US011938201B2
`
`(12) United States Patent
`Yang et al.
`
`(10) Patent No.: US 11,938,201 B2
`(45) Date of Patent:
`*Mar. 26, 2024
`
`(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 0 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 A1
`
`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.
`A61K 51/00
`
`A61K 47/54
`
`A61K 51/04
`
`A61M 36/14
`
`(2006.01)
`(2017.01)
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl.
`CPC ........ A61K 51/0485 (2013.01); A61K 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
`
`U.S. PATENT DOCUMENTS
`
`9,346,814 B2
`2008/0280856 A1
`2010/0098633 A1 *
`
`5/2016 Jansen et al.
`11/2008 Cohen et al.
`4/2010 Zimmerman ........ C07D 401/14
`424/1.85
`
`2014/0357650 A1
`2020/0237936 A1
`2021/0038749 A1
`
`12/2014 Jansen et al.
`7/2020 Low et al.
`2/2021 Haberkorn et al.
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`JP
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`18199641.4
`2021-512949 A
`WO 2010/014933
`WO 2010/108125
`WO 2013/107820
`WO 2014/001538
`WO 2015/114166
`WO 2016/065142
`WO 2016/149188
`WO 2016/196628 A1
`WO 2019/154886
`
`10/2018
`5/2021
`2/2010
`9/2010
`7/2013
`1/2014
`8/2015
`4/2016
`9/2016
`12/2016
`8/2019
`
`OTHER PUBLICATIONS
`
`Tahtis et al. (Mol. Cancer Therap. 2003, 2, 729-737).*
`Terry et al. (J. Nucl. Med. 2016, 57, 467-472).*
`Allinen et al., Molecular characterization of the tumor microenviron-
`ment in breast cancer. Cancer Cell. Jul. 2004;6(1):17-32.
`Bae et al., Fibroblast activation protein alpha identifies mesenchymal
`stromal cells from human bone marrow. Br J Haematol. Sep.
`2008;142(5):827-30.
`Chen et al., Advance of molecular imaging technology and targeted
`imaging agent in imaging and therapy. Biomed Res Int. 2014; 2014
`: 819324. PMCID: PMC3943245.
`Coenen et al., Fluorine-18 radiopharmaceuticals beyond [18F]FDG
`for use in oncology and neurosciences. Nuclear medicine and
`biology. 2010; 37:727-740.
`Cho et al., Biodistribution, tumor detection, and radiation dosimetry
`of 18F-DCFBC, a low-molecular-weight
`inhibitor of prostate-
`specific membrane antigen, in patients with metastatic prostate
`cancer. Journal of nuclear medicine : official publication, Society of
`Nuclear Medicine. 2012; 53:1883-1891.
`Dvorakova et al., Inhibitor-Decorated Polymer Conjugates Target-
`ing Fibroblast Activation Protein. J Med Chem. 2017;60:8385-
`8393.
`Edosada et al., Peptide substrate profiling defines fibroblast activa-
`tion protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving
`specificity. FEBS Lett. Mar. 6, 2006;580(6):1581-6.
`Fischer et al., Radioimmunotherapy of fibroblast activation protein
`positive tumors by rapidly internalizing antibodies. Clin Cancer
`Res. 2012;18:6208-6218.
`Franco et al., Cancer associated fibroblasts in cancer pathogenesis.
`Semin Cell Dev Biol. Feb. 2010;21(1):33-9.
`Garin-Chesa et al., Cell surface glycoprotein of reactive stromal
`fibroblasts as a potential antibody target in human epithelial cancers.
`Proc Natl Acad Sci U S A. Sep. 1990;87(18):7235-9. PMCID:
`PMC54718.
`
`(Continued)
`
`Primary Examiner — Michael G. Hartley
`Assistant Examiner — Melissa J Perreira
`(74) Attorney, Agent, or Firm — Casimir Jones, SC;
`Jeffrey W. Childers
`
`(57)
`
`ABSTRACT
`
`Imaging and radiotherapeutics agents targeting fibroblast-
`activation protein-α (FAP-α) and their use in imaging and
`treating FAP-α 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
`
`

