pH-dependent antibodies targeting the transferrin receptor and methods of use thereof to deliver a therapeutic agent
A pH-dependent antibody that binds an antigen with high affinity at a first pH and rapidly dissociates at a second pH, wherein the antigen is a transferrin receptor (TfR), wherein the association at the second pH/the first pH is less than 20%, and wherein the pH-dependent antibody comprises at least two consecutive histidine residues at a single complementarity determining region (CDR) is disclosed.
1. A pH-dependent antibody that binds an antigen at a first pH and rapidly dissociates at a second pH, wherein the antigen is a transferrin receptor (TfR), wherein the pH-dependent antibody comprises at least two consecutive histidine residues at a single complementarity determining region (CDR) and wherein the CDR is CDRH1, wherein the first pH is a physiological pH and the second pH is an endosomal pH.
2. The pH-dependent antibody of claim 1 , wherein the association at the second pH versus the first pH is less than 20%.
3. The pH-dependent antibody of claim 1 , wherein the pH-dependent antibody comprises at least three consecutive histidine residues.
4. The pH-dependent antibody of claim 1 , where the first pH is about pH 7.4.
5. The pH-dependent antibody of claim 1 , where the second pH is about pH 5.5.
6. The pH-dependent antibody of claim 1 , wherein the pH-dependent antibody comprises an antibody fragment having an amino acid sequence selected from the group consisting of SEQ ID NOs 13-20 (corresponding to M4, M8, M10, M11, M16, M17, M20 and M23 respectively).
7. The pH-dependent antibody of claim 1 , wherein the antibody is linked to a therapeutic agent.
8. The pH-dependent antibody of claim 7 , wherein the linkage is selected from the group consisting of covalent and non-covalent linkages.
9. The pH-dependent antibody of claim 7 , wherein the antibody is part of a fusion protein and wherein the therapeutic agent is a second part of the fusion protein.
10. A kit for delivering a molecule into a cell, the kit comprising a pH-dependent antibody of claim 1 , wherein the antibody is a pH-dependent antibody that binds an antigen at a first pH and rapidly dissociates at a second pH, wherein the antigen is a transferrin receptor (TfR), wherein the pH-dependent antibody is bound to the molecule and the antigen of the transferrin receptor (TfR) is existing within the cell, wherein the first pH is a physiological pH and the second pH is an endosomal pH.
11. A pH-dependent antibody that binds an antigen at a first pH and rapidly dissociates at a second pH, wherein the first pH is a physiological pH and the second pH is an endosomal pH, wherein the antigen is a transferrin receptor (TfR), wherein the pH-dependent antibody comprises at least two consecutive histidine residues at a single complementarity determining region (CDR) and wherein the CDR is CDRH1, wherein the pH-dependent antibody comprises an antibody fragment having an amino acid sequence of SEQ ID NO: 17 (corresponding to M16).
12. A method of delivering a therapeutic agent into a cell, the method comprising the steps of
(a) obtaining a pH-dependent antibody of claim 1 , wherein the antibody is a pH-dependent antibody that binds an antigen at a first pH and rapidly dissociates at a second pH, wherein the antigen is a transferrin receptor (TfR);
(b) exposing the cell to a pharmaceutically effective amount of the pH-dependent antibody; and
(c) exposing the cell to a pharmaceutically effective amount of the therapeutic agent, wherein the molecule is delivered into the cell in an enhanced manner.
13. The method of claim 12 , wherein the therapeutic agent has a molecular weight larger than 500 Da.
14. The method of claim 12 , wherein the cell is exposed to the therapeutic agent at the same time as the cell is exposed to the pH-dependent antibody.
15. The method of claim 12 , wherein the agent is linked to the pH-dependent antibody.
16. The method of claim 12 , wherein the pH-dependent antibody is obtained when an antibody is subjected to histidine-saturation mutagenesis at a single CDR.
17. The method of claim 16 , wherein the CDR is CDRH1.
18. The method of claim 12 , wherein in step (b) the cell is exposed to a pharmaceutically effective amount of the pH-dependent antibody at a pH different from neutral pH.
19. The method of claim 18 , wherein the method is conducted at pH 5.5.
20. The method of claim 12 , wherein the cell is a cancer cell.
21. The method of claim 12 , wherein the method is in vivo.
22. A method of delivering a therapeutic agent across a patient's blood brain barrier, the method comprising the steps of
(a) obtaining a pH-dependent antibody of claim 1
(b) exposing a patient's blood brain barrier to a pharmaceutically effective amount of the pH-dependent antibody; and
(c) exposing the patient's blood brain barrier to a pharmaceutically effective amount of the therapeutic agent, wherein the molecule is delivered across the patient's blood brain barrier in an enhanced manner.
23. The method of claim 22 , wherein the therapeutic agent has a molecular weight larger than 500 Da.
24. The method of claim 22 , wherein the patient's blood brain barrier is exposed to the therapeutic agent at the same time as the barrier is exposed to the pH-dependent antibody.
25. The method of claim 22 , wherein the pH-dependent antibody is obtained when an antibody is subjected to histidine-saturation mutagenesis at a single CDR.
26. The method of claim 25 , wherein the CDR is CDRH1.
27. The method of claim 22 , wherein in step (b) the cell is exposed to a pharmaceutically effective amount of the pH-dependent antibody at a pH different from neutral pH.
28. The method of claim 27 , wherein the method is conducted at pH 5.5.