Method of producing an immunoligand/payload conjugate
The present invention relates to a method of producing an immunoligand/payload conjugate, which method encompasses conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof (Fig. 6 b).
1. An immunoligand/payload conjugate comprising
a) an immunoligand selected from the group consisting of one or more of:
i) an antibody,
ii) an antibody-based binding protein being a protein containing at least one antibody-derived V H , V L , or C H immunoglobulin domain,
iii) an antibody fragment binding to a receptor, antigen, growth factor, cytokine, or hormone, or
iv) an antibody mimetic selected from the group consisting of DARPins, C-type lectins, A-domain proteins of S. aureus , transferrins, lipocalins, 10 th type Ill domains of fibronectin, Kunitz domain protease inhibitors, affilins, gamma crystallin derived binders, cysteine knots or knottins, thioredoxin A scaffold based binders, nucleic acid aptamers, artificial antibodies produced by molecular imprinting of polymers, and stradobodies,
b) at least one payload having a molecular weight not exceeding 2,500 Dalton, and
c) a linker conjugating the immunoligand of (a) or a subunit thereof to the payload, said linker comprising a sortase recognition motif having its C-terminal amino acid replaced with a Gly n , wherein n>1.
2. The immunoligand/payload conjugate of claim 1 , wherein the sortase recognition motif comprises LPXTG (SEQ ID NO: 27) or NPQTN (SEQ ID NO: 28), or wherein the sortase recognition motif is LPXTG (SEQ ID NO: 27) or NPQTN (SEQ ID NO: 28).
3. The immunoligand/payload conjugate of claim 2 , wherein the linker of (c) comprises, from its N-terminus to its C-terminus, LPXTG n , wherein n>1.
4. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand of (a) comprises two or more subunits, and wherein the linker of (c) conjugates one of the two or more subunits to one payload.
5. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand of (a) comprises two or more subunits, wherein a first linker of (c) conjugates a first subunit to a first payload, and wherein a second linker of (c) conjugates a second subunit to a second payload.
6. The immunoligand/payload conjugate of claim 5 , wherein the first payload is different from the second payload, or wherein the first payload is the same as the second payload.
7. The immunoligand/payload conjugate of claim 5 , wherein the first linker is the same as the second linker, or wherein the first linker is different from the second linker.
8. The immunoligand/payload conjugate of claim 5 , wherein the immunoligand comprises two light chains and two heavy chains, and wherein one linker of (c) conjugates each of the two light chains to one payload.
9. The immunoligand/payload conjugate of claim 5 , wherein the immunoligand comprises two light chains and two heavy chains, and wherein one linker of (c) conjugates each of the two light chains and the two heavy chains to one payload.
10. The immunoligand/payload conjugate of claim 9 , wherein the linker conjugating either of the two heavy chains to one payload is different from the linker conjugating either of the two light chains to one payload.
11. The immunoligand/payload conjugate of claim 9 , wherein the linker conjugating one heavy chain is the same as the linker conjugating the other heavy chain.
12. The immunoligand/payload conjugate of claim 9 , wherein the linker conjugating one light chain is the same as the linker conjugating the other light chain.
13. The immunoligand/payload conjugate of claim 9 , wherein the linker conjugating one heavy chain is the same as the linker conjugating the other heavy chain, wherein the linker conjugating one light chain is the same as the linker conjugating the other light chain, wherein the linker conjugating the heavy chain is different from the linker conjugating the light chain, and wherein the payload conjugated to the heavy chain is different or the same from the payload conjugated to the light chain.
14. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand of (a) comprises two or more subunits, and wherein one linker of (c) conjugates each of the two or more subunits to one payload.
15. The immunoligand/payload conjugate of claim 1 comprising, starting from its N-terminus in the following order: the immunoligand (a) or a subunit thereof, the linker of (c), and the payload of (c).
16. The immunoligand/payload conjugate of claim 1 comprising, starting from its C-terminus in the following order: the immunoligand (a) or a subunit thereof, the linker of (c), and the payload of (c).
17. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand of (a) or a subunit thereof further comprises a spacer of at least two amino acids at its C terminus.
18. The immunoligand/payload conjugate of claim 17 , wherein the spacer comprises 2-5 amino acids.
19. The immunoligand/payload conjugate of claim 1 , wherein the ratio of the immunoligand to the payload is stoichiometrically defined.
20. The immunoligand/payload conjugate of claim 1 , wherein the conjugation of the payload to the immunoligand or a subunit thereof is site-specific.
21. The immunoligand/payload conjugate of claim 1 , wherein the payload is a drug or a marker.
22. The immunoligand/payload conjugate of claim 21 , wherein the drug is a cytokine, a radioactive agent, a toxin, or a chemotherapeutic agent.
23. The immunoligand/payload conjugate of claim 21 , wherein the marker is a radiolabel, a fluorescent label, or an enzyme label.