a2 United States Patent
`Yang et al.
`
`US011938201B2
`
`US 11,938,201 B2
`*Mar. 26, 2024
`
`(10) Patent No.:
`45) Date of Patent:
`
`(54) IMAGING AND RADIOTHERAPEUTICS
`AGENTS TARGETING
`FIBROBLAST-ACTIVATION
`PROTEIN-ALPHA (FAP-ALPHA)
`
`(71) Applicant: The Johns Hopkins University,
`Baltimore, MD (US)
`
`(72) Inventors: Xing Yang, Baltimore, MD (US);
`Sridhar Nimmagadda, Baltimore, MD
`(US); Steven Rowe, Parkville, MA
`(US); Stephanie Slania, Baltimore, MD
`(US); Martin G. Pomper, Baltimore,
`MD (US)
`
`(73) Assignee: The Johns Hopkins University,
`Baltimore, MD (US)
`
`(*) Notice: Subject to any disclaimer, the term of this
`
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 18/354,282
`(22) Filed: Jul. 18, 2023
`
`(65) Prior Publication Data
`US 2023/0364274 Al Nov. 16, 2023
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 16/758,182, filed as
`application No. PCT/US2018/057086 on Oct. 23,
`2018.
`
`(60) Provisional application No. 62/575,607, filed on Oct.
`
`23, 2017.
`(51) Int. CL
`AG6IK 51/00 (2006.01)
`AGIK 47/54 (2017.01)
`AG6IK 51/04 (2006.01)
`AG6IM 36/14 (2006.01)
`(52) US. CL
`CPC ...... AGIK 51/0485 (2013.01); A6IK 47/545
`
`(2017.08); A61K 51/0478 (2013.01); A61K
`51/0482 (2013.01)
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`(56) References Cited
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`Chen et al., Advance of molecular imaging technology and targeted
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`Edosada et al., Peptide substrate profiling defines fibroblast activa-
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`
`(Continued)
`
`Primary Examiner — Michael G. Hartley
`
`Assistant Examiner — Melissa J Perreira
`
`(74) Attorney, Agent, or Firm — Casimir Jones, SC;
`Jeftrey W. Childers
`
`(57) ABSTRACT
`Imaging and radiotherapeutics agents targeting fibroblast-
`
`activation protein- (FAP- ) and their use in imaging and
`treating FAP- related diseases and disorders are disclosed.
`
`3 Claims, 9 Drawing Sheets
`(4 of 9 Drawing Sheet(s) Filed in Color)
`
`Petitioner GE Healthcare — Ex. 1001, p. 1
`
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`
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`
`
`
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`US 11,938,201 B2
`Page 2
`
`(56) References Cited
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`Kraman et al., Suppression of antitumor immunity by stromal cells
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`Laverman et al., Immuno-PET and Immuno-SPECT of Rheumatoid
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`Scanlan et al., Molecular cloning of fibroblast activation protein
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`in stromal fibroblasts of epithelial cancers. Proc Natl Acad Sci U S
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`Scott et al., A Phase I dose-escalation study of sibrotuzumab in
`patients with advanced or metastatic fibroblast activation protein-
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`Tsai et al., Substituted 4-carboxymethylpyroglutamic acid diamides
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`Tuxhorn et al., Reactive stroma in human prostate cancer: induction
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`Clin Cancer Res. Sep. 2002;8(9):2912-23.
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`Welt et al., Antibody targeting in metastatic colon cancer: a phase
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`International Search Report and Written Opinion for PCT/US2018/
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`Jan. 10, 2024, 553 pages.
