Pet imaging with pd-l1 binding polypeptides

Abstract

Provided herein are novel 10 Fn3 domains which specifically bind to PD-L1, as well as imaging agents based on the same for diagnostics.

1 . A method of visualizing PD-L1 protein in a subject, comprising:
 (a) administering to the subject a PD-L1 imaging agent at a dose of about 3-10 mCi (100-333 MBq); and   (b) conducting a PET scan of the subject about 30-120 minutes after step (a).   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the subject has at least one tumor. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , for determining whether a subject is likely to respond to a treatment with an immuno-oncology agent. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the subject is being treated with a therapeutic agent. 
     
     
         11 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the PD-L1 imaging agent is a protein binding specifically to human PD-L1, wherein the protein is linked to a detectable agent. 
     
     
         16 . The method of  claim 15 , wherein the PD-L1 imaging agent is an anti-PD-L1 adnectin. 
     
     
         17 . The method of  claim 15 , wherein the detectable agent is a radioactive PET tracer. 
     
     
         18 - 23 . (canceled) 
     
     
         24 . The method of  claim 16 , wherein the anti-human PD-L1 adnectin comprises a fibronectin type III tenth domain ( 10 Fn3), wherein (a) the  10 Fn3 domain comprises AB, BC, CD, DE, EF, and FG loops, (b) the  10 Fn3 has at least one loop selected from loop BC, DE, and FG with an altered amino acid sequence relative to the sequence of the corresponding loop of the human  10 Fn3 domain (SEQ ID NO: 1), and (c) the polypeptide specifically binds to human PD-L1. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . The method of  claim 24 , wherein the BC, DE, and FG loops comprise the amino acid sequences of:
 (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;   (i) 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;   (g) 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;   (ii) 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, 530 and 531, 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.   
     
     
         28 . The method  claim 24 , wherein the polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 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. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method of  claim 24 , wherein the polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NO: 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. 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 24 , wherein the polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop regions of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 9-15, 24-30, 39-45, 54-60, 69-75, 84-91, 100-116-122, 127-133, 138-144, 150-155, 160-166, 171-177, 182-188, 193-199, 204-210, 215-221, 227-232, 237-243, 248-254, 259-265, 271-276, 291-287, 292-298, 303-309, 314-320, 325-331, 337-342, 347-353, 358-364, 369-375, 380-386, 391-397, 402-408, 413-419, 424-430, 435-441, 446-452, 457-463, 468-474, 479-485, 490-496, 501-507, 512-518, 523-529, 534-540, 545-551, and 556-562. 
     
     
         34 . The method of  claim 24 , wherein the polypeptide comprises an N-terminal leader selected from the group consisting of SEQ ID NOs: 112-121, and/or a C-terminal tail selected from the group consisting of SEQ ID NOs: 122-156. 
     
     
         35 . The method of  claim 24 , wherein the polypeptide comprises one or more pharmacokinetic (PK) moieties selected from the group consisting of polyethylene glycol and sialic acid. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 35 , wherein the PK moiety and the polypeptide are linked via a linker with an amino acid sequence selected from the group consisting of SEQ ID NOs: 167-216. 
     
     
         38 . The method of  claim 1 , wherein the imaging agent comprises the 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, or pharmaceutically acceptable salt thereof. 
       
     
     
         39 - 50 . (canceled) 
     
     
         51 . The method of  claim 38 , wherein the  18 F-radiolabeled prosthetic group has the structure 
       
         
           
           
               
               
           
         
       
     
     
         52 - 55 . (canceled) 
     
     
         56 . The method of  claim 38 , wherein the BFC is a cyclooctyne comprising a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein. 
     
     
         57 . The method of  claim 56 , wherein the cyclooctyne is selected from the group consisting of dibenzocyclooctyne (DIBO), biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC) and dibenzocyclooctyne (DBCO). 
     
     
         58 . (canceled) 
     
     
         59 . The method of  claim 38 , wherein the BFC further comprises a polyethylene glycol (PEG) y  spacer arm, wherein y is an integer from 1 to 8. 
     
     
         60 - 63 . (canceled) 
     
     
         64 . The method of  claim 38 , wherein the imaging agent has the structure: 
       
         
           
           
               
               
           
         
       
       wherein X is a polypeptide comprising an anti-human PD-L1 adnectin comprising a fibronectin type III tenth domain ( 10 Fn3), wherein the BC, DE, and FG loops of the  10 Fn3 domain comprise the amino acid sequences of:
 (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; 
 (g) 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, 530 and 531, 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. 
 
     
     
         65 - 66 . (canceled) 
     
     
         67 . The method of  claim 64 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 9-15, 24-30, 39-45, 54-60, 69-75, 84-91, 100-107, 116-122, 127-133, 138-144, 150-155, 160-166, 171-177, 182-188, 193-199, 204-210, 215-221, 227-232, 237-243, 248-254, 259-265, 271-276, 291-287, 292-298, 303-309, 314-320, 325-331, 337-342, 347-353, 358-364, 369-375, 380-386, 391-397, 402-408, 413-419, 424-430, 435-441, 446-452, 457-463, 468-474, 479-485, 490-496, 501-507, 512-518, 523-529, 534-540, 545-551, and 556-562. 
     
     
         68 . The method of  claim 64 , wherein the BFC is conjugated to the polypeptide at a cysteine residue near the C-terminus of the polypeptide.