PD-L1 binding polypeptides for imaging

1. A polypeptide comprising a modified human fibronectin type III tenth ( 10 Fn3) domain, wherein the polypeptide specifically binds to human Programmed Death Ligand-1 (PD-L1), and wherein the BC, DE, and FG loops of the modified 10 Fn3 domain comprise the amino acid sequences set forth in:

(a) SEQ ID NOs: 6, 7 and 8, respectively;

(b) SEQ ID NOs: 21, 22 and 23, respectively;

(c) SEQ ID NOs: 36, 37 and 38, respectively;

(d) SEQ ID NOs: 51, 52 and 53, respectively;

(e) SEQ ID NOs: 66, 67 and 68, respectively;

(f) SEQ ID NOs: 81, 82 and 83, respectively;

(g) SEQ ID NOs: 97, 98 and 99, respectively;

(h) SEQ ID NOs: 113, 114 and 115, respectively;

(i) SEQ ID NOs: 124, 125 and 126, respectively;

(j) SEQ ID NOs: 135, 136 and 137, respectively;

(k) SEQ ID NOs: 146, 147 and 148, respectively;

(l) SEQ ID NOs: 157, 158 and 159, respectively;

(m) SEQ ID NOs: 168, 169 and 170, respectively;

(n) SEQ ID NOs: 179, 180 and 181, respectively;

(o) SEQ ID NOs: 190, 191 and 192, respectively;

(p) SEQ ID NOs: 201, 202 and 203, respectively;

(q) SEQ ID NOs: 212, 213 and 214, respectively;

(r) SEQ ID NOs: 223, 224 and 225, respectively;

(s) SEQ ID NOs: 234, 235 and 236, respectively;

(t) SEQ ID NOs: 245, 246 and 247, respectively;

(u) SEQ ID NOs: 256, 257 and 258, respectively;

(v) SEQ ID NOs: 267, 268 and 269, respectively;

(w) SEQ ID NOs: 278, 279 and 280, respectively;

(x) SEQ ID NOs: 289, 290 and 291, respectively;

(y) SEQ ID NOs: 300, 301 and 302, respectively;

(z) SEQ ID NOs: 311, 312 and 313, respectively;

(aa) SEQ ID NOs: 322, 323 and 324, respectively;

(bb) SEQ ID NOs: 333, 334 and 335, respectively;

(cc) SEQ ID NOs: 344, 345 and 346, respectively;

(dd) SEQ ID NOs: 355, 356 and 357, respectively;

(ee) SEQ ID NOs: 366, 367 and 368, respectively;

(ff) SEQ ID NOs: 377, 378 and 379, respectively;

(gg) SEQ ID NOs: 388, 389 and 390, respectively;

(hh) SEQ ID NOs: 399, 400 and 401, respectively;

(ii) SEQ ID NOs: 410, 411 and 412, respectively;

(jj) SEQ ID NOs: 421, 422 and 423, respectively;

(kk) SEQ ID NOs: 432, 433 and 434, respectively;

(ll) SEQ ID NOs: 443, 444 and 445, respectively;

(mm) SEQ ID NOs: 454, 455 and 456, respectively;

(nn) SEQ ID NOs: 465, 466 and 467, respectively;

(oo) SEQ ID NOs: 476, 477 and 478, respectively;

(pp) SEQ ID NOs: 487, 488 and 489, respectively;

(qq) SEQ ID NOs: 498, 499 and 500, respectively;

(rr) SEQ ID NOs: 509, 510 and 511, respectively;

(ss) SEQ ID NOs: 520, 521 and 522, respectively;

(tt) SEQ ID NOs: 531, 532 and 533, respectively;

(uu) SEQ ID NOs: 542, 543 and 544, respectively;

(vv) SEQ ID NOs: 553, 554 and 555, respectively; or

(ww) SEQ ID NOs: 564, 565 and 566, respectively.

2. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 20, 35, 50, 65, 80, 96, 112, 123, 134, 145, 156, 167, 178, 189, 200, 211, 222, 233, 244, 255, 266, 277, 288, 299, 310, 321, 332, 343, 354, 365, 376, 387, 398, 409, 420, 431, 442, 453, 464, 475, 486, 497, 508, 519, 530, 541, 552 and 563.