`
`* cited by examiner
`
`Petitioner GE Healthcare — Ex. 1001, p. 2
`
`
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`
`
`U.S. Patent Mar. 26,2024 Sheet 1 of 9 US 11,938,201 B2
`
`-
`W ©
`O~ OH 0o N \/go
`HCI
`HO Z [e] HOBT, HBTU, DIPEf HO P
`)N A ]
`N DMF, r.t. 6h N
`TFA
`+ N B
`Boc” D N
`Cs,CO;4
`DMF, r.t. overnight
`-
`OH (o]
`H H
`0. N
`&) N\/go LIOH \/go
`BocHN . _~_.0 / - - BocHN _~_0 Z
`| H20!THF, r.L 6h - |
`N NT TFA
`
`+
`HCI
`L;L
`
`CSN
`HOBT, HBTU, DIPEA &c
`N
`
`DMF, r.t. 6h
`
`BocHN _~_0O
`
`Petitioner GE Healthcare — Ex. 1001, p. 3
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`U.S. Patent Mar. 26,2024 Sheet 2 of 9 US 11,938,201 B2
`
`S0:H
`
`o MK,
`=
`4
`o 4N N
`\ 0-_N
`)\“ COOH | O
`o \N/\\ N _~_© =
`“\“--Iln j o) g |
`>N LN N
`L/
`0™ XY-FAP-02-[In]
`
`Fig. 1C
`
`Petitioner GE Healthcare — Ex. 1001, p. 4
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`U.S. Patent Mar. 26,2024 Sheet 3 of 9 US 11,938,201 B2
`
`FAP Binding
`
`0.4
`
`0.3
`
`(O]
`
`(2]
`
`C
`
`S 0.2-
`
`(D]
`
`(0]
`
`14
`
`0.1
`
`0.0+
`
`102 10" 10 10° 10% 107
`[inhibitor] (M)
`
`Fig. 2
`
`Petitioner GE Healthcare — Ex. 1001, p. 5
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`U.S. Patent Mar. 26,2024 Sheet 4 of 9 US 11,938,201 B2
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`[XY-FAP-01] (nM)
`
`50 25 125 625 313 15 078 0
`
`PC3 100% - F ‘." - PC3
`M - NCIH226
`NCIH226 i,;’f -+ HCT116
`o
`: a B -¥. NCIH2228
`HCT116 [ a i
`o 50% -e- U7
`sl @ @ - SKMEL24
`us7
`ot wlgl aa®
`SKMEL24 . - 10" 10° 10+ 107
`[XYFAP-01] (nM)
`Fig. 34
`ig. 3
`DPPIV + FAP Inhibition DPPIV Inhibition
`£ =109 2210,
`[} =7
`5 >
`- HZZ2E
`Eq 5102 1.5 401 a US7
`g -+ SKMEL24
`% 1=104 =409
`o
`2 A=109 =404
`(1
`c
`10-7 04 104 104 04 10-2 04 10-7 104 104 104 104
`[Talabostaf (hi) [Sitagliptin] (kD)
`
`Fig. 3B
`
`AAAR
`
`% Incubated Dose
`Pad
`=
`
`ua7 PC3
`
`ig. 3C
`
`Petitioner GE Healthcare — Ex. 1001, p. 6
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`U.S. Patent Mar. 26,2024 Sheet 5 of 9 US 11,938,201 B2
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`5 min 05h 2h 6 hr 6 h - blocking 12h
`(n=3) (n=4) (n=3) (n=4) (n=4) (n=4)
`blood 1513062 1064 +1.54 410+039 201018 0.02 +0.001 042+004
`heart 6.68 £ 0.99 495071 261+0.09 1.07£0.09 0.02 +0.002 046+0.04
`lungs 6.99 +1.37 560+0.89 259032 1.36+0.13 0.04 +0.005 039+003
`liver 6.32+0.77 490+059 258009 251+009 033+0.058 166+0.22
`stomach 328+046 262+0.20 169+0.14 1.19+£0.15 0.06 +0.032 034+005
`pancreas 1454 + 166 1214 +£2385 811+034 328027 0.03 +0.005 115052
`spleen 293+025 245047 177027 154+0.19 0.06 + 0.008 1.10+£0.13
`fat 0.74+0.09 073+£0.13 0.61+0.19 045+0.16 0.02+0.017 015+0.08
`kidney 460057 356+0.18 1.95+0.06 1.79+0.21 1.16 £0.158 0.73+005
`sm. int. 8.80+1.97 841135 364+045 166 +0.22 0.09 +0.052 076+0.14
`Irg. int. 467042 457057 298+027 197 +£0.47 036 +0.342 055+0.04
`bladder 296+0.95 13.60+8.80 394+643 341+0584 1.04 + 0.466 225+057
`muscle 3.00+£022 353016 2.30+0.07 179+0.15 0.02 +0.005 060+0.03
`femur 476012 583072 495+ 060 391+070 0.08 +0.021 1.38+0.20
`us7 9.71+0.24° 12,80+ 1.45° 12.28 + 1.952 11.20 £ 1.03* 027 +0.01% 457 +0.54°
`PC3 220+0.20 3.10+057 275011 340+034 0.11 £ 0.024¢ 095+0.06
`Us7:PC3 443031 423072 4.46+062 330+014 261+0.440 482+028
`U87:blood 064 £0.04 1.22+0.07 3.00+£038 558+0.36 18.07 £1.197 1094+ 093
`Ug87:mm 3.25+0.26 3.66+0.44 4.38 + 0.66 6.29 + 0.82 11.81 + 2.086 7.68 +0.97
