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. A method of treating a pathologic condition in a subject in need thereof, comprising
administering to the subject in need thereof an effective amount of an immunoligand/payload conjugate,
wherein the immunoligand/payload conjugate is produced by enzymatically conjugating at least one glycine-modified payload to the immunoligand with a sequence-specific sortase or a catalytic domain thereof,
wherein the immunoligand is 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
(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
wherein the payload is selected from the group consisting of a cytokine, a radioactive agent, an anti-inflammatory drug, a toxin, and a chemotherapeutic agent.
2. The method of claim 1 , wherein the sortase is sortase A.
3. The method of claim 1 , wherein the immunoligand/payload conjugate further comprises at least one linker between the immunoligand and the payload, 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.
4. The method according to claim 3 , wherein the C-terminal amino acid residue of the sortase recognition motif is replaced by a glycine residue.
5. The method according to claim 4 , wherein the sortase recognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28), wherein X represents any amino acid.
6. The method of claim 1 , wherein the immunoligand/payload conjugate comprises an antibody/drug conjugate.
7. The method of claim 1 , wherein the payload comprises a toxin of molecular weight ≥2500 Dalton that is cytotoxic to a mammalian cell.
8. The method of claim 7 , wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins, Pseudomonas exotoxin PE38, monomethyl Auristatin F (MMAF), monomethyl Auristatin E (MMAE), alpha aminitin, and Diphtheria toxin.
9. The method of claim 1 , wherein the payload comprises a chemotherapeutic agent.
10. The method of claim 9 , wherein the chemotherapeutic agent is doxorubicin.
11. The method of claim 1 , wherein the immunoligand comprises at least two subunits, each being conjugated to a payload.
12. The method of claim 11 , wherein the immunoligand with at least two subunits is conjugated to at least two different payloads.
13. The method of claim 12 , wherein the different payloads are toxic payloads, each interfering with one or more cellular pathways.
14. The method of claim 11 , wherein said immunoligand with at least two subunits comprises a peptide spacer of at least two amino acids appended to the C-terminus of at least one of the two subunits.
15. The method of claim 14 , wherein the peptide spacer comprises 2 to 5 amino acids.
16. The method of claim 1 , wherein the pathologic condition is a neoplastic disease.
17. The method of claim 16 , wherein the neoplastic disease is breast cancer.
18. The method of claim 16 , wherein the neoplastic disease is ovarian cancer.
19. The method of claim 1 , wherein the subject in need thereof is a mammal.
20. The method of claim 1 , wherein the subject in need thereof is a human.