Nanobody dimers linked via C-terminally engineered cysteins

Patent metadata

Field	Value
Publication number	US-11021544-B2
Application number	US-201615548830-A
Country	US
Kind code	B2
Filing date	Feb 5, 2016
Priority date	Feb 5, 2015
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

The present invention relates to dimers comprising a first polypeptide and a second polypeptide, wherein each of said first and second polypeptide comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine moiety (preferably at the C-terminus), wherein said first polypeptide and said second polypeptide are covalently linked via a disulfide bond between the cysteine moiety of said first polypeptide and the cysteine moiety of said second polypeptide, in which the dimer outperformed the benchmark constructs, e.g. cognate multivalent and multispecific constructs, in various assays. The present invention provides methods for making the dimers of the invention.

First claim

The invention claimed is:

1. A method for making a dimer, comprising:
(i) providing a first polypeptide, wherein said first polypeptide comprises:
at least one immunoglobulin single variable domain (ISVD) and
a C-terminal extension comprising a cysteine moiety, optionally at the C-terminus;

(ii) providing a second polypeptide, wherein said second polypeptide comprises:
at least one immunoglobulin single variable domain (ISVD) and
a C-terminal extension comprising a cysteine moiety, optionally at the C-terminus; and

(iii) oxidizing the thiol moiety of said cysteine moiety of said first polypeptide and the thiol moiety of said cysteine moiety of said second polypeptide to produce a first dimer comprising a disulfide derivative cystine by adding oxidizing copper ions (Cu 2+ ), optionally at pH 6.5 to pH 7.5;
(iv) reacting the disulfide derivative cystine with a conjugating agent to produce a second dimer comprising a bridge between the two cysteine residues derived from the cystine; and
(v) optionally purifying said second dimer, optionally via size exclusion chromatography;
wherein the integrities of the ISVDs are maintained; and
wherein said conjugating agent comprises: the functional group of formula (I)

in which W represents an electron-withdrawing group; A represents a C 1-5 alkylene or alkenylene chain; B represents a bond or a C 1-4 alkylene or alkenylene chain; and each L independently represents a leaving group;
the functional group of formula (Ia):

in which W, A, B, and L have the meanings given for the formula I, Q represents a linking group and D represents a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent;

the functional group of formula (Ib):

in which A, B, and L have the meanings given for the formula I, CN represents a cyano group, Q represents a linking group and D represents a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent;

the functional group of formula (II):

in which one of X and X′ represents a polymer and the other represents a hydrogen atom;
Q represents a linking group;
W represents an electron-withdrawing group; or, if X′ represents a polymer, X-Q-W together may represent an electron withdrawing group;
A represents a C 1-5 alkylene or alkenylene chain;
B represents a bond or a C 1-4 alkylene or alkenylene chain; and
each L independently represents a leaving group;

the functional group of formula (III):

in which X, X′, Q, W, A and L have the meanings given for the general formula II, and in addition if X represents a polymer, X′ and electron-withdrawing group W together with the interjacent atoms may form a ring, and m represents an integer 1, 2, 3 or 4; or

the functional group of formula (IV):
X-Q-W—CR 1 R 1′ —CR 2 .L.L′  (IV)

in which X, Q and W have the meanings given for the general formula II, and either
R 1 represents a hydrogen atom or a C 1-4 alkyl group, R 1′ represents a hydrogen atom, and each of L and L′ independently represents a leaving group; or
R 1 represents a hydrogen atom or a C 1-4 alkyl group, L represents a leaving group, and
R 1 and L′ together represent a bond; or
R 1 and L together represent a bond and R 1 and L′ together represent a bond; and
R 2 represents a hydrogen atom or a C 1-4 alkyl group.

2. The method according to claim 1 , wherein the conjugating agent comprises a drug, and wherein said drug is a cytostatic agent, a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a toxin moiety, or a radioactive isotope; and optionally wherein said drug is monomethyl auristatin E (MMAE).

3. The method according to claim 1 , wherein the conjugating agent comprises a binding agent, wherein said binding agent is a linker for a diagnostic, therapeutic or labelling agent.

4. The method according to claim 1 , wherein said first polypeptide and/or said second polypeptide further comprises maleimide-val-cit-MMAE.

5. The method according to claim 3 , wherein said linker is a non-cleavable linker.

6. The method according to claim 1 , wherein the conjugating agent comprises a drug, and wherein the drug to dimer ratio (DAR) is 1.

7. The method according to claim 1 , wherein said first polypeptide and/or said second polypeptide comprises a C-terminal extension of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s) comprising a cysteine moiety, optionally at the C-terminus, optionally said C-terminal extension is genetically fused to the C-terminal end of the most C-terminally located ISVD in said polypeptide.

8. A dimer comprising a first polypeptide and a second polypeptide, wherein said first polypeptide comprises
at least one immunoglobulin single variable domain (ISVD) and
a C-terminal extension comprising a cysteine moiety, optionally at the C-terminus;
wherein said second polypeptide comprises
at least one immunoglobulin single variable domain (ISVD) and
a C-terminal extension comprising a cysteine moiety, optionally at the C-terminus; and
wherein said first polypeptide and said second polypeptide are covalently linked via a thioether bond of the cysteine moiety (C) in the C-terminal extension of said first polypeptide and a thioether bond of the cysteine moiety (C) in the C-terminal extension of said second polypeptide with the compound according to formula (V) or formula (VI)

in which
“—C—” represents a cysteine moiety in the C-terminal extension of said first polypeptide and said second polypeptide;
“—C” represents cysteine moiety at the C-terminus of a C-terminal extension of said first polypeptide and said second polypeptide;
R 1 represents a C 1-4 alkyl group; and D represents a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent.

9. The dimer according to claim 8 , wherein D represents a therapeutic that is a drug, and wherein said drug is a cytostatic agent, a cytotoxic agent, a chemotherapeutic agent a growth inhibitory agent, a toxin, a toxin moiety, or a radioactive isotope; and optionally wherein said drug is monomethyl auristatin E (MMAE).

10. The dimer according to claim 8 , comprising maleimide-val-cit-MMAE.

11. The dimer according to claim 8 , wherein said binding agent is a linker for a diagnostic, therapeutic or labelling agent.

12. The dimer according to claim 11 , wherein said linker is a non-cleavable linker.

13. The dimer according to claim 9 , wherein the drug to dimer ratio (DAR) is 1.

14. The dimer according to claim 8 , wherein said first polypeptide and/or said second polypeptide comprises a C-terminal extension of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s) comprising a cysteine moiety, wherei

Assignees

• Ablynx Nv

Inventors

• Boutton Carlo
• Janssen Daniel
• Casteels Peter
• Schotte Peter
• Descamps Francis

Classifications

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K16/30 Primary

  from tumour cells · CPC title

• A61K47/68031

  the drug being an auristatin · CPC title

• C07K16/3007

  Carcino-embryonic Antigens · CPC title

• A61K51/103

  against receptors for growth factors or receptors for growth regulators · CPC title