`
`Fig. 4
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`Petitioner GE Healthcare — Ex. 1001, p. 7
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`US 11,938,201 B2
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`U.S. Patent Mar. 26, 2024 Sheet 6 of 9
`20-
`-8- S7 tumor (FAPH
`15 @ PC3tumor (FAF-)
`% -+ Blood
`_E:'I -
`=
`=
`o 1 B7PC3
`& |57 blood
`-&- 1B mm
`2
`"
`=5
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`Petitioner GE Healthcare — Ex. 1001, p. 8
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`U.S. Patent Mar. 26,2024 Sheet 7 of 9 US 11,938,201 B2
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`wo- 8.06E-2
`- 1.00E-2
`
`- 9.00E-2
`
`- 3.00E-2
`
`- 7.00E-3
`* 0.00E0
`
`0.5h 2.5h
`
`Fig. 6
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`Petitioner GE Healthcare — Ex. 1001, p. 9
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`U.S. Patent Mar. 26,2024 Sheet 8 of 9 US 11,938,201 B2
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`30 min 2 hr 6 hr
`
`Fig. 7
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`Petitioner GE Healthcare — Ex. 1001, p. 10
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`U.S. Patent Mar. 26,2024 Sheet 9 of 9 US 11,938,201 B2
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`30 min 2 hr 6 hr 12 hr
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`Fig. 8
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`Petitioner GE Healthcare — Ex. 1001, p. 11
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`US
`
`1
`IMAGING AND RADIOTHERAPEUTICS
`AGENTS TARGETING
`FIBROBLAST-ACTIVATION
`PROTEIN-ALPHA (FAP-ALPHA)
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of U.S. patent applica-
`tion Ser. No. 16/758,182, filed Apr. 22, 2020, which is a U.S.
`§ 371 National Entry Application of PCT/US2018/057086,
`filed Oct. 23, 2018, which claims the benefit of U.S. Pro-
`visional Application No. 62/575,607, filed Oct. 23, 2017,
`each of which is incorporated herein by reference in its
`entirety.
`
`FEDERALLY SPONSORED RESEARCH OR
`DEVELOPMENT
`
`This invention was made with government support under
`CA197470 awarded by the National Institutes of Health. The
`government has certain rights in the invention.
`
`BACKGROUND
`
`Fibroblast-activation protein- (FAP- ) expression has
`been detected on the surface of fibroblasts in the stroma
`surrounding >90% of the epithelial cancers examined,
`including malignant breast, colorectal, skin, prostate and
`pancreatic cancers. (Garin-Chesa, et al., 1990; Rettig, et al.,
`1993; Tuxhorn, et al., 2002; Scanlan, et al., 1994). It is a
`characteristic marker for carcinoma-associated-fibroblast
`(CAF), which plays a critical role in promoting angiogen-
`esis, proliferation, invasion, and inhibition of tumor cell
`death. (Allinen, et al., 2004; Franco, et al., 2010). In healthy
`adult tissues, FAP- expression is only limited to areas of
`tissue remodeling or wound healing. (Scanlan, et al., 1994;
`Yu, et al., 2010; Bae, et al., 2008; Kraman, et al., 2010). In
`addition, FAP- -positive cells are observed during embryo-
`genesis in areas of chronic inflammation, arthritis, and
`fibrosis, as well as in soft tissue and bone sarcomas. (Scan-
`lan, et al., 1994; Yu, et al., 2010). These characteristics make
`FAP- a potential imaging and radiotherapeutic target for
`cancer and inflammation diseases.