`

`US 11,938,201 B2
`Page 2
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`Jansen et al., Selective Inhibitors of Fibroblast Activation Protein
`(FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold.
`ACS Med Chem Lett. Mar. 18, 2013;4(5):491-6.
`Jansen et al., Extended structure-activity relationship and
`pharmacokinetic investigation of
`(4-quinolinoyl)glycyl-2-
`cyanopyrrolidine inhibitors of fibroblast activation protein (Fap). J
`Med Chem. Apr. 10, 2014;57(7):3053-74.
`Kelly, Fibroblast activation protein-alpha and dipeptidyl peptidase
`IV (CD26): cell-surface proteases that activate cell signaling and are
`potential targets for cancer therapy. Drug Resist Updat. Feb.-Apr.
`2005;8(1-2):51-8.
`Kraman et al., Suppression of antitumor immunity by stromal cells
`expressing fibroblast activation protein-alpha. Science. Nov. 5,
`2010;330(6005):827-30.
`Laverman et al., Immuno-PET and Immuno-SPECT of Rheumatoid
`Arthritis with Radiolabeled Anti-Fibroblast Activation Protein Anti-
`body Correlates with Severity of Arthritis. J Nucl Med. May
`2015;56(5):778-83.
`Li et al., Activatable Near-Infrared Fluorescent Probe for In Vivo
`Imaging of Fibroblast Activation Protein-alpha. Bioconjugate Chem.
`Jul. 19, 2012;23:1704-11.
`Lo et al., Photodynamic molecular beacon triggered by fibroblast
`activation protein on cancer-associated fibroblasts for diagnosis and
`treatment of epithelial cancers. J Med Chem. Jan. 22, 2009;52(2):358-
`68.
`Poplawski et al., Identification of selective and potent inhibitors of
`fibroblast activation protein and prolyl oligopeptidase. J Med Chem.
`May 9, 2013;56(9):3467-77.
`Reilly et al., Advancing Novel Molecular Imaging Agents from
`Preclinical Studies to First-in-Humans Phase I Clinical Trials in
`Academia—A Roadmap for Overcoming Perceived Barriers.
`Bioconjugate chemistry. 2015; 26:625-632.
`Rettig et al., Regulation and heteromeric structure of the fibroblast
`activation protein in normal and transformed cells of mesenchymal
`and neuroectodermal origin Cancer Res. Jul. 15, 1993;53(14):3327-
`35.
`Ryabtsova et al., Acylated Gly-(2-cyano)pyrrolidines as inhibitors
`of fibroblast activation protein (FAP) and the issue of FAP/prolyl
`oligopeptidase (PREP)-selectivity. Bioorg Med Chem Lett. May 15,
`2012;22(10):3412-7.
`Scanlan et al., Molecular cloning of fibroblast activation protein
`alpha, a member of the serine protease family selectively expressed
`in stromal fibroblasts of epithelial cancers. Proc Natl Acad Sci U S
`A. Jun. 7, 1994;91(12):5657-61.
`Scott et al., A Phase I dose-escalation study of sibrotuzumab in
`patients with advanced or metastatic fibroblast activation protein-
`positive cancer. Clin Cancer Res. May 2003;9(5):1639-47.
`
`Tsai et al., Substituted 4-carboxymethylpyroglutamic acid diamides
`as potent and selective inhibitors of fibroblast activation protein. J
`Med Chem. Sep. 23, 2010;53(18):6572-83.
`Tuxhorn et al., Reactive stroma in human prostate cancer: induction
`of myofibroblast phenotype and extracellular matrix remodeling.
`Clin Cancer Res. Sep. 2002;8(9):2912-23.
`Welt et al., Antibody targeting in metastatic colon cancer: a phase
`I study of monoclonal antibody F19 against a cell-surface protein of
`reactive tumor stromal fibroblasts. J Clin Oncol. Jun. 1994;12(6):
`1193-203.
`Youn et al., In vivo noninvasive small animal molecular imaging.
`Osong Public Health Res Perspect. 2012; 3 :48-59.
