High affinity PD-1 agents and methods of use
High affinity PD-1 mimic polypeptides are provided, which (i) comprise at least one amino acid change relative to a wild-type PD-1 protein; and (ii) have an increased affinity for PD-L1 relative to the wild-type protein. Compositions and methods are provided for modulating the activity of immune cells in a mammal by administering a therapeutic dose of a pharmaceutical composition comprising a high affinity PD-1 mimic polypeptide, which blocks the physiological binding interaction between PD-1 and its ligand PD-L1 and/or PD-L2.
1. A method of imaging PD-L1-expressing cells, the method comprising contacting cells expressing programmed cell death 1 ligand 1 (PD-L1) with a high affinity PD-1 mimic polypeptide comprising an amino acid sequence that is a variant of a human wild-type PD-1 ectodomain sequence, wherein the high affinity PD-1 mimic polypeptide:
(a) comprises a detectable label,
(b) lacks a wild-type PD-1 transmembrane domain, and
(c) comprises the amino acid sequence set forth in any of SEQ ID NOs: 3-25, and 44-46.
2. A method of imaging PD-L1-expressing cells, the method comprising contacting cells expressing programmed cell death 1 ligand 1 (PD-L1) with a high affinity PD-1 mimic polypeptide comprising an amino acid sequence that is a variant of a human wild-type PD-1 ectodomain sequence, wherein the high affinity PD-1 mimic polypeptide:
(a) comprises a detectable label,
(b) lacks a wild-type PD-1 transmembrane domain,
(c) comprises an amino acid sequence having 85% or more sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 2-25 and 44-46, and
(d) relative to the amino sequence set forth in SEQ ID NO: 2, comprises one or more amino acid changes that cause an increased affinity for human and/or mouse Pd-L1.
3. The method of claim 2 , wherein the high affinity PD-1 mimic polypeptide has a K d of 1×10 −7 M or less for human and/or mouse PD-L1.
4. The method of claim 2 , wherein the one or more amino acid changes is located at an amino acid position of PD-1 that contacts human and/or mouse PD-L1.
5. The method of claim 2 , wherein the one or more amino acid changes is 2 or more amino acid changes located at amino acid positions, relative to the sequence set forth in SEQ ID NO: 2, selected from: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, M83, R90, Y96, L97, A100, S102, L103, A104, P105, and A107.
6. The method of claim 2 , wherein the one or more amino acid changes are located at amino acid positions, relative to the sequence set forth in SEQ ID NO: 2, selected from: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104, P105, K106, and A107.
7. The method of claim 6 , wherein the one or more amino acid changes is 5 or more amino acid changes that are located at amino acid positions, relative to the sequence set forth in SEQ ID NO: 2, selected from: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104, P105, K106, and A107.
8. The method of claim 6 , wherein the one or more amino acid changes is 3 or more amino acid changes selected from: (1)-(29), wherein (1) is V39H or V39R; (2) is L40V or L40I; (3) is N41I or N41V; (4) is Y43F or Y43H; (5) is R44Y or R44L; (6) is M45Q, M45E, M45L, or M45D; (7) is S48D, S48L, S48N, S48G, or 548V; (8) is N49C, N49G, N49Y, or N49S; (9) is Q50K, Q50E, or Q50H; (10) is T51V, T51L, or T51A; (11) is D52F, D52R, D52Y, or D52V; (12) is K53T or K53L; (13) is A56S or A56L; (14) is Q63T, Q63I , Q63E, Q63L, or Q63P; (15) is G65N, G65R, G65I, G65L, G65F, or G65V; (16) is Q66P; (17) is V72I; (18) is H82Q; (19) is M83L or M83F; (20) is R90K; (21) is Y96F; (22) is L97Y, L97V, or L97I; (23) is A100I or A100V; (24) is S102T or S102A; (25) is L103I, L103Y, or L103F; (26) is A104S, A104H, or A104D; (27) is P105A; (28) is K106G, K106E, K106I, K106V, K106R, or K106T; and (29) is A107P, A107I, or A107V.