`
`Because FAP- is expressed in tumor stroma, anti-FAP
`antibodies have been investigated for radioimmunotargeting
`of malignancies, including murine F19, sibrotuzumab (a
`humanized version of the F19 antibody), ESC11, ESC14,
`and others. (Welt, et al., 1994; Scott, et al., 2003; Fischer, et
`al., 2012). Antibodies also demonstrated the feasibility of
`imaging inflammation, such as rheumatoid arthritis. (Laver-
`man, et al., 2015). The use of antibodies as molecular
`imaging agents, however, suffers from pharmacokinetic
`limitations, including slow blood and non-target tissue clear-
`ance (normally 2-5 days or longer) and non-specific organ
`uptake. Low molecular weight (LMW) agents demonstrate
`faster pharmacokinetics and a higher specific signal within
`clinically convenient times after administration. They also
`can be synthesized in radiolabeled form more easily and
`may offer a shorter path to regulatory approval. (Coenen, et
`al., 2010; Coenen, et al., 2012; Reilly, et al., 2015). To date,
`however, no LMW ligand has been reported with ideal
`properties for nuclear imaging of FAP- .
`
`20
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`25
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`30
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`35
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`40
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`45
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`50
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`11,938,201 B2
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`2
`SUMMARY
`
`In some aspects, the presently disclosed subject matter
`provides a compound of Formula (I):
`
`B-L-A @
`
`wherein: A is a targeting moiety for FAP- ; B is any optical
`or radiolabeled functional group suitable for optical imag-
`ing, PET imaging, SPECT imaging, or radiotherapy; and L
`is a linker having bi-functionalization adapted to form a
`chemical bond with B and A.
`
`In particular aspects, A is an FAP-
`having the structure of:
`
`targeting moiety
`
`)
`SRy (o),
`(Rs
`TN T R
`H
`0. N
`/ CHy), Ry
`
`Rs,’ Ry
`
`Rex
`
`wherein each y is independently an integer selected from
`the group consisting of 0, 1, and 2; R,,, R,,, and R;_., are
`each independently selected from the group consisting of H,
`OH, halogen, C, qalkyl, —O—C, alkyl, and
`—S—~C, _salkyl; R; is selected from the group consisting of
`H, —CN, —B(OH),, —C(O)alkyl, —C(O)aryl-, —C—C—
`C(Ojaryl, —C—=—C—S(O),aryl, —CO,H, —SO;H,
`—SO,NH,, —PO;H,, and 5-tetrazolyl; R, is H; Rs,, Rq,,
`and R, are each independently selected from the group
`consisting of H, —OH, oxo, halogen, —C, calkyl,
`—NR;, Ry, —OR,,,, —Het, and —Ar,; each of C, salkyl
`being optionally substituted with from 1 to 3 substituents
`selected from —OH and halogen; Rg,, and Ry, and R, are
`each independently selected from the group consisting of H,
`—OH, halo, —C, _¢alkyl, —O—C, 4alkyl, —S—C, qalkyl,
`and —Ar;; R, Ry Ry5, and R, are each independently
`selected from the group consisting of H, —OH, halogen,
`—C, _¢alkyl, —O—C, 4alkyl, and —S—C, ¢alkyl; Ar,, Ar,
`and Ar; are each independently a 5- or 6-membered aromatic
`monocycle optionally comprising 1 or 2 heteroatoms
`selected from O, N and S; each of Ar,, Ar, and Ar; being
`optionally and independently substituted with from 1 to 3
`substituents selected from —NR,,R;,,, —C, ¢alkyl,
`—O0—C, ¢alkyl, and —S—C, _qalkyl; Het, is a 5- or 6-mem-
`bered non-aromatic monocycle optionally comprising 1 or 2
`heteroatoms selected from O, N and S; Het, being optionally
`substituted with from 1 to 3 substituents selected from
`—NR 5, R4, —C, ealkyl, —O—C, qalkyl, and —S—C,_
`salkyl; v is 0, 1, 2, or 3; and
`
`N
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`Petitioner GE Healthcare — Ex. 1001, p. 12
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`US 11,938,201 B2
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`3
`
`represents a 5 to 10-membered N-containing aromatic or
`non-aromatic mono- or bicyclic heterocycle, said hetero-
`cycle optionally further comprising 1, 2 or 3 heteroatoms
`selected from O, N and S; wherein
`
`10
`
`indicates a point of attachment of the FAP- binding ligand
`to the linker, L, or the reporter moiety, B, wherein the point
`of attachment can be through any of the carbon atoms of the
`5 to 10-membered N-containing aromatic or non-aromatic
`mono- or bicyclic heterocycle thereof; and stereoisomers
`and pharmaceutically acceptable salts thereof.