`Yu et al., The dipeptidyl peptidase IV family in cancer and cell
`biology. FEBS J. Mar. 2010;277(5):1126-44.
`International Search Report and Written Opinion for PCT/US2018/
`057086, dated May 10, 2019, 12 pages.
`Extended EP Search Report for EP 18871298.8, dated May 20,
`2021, 7 pages.
`Third Party Preissuance Submission filed in U.S. Appl. No. 16/758,182,
`filed Feb. 23, 2022, 6 pages.
`Third Party Observations Japanese Patent Application No. 2020-
`523010. Dated Oct. 24, 2022. 7 pages.
`Third Party Observations for application No. EP20180871298.
`Dated Apr. 3, 2022. 7 pages.
`Bernhard et al., DOTAGA-anhydride: a valuable building block for
`the preparation of DOTA-like chelating agents. Chemistry. Jun. 18,
`2012;18(25):7834-41.
`Jamous et al., Synthesis of peptide radiopharmaceuticals for the
`therapy and diagnosis of tumor diseases. Molecules. Mar. 14,
`2013;18(3):3379-409.
`Beebe et al., “Understanding the Apothecaries Within: The Neces-
`sity of a Systematic Approach for Defining the Chemical Output of
`the Human Microbiome”. Clin Transl Sci. Feb. 2014; 7(1): 74-81.
`Ferreira et al., “Monitoring Alcoholic Fermentation: An Untargeted
`Approach”. Journal of Agricultural and Food Chemistry, 2014, 62,
`6784-6793.
`Llewellyn et al., “Using community metabolomics as a new approach
`to discriminate marine microbial particulate organic matter in the
`western English Channel”. Progress in Oceanography, vol. 137, Part
`B, Sep. 2015, p. 421-433.
`Meletta et al., “Evaluation of the radiolabeled boronic acid-based
`FAP inhibitor MIP-1232 for atherosclerotic plaque imaging”. Mol-
`ecules, Jan. 27, 2015;20(2):2081-99.
`Metabolomics—EMBL-EBI, https://www.ebi.ac.uk/training/online/
`courses/metabolomics-introduction/what-is/small-molecules/, retrieved
`on Dec. 12, 2023. 5 pages.
`Azhdarinia et al., “Characterization of chemical, radiochemical and
`optical properties of a dual-labeled MMP-9 targeting peptide”.
`Bioorganic & Medicinal Chemistry, vol. 19, Issue 12, May 6, 2011,
`3769-3776.
`Third Party Observations for application No. EP18871298.8 dated
`Jan. 10, 2024, 553 pages.
`
`* cited by examiner
`
`Petitioner GE Healthcare – Ex. 1001, p. 2
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 1 of 9
`
`US 11,938,201 B2
`
`Petitioner GE Healthcare – Ex. 1001, p. 3
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 2 of 9
`
`US 11,938,201 B2
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`Petitioner GE Healthcare – Ex. 1001, p. 4
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 3 of 9
`
`US 11,938,201 B2
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`Petitioner GE Healthcare – Ex. 1001, p. 5
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 4 of 9
`
`US 11,938,201 B2
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`Petitioner GE Healthcare – Ex. 1001, p. 6
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 5 of 9
`
`US 11,938,201 B2
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`Petitioner GE Healthcare – Ex. 1001, p. 7
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 6 of 9
`
`US 11,938,201 B2
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`Petitioner GE Healthcare – Ex. 1001, p. 8
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 7 of 9
`
`US 11,938,201 B2
`
`Petitioner GE Healthcare – Ex. 1001, p. 9
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 8 of 9
`
`US 11,938,201 B2
`
`Petitioner GE Healthcare – Ex. 1001, p. 10
`
`

