminal amino acid extension over a cysteine residue not in the C-terminal amino acid extension, or (b) reducing one or more cysteine residues in the C-terminal amino acid extension and not reducing any cysteine residues not in the C-terminal amino acid extension.

28. The method of claim 25 , wherein the agent is a therapeutic agent or a labeling agent.

29. The antibody or antigen-binding fragment of claim 2 , wherein the C-terminal amino acid extension comprises from 2 to 10 spacers and wherein at least two of the spacers comprise the sequence GGGS (SEQ ID NO:1).

30. The conjugate of claim 12 , wherein the C-terminal amino acid extension comprises from 2 to 10 spacers and wherein at least two of the spacers comprise the sequence GGGS (SEQ ID NO:1).

31. The conjugate of claim 18 , wherein the cytotoxic agent is an auristatin or analog thereof, or a hemiasterlin or analog thereof.

32. The antibody or antigen-binding fragment of claim 1 , wherein the C-terminal amino acid extension is 25 amino acids or less in length.

33. The antibody or antigen-binding fragment of claim 1 , wherein the antibody or antigen-binding fragment thereof is bispecific.

34. The conjugate of claim 10 , wherein the C-terminal amino acid extension is 25 amino acids or less in length.

35. The conjugate of claim 10 , wherein the antibody or antigen-binding fragment thereof is bispecific.

36. The antibody or antigen-binding fragment of claim 1 , wherein the human amino acid sequence is an amino acid sequence of a human TCR hinge region, or a portion thereof.

37. The antibody or antigen-binding fragment of claim 1 , wherein the IgG is a human IgG1, IgG2 or IgG4.

38. The antibody or antigen-binding fragment of claim 1 , wherein the human TCR hinge region is a human TCR α, δ or γ hinge region.

39. The antibody or antigen-binding fragment of claim 36 , wherein the human TCR hinge region is a human TCR α, δ or γ hinge region.

Assignees

• Zymeworks Inc

Inventors

• Babcook John
• Rich James R
• Bergqvist Jan Peter
• Barnscher Stuart Daniel

Classifications

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K2317/522

  CH1 domain · CPC title

• C07K2317/73

  Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation · CPC title

• C07K2317/92

  Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value · CPC title

• A61K2039/505

  comprising antibodies · CPC title