`
`15
`
`In more particular aspects, A is an FAP- targeting moiety
`
`having the structure of: 20
`
`y(R1) (Ray)y
`
`25
`
`w(Rax)
`
`H
`o N\/K
`(0]
`
`30
`
`8 35
`
`wherein
`
`40
`
`45
`
`indicates a point of attachment of the FAP- binding ligand
`to the linker, L, or the reporter moiety, B, wherein the point
`of attachment can be through any of carbon atoms 5, 6, 7, or
`8 of the quinolinyl ring thereof; and stereoisomers and
`
`50
`pharmaceutically acceptable salts thereof.
`
`In yet more particular aspects, A is selected from the
`group consisting of:
`
`55
`Al
`
`60
`
`| 65
`
`/
`
`-continued
`A2
`Q\C§ N; and
`H
`[¢) N \/K
`o
`/ ‘
`AN
`N
`A3
`
`Q\C$ N.
`H
`N\/K
`o
`
`/
`
`In other aspects, the presently disclosed subject matter
`provides a pharmaceutical composition comprising a com-
`pound of formula (I).
`
`In some aspects, the presently disclosed subject matter
`provides a method for imaging a disease or disorder asso-
`ciated with fibroblast-activation protein- (FAP- ), the
`method comprising administering a compound of formula
`(I), wherein the compound of formula (I) comprises an
`optical or radiolabeled functional group suitable for optical
`imaging, PET imaging, or SPECT imaging; and obtaining an
`image.
`
`In other aspects, the presently disclosed subject matter
`provides a method for inhibiting fibroblast-activation pro-
`tein- (FAP- ), the method comprising administering to a
`subject in need thereof an effective amount of a compound
`of formula (I).
`
`In yet other aspects, the presently disclosed subject matter
`provides a method for treating a fibroblast-activation pro-
`tein- (FAP- )-related disease or disorder, the method com-
`prising administering to a subject in need of treatment
`thereof an effective amount of a compound of formula (I),
`wherein the compound of formula (I) comprises a radiola-
`beled functional group suitable for radiotherapy.
`
`In certain aspects, the (FAP- )-related disease or disorder
`is selected from the group consisting of a proliferative
`disease, including, but not limited to, breast cancer, colorec-
`tal cancer, ovarian cancer, prostate cancer, pancreatic cancer,
`kidney cancer, lung cancer, melanoma, fibrosarcoma, bone
`and connective tissue sarcomas, renal cell carcinoma, giant
`cell carcinoma, squamous cell carcinoma, and adenocarci-
`noma; diseases characterized by tissue remodeling and/or
`chronic inflammation; disorders involving endocrinological
`dysfunction; and blood clotting disorders.
`
`Certain aspects of the presently disclosed subject matter
`having been stated hereinabove, which are addressed in
`whole or in part by the presently disclosed subject matter,
`other aspects will become evident as the description pro-
`ceeds when taken in connection with the accompanying
`Examples and Figures as best described herein below.
