Nanobody dimers linked via c-terminally engineered cysteins

Method for making dimers, comprising at least the steps of:

• (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, preferably 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, preferably at the C-terminus;
• and
• (iii) oxidizing the thiol moiety of said cysteine moiety at the C-terminus of said first polypeptide and the thiol moiety of said cysteine moiety at the C-terminus of said second polypeptide optionally by adding oxidizing copper ions (Cu2+), and preferably at pH 6.5 to pH 7.5 to a disulfide derivative cystine;
• (iv) reacting the disulfide derivative cystine with a conjugating agent that forms a bridge between the two cysteine residues derived from the cystine;
• and
• (v) optionally purifying said dimers, preferably via size exclusion chromatography;
• characterized in that the integrity of the ISVDs is maintained,
• wherein preferably at least 80%, such as 85%, 90%, 95%, 99% or even more, such as 100% of said first and said second polypeptide are dimerized, for instance as determined by mass spectrometry;
• and
• wherein said conjugating agent is chosen from the group consisting of conjugating agents comprising the functional group of formula (I)
• in which W represents an electron-withdrawing group;
• A represents a C1-5 alkylene or alkenylene chain;
• B represents a bond or a C1-4 alkylene or alkenylene chain;
• and each L independently represents a leaving group;
• conjugating agents comprising 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, such as, for instance, a drug, or a binding agent, e.g. a linker, for a diagnostic, therapeutic or labelling agent, such as, for instance, a drug;
• conjugating agents comprising 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, such as, for instance, a drug, or a binding agent, e.g. a linker, for a diagnostic, therapeutic or labelling agent, such as, for instance, a drug;
• conjugating agents comprising 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 C1-5 alkylene or alkenylene chain;
• B represents a bond or a C1-4 alkylene or alkenylene chain;
• and
• each L independently represents a leaving group;
• conjugating agents comprising 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
• conjugating agents comprising the functional group of formula (IV):
• X-Q-W—CR1R1′—CR2.L.L′ (IV)
• in which X, Q and W have the meanings given for the general formula II, and either
• R1 represents a hydrogen atom or a C1-4 alkyl group, R1′ represents a hydrogen atom, and each of L and L′ independently represents a leaving group;
• or
• R1 represents a hydrogen atom or a C1-4 alkyl group, L represents a leaving group, and
• R1 and L′ together represent a bond;
• or
• R1 and L together represent a bond and R1 and L′ together represent a bond;
• and
• R2 represents a hydrogen atom or a C1-4 alkyl group.