3. The polypeptide of claim 1 , wherein the polypeptide further comprises one or more pharmacokinetic (PK) moieties selected from the group consisting of polyethylene glycol, sialic acid, Fc, Fc fragment, transferrin, serum albumin, a serum albumin binding protein, and a serum immunoglobulin binding protein.

4. The polypeptide of claim 1 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain comprise the amino acid sequences set forth in:

(a) SEQ ID NOs: 6, 7 and 8, respectively;

(b) SEQ ID NOs: 21, 22 and 23, respectively;

(c) SEQ ID NOs: 36, 37 and 38, respectively;

(d) SEQ ID NOs: 51, 52 and 53, respectively;

(e) SEQ ID NOs: 66, 67 and 68, respectively;

(f) SEQ ID NOs: 81, 82 and 83, respectively; or

(g) SEQ ID NOs: 97, 98 and 99, respectively.

5. The polypeptide of claim 4 , wherein the modified human 10 Fn3 domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 20, 35, 65, 80 and 96.

6. The polypeptide of claim 1 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 81, 82 and 83, respectively.

7. The polypeptide of claim 6 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 80.

8. The polypeptide of claim 7 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 84.

9. The polypeptide of claim 7 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 88.

10. The polypeptide of claim 1 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 97, 98 and 99, respectively.

11. The polypeptide of claim 10 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 96.

12. The polypeptide of claim 10 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 100.

13. The polypeptide of claim 10 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 104.

14. A composition comprising the polypeptide of claim 1 , and a carrier.

15. A kit comprising the polypeptide of claim 1 , and instructions for use.

16. An imaging agent comprising the polypeptide of claim 1 , and a detectable label.

17. The imaging agent of claim 16 , wherein the detectable label is a radionuclide.

18. An imaging agent comprising a protein, an 18 F-radiolabeled prosthetic group, and a bifunctional conjugating (BFC) moiety, wherein the imaging agent has the following structure:

wherein the 18 F is ortho to the N atom, x is an integer from 1 to 8, and the protein comprises a polypeptide of claim 1 .

19. The imaging agent of claim 18 , wherein the 18 F-radiolabeled prosthetic group has the structure:

20. The imaging agent of claim 18 , wherein the BFC is dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS-Ester), dibenzocyclooctyne-Sulfo-N-hydroxysuccinimidyl ester (DBCO-Sulfo-NHS-Ester), dibenzocyclooctyne-PEG4-acid (DBCO-PEG4-Acid), dibenzocyclooctyne-PEG4-amine (DBCO-PEG4-Amine) or dibenzocyclooctyne-PEG4-maleimide (DBCO-PEG4-Maleimide).

21. The imaging agent of claim 18 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 81, 82 and 83, respectively.

22. The imaging agent of claim 21 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 80.

23. The imaging agent of claim 18 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain comprise the amino acid sequences set forth in SEQ ID NOs: 97, 98 and 99, respectively.

24. The imaging agent of claim 23 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 96.

25. An imaging agent comprising a protein, an 18 F-radiolabeled prosthetic group, and a bifunctional conjugating (BFC) moiety, wherein the imaging agent has the following structure:

wherein “OPEG” is [O(CH 2 ) 2 ] x , x is an integer from 1 to 8, and the protein comprises a polypeptide of claim 1 .

26. An imaging agent comprising the structure:

wherein X is a polypeptide of claim 1 .

27. The imaging agent of claim 26 , wherein X is a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 13, 28, 43, 58, 73, 88 and 104.

28. The imaging agent of claim 26 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain in X comprise the amino acid sequences set forth in SEQ ID NOs: 81, 82 and 83, respectively.

29. The imaging agent of claim 28 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 80.

30. The imaging agent of claim 28 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 88.

31. The imaging agent of claim 30 , wherein the C-terminal cysteine residue of SEQ ID NO: 88 is covalently bound to the maleimide moiety.

32. The imaging agent of claim 26 , wherein the BC, DE and FG loops of the modified human 10 Fn3 domain in X comprise the amino acid sequences set forth in SEQ ID NOs: 97, 98 and 99, respectively.

33. The imaging agent of claim 32 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 96.

34. The imaging agent of claim 32 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 104.

35. The imaging agent of claim 34 , wherein the C-terminal cysteine residue of SEQ ID NO: 104 is covalently bound to the maleimide moiety.