`
`Petitioner GE Healthcare — Ex. 1001, p. 13
`
`
`
`
`
`
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`US 11,938,201 B2
`
`5
`BRIEF DESCRIPTION OF THE FIGURES
`
`The patent or application file contains at least one drawing
`executed in color. Copies of this patent or patent application
`publication with color drawings will be provided by the
`Office upon request and payment of the necessary fee.
`
`Having thus described the presently disclosed subject
`matter in general terms, reference will now be made to the
`accompanying Figures, which are not necessarily drawn to
`scale, and wherein:
`
`FIG. 1A, FIG. 1B, and FIG. 1C show the synthetic
`pathway and structures of representative FAP-targeted
`agents, XY-FAP-01 and ['''In]-XY-FAP-02. FIG. 1A shows
`the multi-step synthesis of the ligand precursor, tert-butyl
`(S)-(3-((4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbam-
`oyl)quinolin-6-yl)oxy)propyl)carbamate. After each step,
`the reaction mixture was loaded onto a 25-g C18 cartridge
`and purified with a MeCN/water/TFA gradient. Identity of
`intermediate products was confirmed with "H NMR. FIG.
`1B shows the full structure of optical imaging agent, XY-
`FAP-01. XY-FAP-01 was produced with a one step reaction
`between the precursor and IRDye800CW-NHS. The major
`product was obtained at a yield of 85% after purification
`with HPLC. FIG. 1C shows the full structure of the SPECT
`imaging agent, ['''In]-X Y-FAP-02. First, the precursor was
`functionalized with DOTA via a one step reaction between
`the precursor and DOTA-GA(t-Bu),-NHS. Unlabeled prod-
`uct was purified via HPLC to produce XY-FAP-02. Subse-
`quent radiolabeling with '"'In and HPLC purification
`resulted in the radiolabeled product, ['''In]-XY-FAP-02;
`
`FIG. 2 shows the inhibitory activity of XY-FAP-01 on
`human recombinant FAP. The inhibitory activity of XY-
`FAP-01 was determined using a fluorogenic FAP assay Kkit.
`Enzymatic activity of human recombinant FAP on a native
`substrate was inhibited in a concentration dependent fashion
`by XY-FAP-01. Semi-log inhibitory curves of XY-FAP-01
`activity were generated and the determined Ki value of
`XY-FAP-01 was 1.26 nM;
`
`FIG. 3A, FIG. 3B, and FIG. 3C show the assessment of
`the in vitro binding ability and specificity of XY-FAP-01 and
`['"In]-XY-FAP-02. FIG. 3A shows the concentration
`dependent uptake of XY-FAP-01 in various cell lines. Cells
`incubated with various concentrations (range: 50 nM to 0.78
`nM) of XY-FAP-01 were imaged with the LI-COR Pearl
`Impulse Imager to assess uptake of agent in various FAP-
`positive and FAP-negative cell lines (left). Dose-response
`curves of XY-FAP-O1 uptake in FAP-positive cell lines
`(NCIH2228, U87, and SKMEL24) and FAP-negative cell
`lines (PC3, NCIH226, and HCT116) were generated (right).
