Method of assessing the efficacy of a test agent for modulating programmed death 1 (PD-1) signaling by using anti-phosphotyrosinylated PD-1 monoclonal antibodies

12258,409

17/934441

September 22, 2022

The present invention is based, in part, on the discovery of monoclonal and polyclonal antibodies that specifically bind to phosphorylated PD-1, as well as immunoglobulins, polypeptides, nucleic acids thereof, and methods of using such antibodies for diagnostic, prognostic, and therapeutic purposes.

Dana-Farber Cancer Institute, Inc. (Boston, MA, US); Beth Israel Deaconess Medical Center (Boston, MA, US); President and Fellows of Harvard College (Cambridge, MA, US)

Dana-Farber Cancer Institute, Inc. (Boston, MA, US), Beth Israel Deaconess Medical Center (Boston, MA, US), President and Fellows of Harvard College (Cambridge, MA, US)

1. A method of assessing the efficacy of a test agent for modulating PD-1 signaling, the method comprising: a) contacting a cell expressing PD-1 with a test agent; and b) determining the ability of the test agent to modulate the level of phosphorylated PD-1 using at least one monoclonal antibody, or antigen-binding fragment thereof, that binds to a phosphorylation site at tyrosine residue 248 of human PD-1, wherein the monoclonal, or antigen-binding fragment thereof, comprises: i) a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 6, CDR-L2 having the sequence of SEQ ID NO: 9, and CDR-L3 having the sequence of SEQ ID NO: 12, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 19, CDR-H2 having the sequence of SEQ ID NO: 22, and CDR-H3 having the sequence of SEQ ID NO: 25; ii) a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 34, CDR-L2 having the sequence of SEQ ID NO: 38, and CDR-L3 having the sequence of SEQ ID NO: 43, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 52, CDR-H2 having the sequence of SEQ ID NO: 56, and CDR-H3 having the sequence of SEQ ID NO: 60; iii) a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 70, CDR-L2 having the sequence of SEQ ID NO: 74, and CDR-L3 having the sequence of SEQ ID NO: 78, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 88, CDR-H2 having the sequence of SEQ ID NO: 92, and CDR-H3 having the sequence of SEQ ID NO: 96; or iv) a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 106, CDR-L2 having the sequence of SEQ ID NO: 110, and CDR-L3 having the sequence of SEQ ID NO: 114, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 124, CDR-H2 having the sequence of SEQ ID NO: 128, and CDR-H3 having the sequence of SEQ ID NO: 132; wherein a modulated level of phosphorylated PD-1 resulting from contacting with the test agent identifies the test agent as a modulator of PD-1 signaling.

2. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 6, CDR-L2 having the sequence of SEQ ID NO: 9, and CDR-L3 having the sequence of SEQ ID NO: 12, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 19, CDR-H2 having the sequence of SEQ ID NO: 22, and CDR-H3 having the sequence of SEQ ID NO: 25.

3. The method of claim 2 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising a sequence at least 90% identical to SEQ ID NO: 1 and a heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 14.

4. The method of claim 3 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising the sequence of SEQ ID NO: 1 and a heavy chain comprising the sequence of SEQ ID NO: 14.

5. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 34, CDR-L2 having the sequence of SEQ ID NO: 38, and CDR-L3 having the sequence of SEQ ID NO: 43, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 52, CDR-H2 having the sequence of SEQ ID NO: 56, and CDR-H3 having the sequence of SEQ ID NO: 60.

6. The method of claim 5 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising a sequence at least 90% identical to SEQ ID NO: 30 and a heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 46.

7. The method of claim 6 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising the sequence of SEQ ID NO: 30 and a heavy chain comprising the sequence of SEQ ID NO: 46.

8. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 70, CDR-L2 having the sequence of SEQ ID NO: 74, and CDR-L3 having the sequence of SEQ ID NO: 78, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 88, CDR-H2 having the sequence of SEQ ID NO: 92, and CDR-H3 having the sequence of SEQ ID NO: 96.

9. The method of claim 8 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising a sequence at least 90% identical to SEQ ID NO: 64 and a heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 82.

10. The method of claim 9 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising the sequence of SEQ ID NO: 64 and a heavy chain comprising the sequence of SEQ ID NO: 82.

11. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising CDR-L1 having the sequence of SEQ ID NO: 106, CDR-L2 having the sequence of SEQ ID NO: 110, and CDR-L3 having the sequence of SEQ ID NO: 114, and a heavy chain comprising CDR-H1 having the sequence of SEQ ID NO: 124, CDR-H2 having the sequence of SEQ ID NO: 128, and CDR-H3 having the sequence of SEQ ID NO: 132.

12. The method of claim 11 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising a sequence at least 90% identical to SEQ ID NO: 100 and a heavy chain comprising a sequence at least 90% identical to SEQ ID NO: 118.

13. The method of claim 12 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain comprising the sequence of SEQ ID NO: 100 and a heavy chain comprising the sequence of SEQ ID NO: 118.

14. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, is chimeric, humanized, murine, or rabbit.

15. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, is detectably labeled.

16. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises an effector domain.

17. The method of claim 1 , wherein the monoclonal antibody, or antigen-binding fragment thereof, comprises an Fc domain.

18. The method of claim 1 , wherein the antigen-binding fragment thereof, is selected from the group consisting of Fv, Fav, F (ab′)2), Fab′, dsFv, scFv, sc (Fv)2, and diabodies fragments.

11492403 November 2022 Freeman et al.
2015/0210769 July 2015 Freeman et al.
2020/0115452 April 2020 Freeman et al.
WO-2015/035606 March 2015
WO-2016/014688 January 2016
WO-2017/055443 April 2017
WO-2018/231339 December 2018

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