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.
1. An immunoligand/payload conjugate produced by a method comprising
a) providing an immunoligand selected from the group consisting 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 and/or hormone, and/or
(iv) an antibody mimetic selected from the group consisting of DARPins, C-type lectins, A-domain proteins of S. aureus , transferrins, lipocalins, 10th type III 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; and
b) enzymatically conjugating at least one payload to the immunoligand by a sequence-specific sortase or a catalytic domain thereof, wherein the payload has a molecular weight not exceeding 2,500 Dalton,
wherein either
the immunoligand comprises a sortase recognition motif and the payload is modified with a Gly n -modification, wherein n>1, or
the payload comprises a sortase recognition motif and the immunoligand is modified with a Gly n -modification, wherein n>1.
2. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand binds at least one entity selected from the group consisting of a receptor, an antigen, a growth factor, a cytokine, and a hormone.
3. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand is conjugated to at least two different payloads, and wherein said immunoligand has at least two subunits each being conjugated to a payload.
4. The immunoligand/payload conjugate of claim 1 , wherein the immunoligand comprises two subunits, at least one of which has a peptide spacer appended to the C-terminus thereof, said spacer comprising at least two amino acids.
5. The immunoligand/payload conjugate of claim 4 , wherein the spacer is 2-5 amino acids.
6. The immunoligand/payload conjugate of claim 1 , wherein the ratio of immunoligand and payload is stoichiometrically defined.
7. The immunoligand/payload conjugate of claim 1 , wherein the conjugation of the payload to the immunoligand is site-specific.
8. The immunoligand/payload conjugate of claim 1 , wherein the sortase recognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28).
9. An immunoligand/payload conjugate comprising
a) an immunoligand selected from the group consisting 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 and/or hormone, and/or
(iv) an antibody mimetic selected from the group consisting of DARPins, C-type lectins, A-domain proteins of S. aureus , transferrins, lipocalins, 10th type III 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, and,
b) at least one payload having a molecular weight not exceeding 2,500 Dalton, wherein the conjugate further comprises at least one linker between the payload and the immunoligand, said linker comprising a peptide motif that is a sortase recognition motif, and said linker being conjugated to the C-terminus of at least one peptide chain of the immunoligand,
wherein either
the immunoligand comprises a sortase recognition motif and the payload is modified with a Gly n -modification, wherein n>1, or
the payload comprises a sortase recognition motif and the immunoligand is modified with a Gly n -modification, wherein n>1.
10. The immunoligand/payload conjugate according to claim 9 , wherein the C-terminal amino acid residue of the original sortase recognition motif is replaced by a G residue.
11. The immunoligand/payload conjugate according to claim 10 , wherein the sortase recognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28).
12. The immunoligand/payload conjugate of claim 9 , wherein the immunoligand binds at least one entity selected from the group consisting of a receptor, an antigen, a growth factor, a cytokine, and a hormone.
13. The immunoligand/payload conjugate of claim 9 , wherein the immunoligand is conjugated to at least two different payloads, and wherein said immunoligand has at least two subunits each being conjugated to a payload.
14. The immunoligand/payload conjugate of claim 9 , wherein the immunoligand comprises two subunits, at least one of which has a peptide spacer appended to the C-terminus thereof, said spacer comprising at least two amino acids.
15. The immunoligand/payload conjugate of claim 14 , wherein the spacer is 2-5 amino acids.
16. The immunoligand/payload conjugate of claim 9 , wherein the ratio of immunoligand and payload is stoichiometrically defined.
17. The immunoligand/payload conjugate of claim 9 , wherein the conjugation of the payload to the immunoligand is site-specific.
18. The immunoligand/payload conjugate of claim 9 , wherein the sortase recognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28).
19. The immunoligand/payload conjugate of claim 1 , wherein the payload is a drug.
20. The immunoligand/payload conjugate of claim 19 , wherein the drug is a cytokine.
21. The immunoligand/payload conjugate of claim 20 , wherein the drug is a radioactive agent.
22. The immunoligand/payload conjugate of claim 20 , wherein the drug is a toxin.
23. The immunoligand/payload conjugate of claim 20 , wherein the drug is a chemotherapeutic agent.
24. The immunoligand/payload conjugate of claim 1 , wherein the payload is a marker.
25. The immunoligand/payload conjugate of claim 24 , wherein the marker is a radiolabel.
26. The immunoligand/payload conjugate of claim 24 , wherein the marker is a fluorescent label.
27. The immunoligand/payload conjugate of claim 24 , wherein the marker is an enzyme label.
28. The immunoligand/payload conjugate of claim 9 , wherein the payload is a drug.
29. The immunoligand/payload conjugate of claim 28 , wherein the drug is a cytokine.
30. The immunoligand/payload conjugate of claim 28 , wherein the drug is a radioactive agent.
31. The immunoligand/payload conjugate of claim 28 , wherein the drug is a toxin.
32. The immunoligand/payload conjugate of claim 28 , wherein the drug is a chemotherapeutic agent.
33. The immunoligand/payload conjugate of claim 28 , wherein the payload is a marker.
34. The immunoligand/payload conjugate of claim 33 , wherein the marker is a radiolabel.
35. The immunoligand/payload conjugate of claim 33 , wherein the marker is a fluorescent label.
36. The immunoligand/payload conjugate of claim 33 , wherein the marker is an enzyme label.