`FIG. 3B shows the inhibition of XY-FAP-01 uptake in
`FAP-positive cell-lines. Cells incubated with 25-nM XY-
`FAP-01 were incubated with various concentrations of either
`a DPPIV and FAP inhibitor, Talabostat, or a DPPIV-only
`inhibitor, Sitagliptin. Uptake of XY-FAP-01 was measured
`and semi-log inhibitor-response curves were generated for
`both Talabostat and Sitagliptin. FIG. 3C shows the uptake of
`["''In]-XY-FAP-02 in FAP-positive U87 and FAP-negative
`PC3 cell lines. Cells were incubated with 1 uCi [*''In]-XY-
`FAP-02 and were washed with cold PBS. Radioactivity of
`the cell pellets was measured and normalized to the incu-
`bated dose;
`
`FIG. 4 is a table showing the ex vivo tissue biodistribution
`of ["''In]-XY-FAP-01 in tumor bearing mice. At 5 min, 0.5
`h, 2 h, 6 h, and 12 h after injection of 10 uCi ['"In]-XY-
`FAP-01, NOD/SKID mice bearing U87 and PC3 tumor
`xenografts were sacrificed and tissues were collected for
`biodistribution analysis. Additionally, mice co-injected with
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`20
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`25
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`30
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`unlabeled XY-FAP-02 and 10 puCi ['''In]-XY-FAP-01 were
`sacrificed at 6 h post-injection to study the effect of blocking
`on uptake of the radiolabeled compound. Data presented as
`mean standard deviation. “Student’s t test comparison of
`mean % ID/g of PC3 tumor versus U87 tumor demonstrated
`significant difference between the two groups at 5 min, 0.5
`h, 2 h, and 6 h post injection (p<0.0001). No significant
`difference between the two groups were seen in the blocking
`study at 6 h. ”Student’s t test comparison of mean % ID/g of
`PC3 tumor versus U87 tumor demonstrated significant dif-
`ference between the two groups at 12 h post injection
`(p=0.0006). “Student’s t test comparing % ID/g between
`PC3 tumor and U87 tumors at 6 h post injection showed
`significant difference between % ID/g tumors in the block-
`ing study at 6 h versus the normal biodistribution results at
`6 h (p<0.0001);
`
`FIG. 5A and FIG. 5B show the time-activity relationship
`of the ex vivo biodistribution of ['''In]-XY-FAP-02. FIG.
`5A shows tissue time activity curves (TACs) of ['''In]-XY-
`FAP-02 activity in U87 tumor, PC3 tumor, and blood. FIG.
`5B shows the ratios of % ID/g between U87 tumor and PC3
`tumor, blood, and muscle (mm) versus time;
`
`FIG. 6 shows serial NIRF-imaging of XY-FAP-0O1 in
`tumor bearing mice. NOD/SKID mice bearing FAP-positive
`U87 (yellow circle) and FAP-negative PC3 (red circle)
`tumor xenografts were injected with 10 nmol of XY-FAP-01
`via the tail vein followed by serial NIRF-imaging on the
`LI-COR Pearl Impulse Imager. Representative images at 0.5
`h, 1 h, 2.5 h, and 4 h after injection are shown;
`
`FIG. 7 shows SPECT-CT images of ['''In]-XY-FAP-02 at
`30 min, 2 h, 6 h, and 24 h after injection in NOD/SKID
`female mice bearing U87 and PC3 tumor xenografts in the
`upper flanks; and
`
`FIG. 8 show three-dimensional SPECT-CT images of
`['"In]-XY-FAP-02 at 30 min, 2 h, 6 h, and 24 h after
`injection in NOD/SKID female mice bearing U87 and PC3
`tumor xenografts in the upper flanks.
`
`DETAILED DESCRIPTION
`
`The presently disclosed subject matter now will be
`described more fully hereinafter with reference to the
`accompanying Figures, in which some, but not all embodi-
`ments of the presently disclosed subject matter are shown.
`Like numbers refer to like elements throughout. The pres-
`ently disclosed subject matter may be embodied in many
`different forms and should not be construed as limited to the
`embodiments set forth herein; rather, these embodiments are
`provided so that this disclosure will satisfy applicable legal
`requirements. Indeed, many modifications and other
`embodiments of the presently disclosed subject matter set
`forth herein will come to mind to one skilled in the art to
`which the presently disclosed subject matter pertains having
`the benefit of the teachings presented in the foregoing
`descriptions and the associated Figures. Therefore, it is to be
`understood that the presently disclosed subject matter is not
`to be limited to the specific embodiments disclosed and that
`modifications and other embodiments are intended to be
`included within the scope of the appended claims.
`
`I. Imaging and Radiotherapeutics Agents Targeting
`Fibroblast-Activation Protein- (FAP- )
`
`FAP- is a type II integral membrane serine protease of
`the prolyl oligopeptidase family, which are distinguished by
`their ability to cleave the Pro-AA peptide bond (where AA
`represents any amino acid). It has been shown to play a role
`
`Petitioner GE Healthcare — Ex. 1001, p. 14
`
`
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`US 11,938,201 B2
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`7
`in cancer by modifying bioactive signaling peptides through
`this enzymatic activity (Kelly, et al., 2005; Edosada, et al.,
`2006). FAP- expression has been detected on the surface of
`fibroblasts in the stroma surrounding greater than 90% of the
`epithelial cancers, including, but not limited to, malignant
`breast, colorectal, skin, prostate, pancreatic cancers, and the
`like, and inflammation diseases, including, but not limited
`to, arthritis, fibrosis, and the like, with nearly no expression
`in healthy tissues. Accordingly, imaging and radiotherapeu-
`
`tic agents specifically targeting FAP- is of clinical impor-
`tance.