9. The method of claim 2 , wherein the high affinity PD-1 mimic polypeptide comprises amino acid changes located at each of the amino acid positions listed for any one of (a)-(h), relative to the sequence set forth in SEQ ID NO: 2, wherein:
(a) is V39, N41, Y43, M45, S48, N49, Q50, K53, A56, Q63, G65, Q66, L97, S102, L103, A104, K106, and A107;
(b) is V39, N41, Y43, M45, S48, Q50, T51, D52F, K53, A56, Q63, G65, Q66, L97, S102, L103, A104, K106, and A107;
(c) is V39, L40, N41, Y43, R44, M45, N49, K53, M83, L97, A100, and A107;
(d) is V39, L40, N41, Y43, M45, N49, K53, Q66, M83, L97, and A107;
(e) is V39, L40, N41, Y43, M45, N49, K53, Q66, H82, M83, L97, A100, and A107;
(f) is V39, L40, N41, Y43, M45, N49, K53, M83, L97, A100, and A107;
(g) is V39, L40, N41, Y43, R44, M45, N49, K53, L97, A100, and A107; and
(h) is V39, L40, N41, Y43, M45, N49, K53, L97, A100, and A107.
10. The method of claim 2 , wherein the high affinity PD-1 mimic polypeptide comprises the amino acid changes, relative to the sequence set forth in SEQ ID NO: 2, listed for any one of (a)-(h), wherein:
(a) is {V39H or V39R}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {S48D, S48L, S48N, S48G, or 548V}, {N49C, N49G, N49Y, or N49S}, {Q50K, Q50E, or Q50H}, {K53T or K53L}, {A56S or A56L}, {Q63T, Q63I, Q63E, Q63L, or Q63P}, {G65N, G65R, G65I, G65L, G65F, or G65V}, {Q66P}, {L97Y, L97V, or L97I}, {S102T or S102A}, {L103I, L103Y, or L103F}, {A104S, A104H, or A104D}, {K106G, K106E, K106I, K106V, K106R, or K106T}, and {A107P, A107I, or A107V};
(b) is {V39H or V39R}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {548D, S48L, S48N, 548G, or 548V}, {Q50K, Q50E, or Q50H}, {T51V, T51L, or T51A}, {D52F, D52R, D52Y, or D52V}, {K53T or K53L}, {A56S or A56L}, {Q63T, Q63I, Q63E, Q63L, or Q63P}, {G65N, G65R, G65I, G65L, G65F, or G65V}, {Q66P}, {L97Y, L97V, or L97I}, {S102T or S102A}, {L103I, L103Y, or L103F}, {A104S, A104H, or A104D}, {K106G, K106E, K106I, K106V, K106R, or K106T}, and {A107P, A107I, or A107V};
(c) is {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P, A107I, or A107V};
(d) is {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T or K53L}, {Q66P}, {M83L or M83F}, {L97Y, L97V, or L97I}, and {A107P, A107I, or A107V};
(e) is {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T or K53L}, {Q66P}, {H82Q}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P, A107I, or A107V};
(f) is {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T or K53L}, {M83L or M83F}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P, A107I, or A107V};
(g) is {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H}, {R44Y or R44L}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P, A107I, or A107V}; and
(h) is {V39H or V39R}, {L40V or L40I}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L, or M45D}, {N49C, N49G, N49Y, or N49S}, {K53T or K53L}, {L97Y, L97V, or L97I}, {A100I or A100V}, and {A107P, A107I, or A107V}.
11. The method of claim 2 , wherein the high affinity PD-1 mimic polypeptide comprises each of the amino acid changes, relative to the sequence set forth in SEQ ID NO: 2, listed for any one of (a)-(i), wherein:
(a) is V39R, N41V, Y43H, M45E, 548G, N49Y, Q50E, K53T, A56S, Q63T, G65L, Q66P, L97V, S102A, L103F, A104H, K106V, and A107I;
(b) is V39R, N41V, Y43H, M45E, S48N, Q50H, T51A, D52Y, K53T, A56L, Q63L, G65F, Q66P, L97I, S102T, L103F, A104D, K106R, and A107I;
(c) is V39H, L40V, N41V, Y43H, R44Y, M45E, N49G, K53T, M83L, L97V, A100I, and A107I;
(d) is V39H, L40V, N41V, Y43H, M45E, N49G, K53T, Q66P, M83L, L97V, and A107I;
(e) is V39H, L40V, N41V, Y43H, M45E, N49S, K53T, Q66P, H82Q, M83L, L97V, A100V, and A107I;
(f) is V39H, L40I, N41I, Y43H, M45E, N49G, K53T, M83L, L97V, A100V, and A107I;
(g) is V39H, L40V, N41I, Y43H, R44L, M45E, N49G, K53T, L97V, A100V, and A107I;
(h) is V39H, L40V, N41I, Y43H, M45E, N49G, K53T, L97V, A100V, and A107I; and
(i) is V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, and A107I.