`FAP- exists as a homodimer to carry out its enzymatic
`
`function. Inhibitors selectively targeting FAP- has been
`reported (Lo, et al., 2009; Tsai, et al., 2010; Ryabtsova, et al.,
`2012; Poplawski, et al., 2013; Jansen, et al., 2013; Jansen, et
`al., 2014). The presently disclosed subject matter provides,
`in part, a FAP- selective targeting moiety that can be
`modified with an optical dye, a radiometal chelation com-
`plex, and other radiolabeled prosthetic groups, thus provid-
`ing a platform for the imaging and radiotherapy targeting
`FAP- .
`
`Radionuclide molecular imaging, including positron
`emission tomography (PET), is the most mature molecular
`imaging technique without tissue penetration limitations.
`Due to its advantages of high sensitivity and quantifiability,
`radionuclide molecular imaging plays an important role in
`clinical and preclinical research (Youn, et al., 2012; Chen, et
`al., 2014). Many radionuclides, primarily - and alpha
`emitters, have been investigated for targeted radioimmuno-
`therapy and include both radiohalogens and radiometals (see
`Table 1 for representative therapeutic radionuclides).
`
`TABLE 1
`
`Representative Therapeutic Radionuclides
`
`QOYV’ 131[, ]77Lu, ISSSm’ 186RC,
`IXRRe 67Cu 212Pb
`
`ZZSAC, 2I3BiY 2]2Bi 2”A[ ZIZPb
`125[ 1’231 67’(;2‘1 IH’IH ’
`
`-particle emitters
`
`-particle emitters
`Auger electron emitters
`
`The highly potent and specific binding moiety targeting
`FAP- enables its use in nuclear imaging and radiotherapy.
`The presently disclosed subject matter provides the first
`synthesis of nuclear imaging and radiotherapy agents based
`on this dual-targeting moiety to FAP- .
`
`Accordingly, in some embodiments, the presently dis-
`closed subject matter provides potent and selective low-
`molecular-weight (LMW) ligands of FAP- , i.e., an FAP-
`selective inhibitor, conjugated with a targeting moiety fea-
`sible for modification with optical dyes and radiolabeling
`groups, including metal chelators and metal complexes,
`which enable in vivo optical imaging, nuclear imaging
`(optical, PET and SPECT), and radiotherapy targeting FAP-
`
`. Importantly, the presently disclosed compounds can be
`modified, e.g., conjugated with, labeling groups without
`significantly losing their potency. The presently disclosed
`approach allows for the convenient labeling of the FAP-
`ligand with optical dyes and PET or SPECT isotopes,
`including, but not limited to, °®Ga, **Cu, '®F, #°Y, *°Y, %Zr,
`"n, #9Tc, 121, 121, for FAP- related imaging applica-
`tions. Further, the presently disclosed approach allows for
`the radiolabeling of the FAP- ligand with radiotherapeutic
`isotopes, including but not limited to, *°Y, '""Lu, '*I, '3'I,
`21 A 1, 153Gy 186Re 188Re 67Cy 212pp 225A¢ 213Bj,
`212Bj, 212Pb, and ®’Ga, for FAP- related radio-therapy.
`
`In a particular embodiment, an optical agent conjugated
`with IRDye-800CW (XY-FAP-01) was synthesized and
`
`20
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`30
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`40
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`showed selective uptake in vitro on a FAP- U87 cell line
`and in vivo on a FAP- U87 tumor and clearly detected the
`tumor. In another particular embodiment, an '''In labeled
`ligand (XY-FAP-02-['''In]) was successfully obtained in
`high yield and purity from its precursor with a metal
`chelator. The