12. The method of claim 2 , wherein the high affinity PD-1 mimic polypeptide comprises a fusion partner.
13. The method of claim 12 , wherein the fusion partner is a fragment of a human immunoglobulin polypeptide sequence.
14. The method of claim 13 , wherein the fragment is selected from: (a) a CH3 domain; and (b) part or whole of an Fc region.
15. The method of claim 2 , wherein the high affinity PD-1 mimic polypeptide comprises one or more amino acid changes, relative to the sequence set forth in SEQ ID NO: 2, selected from: R87C, N91C, and R122C.
16. The method of claim 2 , wherein the detectable label is a positron-emission tomography (PET) imaging label.
17. The method of claim 2 , wherein said contacting comprises administering the high affinity PD-1 mimic polypeptide to an individual.
18. The method of claim 7 , wherein the high affinity PD-1 mimic polypeptide comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 2-25 and 44-46.
19. The method of claim 15 , wherein the high affinity PD-1 mimic polypeptide comprises the N91C amino acid change.
20. The method of claim 19 , wherein the high affinity PD-1 mimic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 44.
21. The method of claim 1 , wherein the high affinity PD-1 mimic polypeptide comprises one or more amino acid changes, relative to the sequence set forth in SEQ ID NO: 2, selected from: R87C, N91C, and R122C.
22. The method of claim 21 , wherein the high affinity PD-1 mimic polypeptide comprises the N91C amino acid change.
23. The method of claim 1 , wherein said contacting comprises administering the high affinity PD-1 mimic polypeptide to an individual.
24. The method of claim 8 , wherein the 3 or more amino acid changes is 5 or more amino acid changes selected from (1)-(29), wherein: (1) is V39H or V39R; (2) is L40V or L40I; (3) is N41I or N41V; (4) is Y43F or Y43H; (5) is R44Y or R44L; (6) is M45Q, M45E, M45L, or M45D; (7) is S48D, S48L, S48N, 548G, or 548V; (8) is N49C, N49G, N49Y, or N49S; (9) is Q50K, Q50E, or Q50H; (10) is T51V, T51L, or T51A; (11) is D52F, D52R, D52Y, or D52V; (12) is K53T or K53L; (13) is A56S or A56L; (14) is Q63T, Q63I, Q63E, Q63L, or Q63P; (15) is G65N, G65R, G65I, G65L, G65F, or G65V; (16) is Q66P; (17) is V72I; (18) is H82Q; (19) is M83L or M83F; (20) is R90K; (21) is Y96F; (22) is L97Y, L97V, or L97I; (23) is A100I or A100V; (24) is S102T or S102A; (25) is L103I, L103Y, or L103F; (26) is A104S, A104H, or A104D; (27) is P105A; (28) is K106G, K106E, K106I, K106V, K106R, or K106T; and (29) is A107P, A107I, or A107V.
25. The method of claim 2 , wherein said increased affinity is a 2-fold or more increased affinity.
26. The method of claim 2 , wherein the one or more amino acid changes, relative to the sequence set forth in SEQ ID NO: 2, are selected from: (1)-(28), wherein: (1) is V39H or V39R; (2) is L40V or L40I; (3) is N41I or N41V; (4) is Y43F or Y43H; (5) is R44Y or R44L; (6) is M45Q, M45E, M45L, or M45D; (7) is S48D, S48L, S48N, 548G, or 548V; (8) is N49C, N49G, N49Y, or N49S; (9) is Q50K, Q50E, or Q50H; (10) is T51V, T51L, or T51A; (11) is D52F, D52R, D52Y, or D52V; (12) is K53T or K53L; (13) is A56S or A56L; (14) is Q63T, Q63I, Q63E, Q63L, or Q63P; (15) is G65N, G65R, G65I, G65L, G65F, or G65V; (16) is V72I; (17) is H82Q; (18) is M83L or M83F; (19) is R90K; (20) is Y96F; (21) is L97Y, L97V, or L97I; (22) is A100I or A100V; (23) is S102T or S102A; (24) is L103I, L103Y, or L103F; (25) is A104S, A104H, or A104D; (26) is P105A; (27) is K106G, K106E, K106I, K106V, or K106R; and (28) is A107P, A107I, or A107V.
27. The method of claim 5 , wherein the 2 or more amino acid changes is 5 or more amino acid changes located at amino acid positions, relative to the sequence set forth in SEQ ID NO: 2, selected from: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, M83, R90, Y96, L97, A100, S102, L103, A104, P105, and A107.