`

`U.S. Patent
`
`Mar. 26, 2024
`
`Sheet 9 of 9
`
`US 11,938,201 B2
`
`Petitioner GE Healthcare – Ex. 1001, p. 11
`
`

`

`US 11,938,201 B2
`
`2
`SUMMARY
`
`In some aspects, the presently disclosed subject matter
`provides a compound of Formula (I):
`
`B-L-A
`
`(I)
`
`wherein: A is a targeting moiety for FAP-α; 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-α targeting moiety
`having the structure of:
`
`y(R1x)
`
`(R2x)y
`
`(X’)
`
`N
`
`(R3x)y;
`
`O
`
`y(R3x′)
`
`HN
`
`O
`
`(
`
`CH2)v
`
`R4x
`
`N
`
`R5x
`
`R7x
`
`R6x
`
`wherein each y is independently an integer selected from
`the group consisting of 0, 1, and 2; R1x, R2x, and R3x', are
`each independently selected from the group consisting of H,
`and
`C1-6alkyl, —O—C1-6alkyl,
`OH,
`halogen,
`—S—C1-6alkyl; R3x is selected from the group consisting of
`H, —CN, —B(OH)2, —C(O)alkyl, —C(O)aryl-, —C~C—
`C(O)aryl, —C~C—S(O)2aryl, —CO2H, —SO3H,
`—SO2NH2, —PO3H2, and 5-tetrazolyl; R4x is H; R5x, R6x,
`and R7x are each independently selected from the group
`consisting of H, —OH, oxo, halogen, —C1-6alkyl,
`—NR8xR9x, —OR12x, —Het2 and —Ar2; each of C1-6alkyl
`being optionally substituted with from 1 to 3 substituents
`selected from —OH and halogen; R8x, and R9x, and R12x are
`each independently selected from the group consisting of H,
`—OH, halo, —C1-6alkyl, —O—C1-6alkyl, —S—C1-6alkyl,
`and —Ar3; R10x, R11x, R13x and R14x are each independently
`selected from the group consisting of H, —OH, halogen,
`—C1-6alkyl, —O—C1-6alkyl, and —S—C1-6alkyl; Ar1, Ar2
`and Ar3 are each independently a 5- or 6-membered aromatic
`monocycle optionally comprising 1 or 2 heteroatoms
`selected from O, N and S; each of Ar1, Ar2 and Ar3 being
`optionally and independently substituted with from 1 to 3
`selected from —NR10xR11x, —C1-6alkyl,
`substituents
`—O—C1-6alkyl, and —S—C1-6alkyl; Het2 is a 5- or 6-mem-
`bered non-aromatic monocycle optionally comprising 1 or 2
`heteroatoms selected from O, N and S; Het2 being optionally
`substituted with from 1 to 3 substituents selected from
`—NR13xR14x, —C1-6alkyl, —O—C1-6alkyl, and —S—C1-
`6alkyl; v is 0, 1, 2, or 3; and
`
`N
`
`*
`
`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-α (FAP-α) 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-α 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-α-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-α a potential imaging and radiotherapeutic target for
`cancer and inflammation diseases.
`Because FAP-α 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-α.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Petitioner GE Healthcare – Ex. 1001, p. 12
`
`

`

`US 11,938,201 B2
`
`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
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`indicates a point of attachment of the FAP-α 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.
`In more particular aspects, A is an FAP-α targeting moiety
`having the structure of:
`
`y(R1x)
`
`(R2x)y
`
`N
`
`C
`
`N;
`
`O
`
`y(R3x′)
`
`HN
`
`O
`
`6
`
`7
`
`5
`
`8
`
`N
`
`wherein
`
`indicates a point of attachment of the FAP-α 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
`pharmaceutically acceptable salts thereof.
`
`In yet more particular aspects, A is selected from the
`group consisting of:
`
`55
`
`A1
`
`60
`
`65
`
`N
`
`C
`
`N;
`
`O
`
`HN
`
`O
`
`N
`
`A2
`
`A3
`
`4
`-continued
`
`N
`
`C
`
`N;
`
`and
`
`O
`
`HN
`
`O
`
`N
`
`N
`
`C
`
`N.
`
`O
`
`HN
`
`O
`
`N
`
`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-α (FAP-α),
`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-α (FAP-α), 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-α (FAP-α)-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-α)-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
`
`

`

`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 [111In]-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 1H 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, [111In]-XY-FAP-02. First, the precursor was
`functionalized with DOTA via a one step reaction between
`the precursor and DOTA-GA(t-Bu)4-NHS. Unlabeled prod-
`uct was purified via HPLC to produce XY-FAP-02. Subse-
`radiolabeling with 111In and HPLC purification
`quent
`resulted in the radiolabeled product, [111In]-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 kit.
`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
`[111In]-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-01 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
`[111In]-XY-FAP-02 in FAP-positive U87 and FAP-negative
`PC3 cell lines. Cells were incubated with 1 µCi [111In]-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 [111In]-XY-FAP-01 in tumor bearing mice. At 5 min, 0.5
`h, 2 h, 6 h, and 12 h after injection of 10 µCi [111In]-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
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`unlabeled XY-FAP-02 and 10 µCi [111In]-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. aStudent’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. bStudent’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). cStudent’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 [111In]-XY-FAP-02. FIG.
`5A shows tissue time activity curves (TACs) of [111In]-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-01 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 [111In]-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
`[111In]-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-α (FAP-α)
`FAP-α 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
`
`

`

`US 11,938,201 B2
`
`8
`showed selective uptake in vitro on a FAP-α+ U87 cell line
`and in vivo on a FAP-α+ U87 tumor and clearly detected the
`tumor. In another particular embodiment, an 111In labeled
`ligand (XY-FAP-02-[111In]) was successfully obtained in
`high yield and purity from its precursor with a metal
`chelator. The in vivo study showed clear tumor radiotracer
`uptake in mice bearing FAP-α-positive U87 tumors with
`minimum non-specific organ uptake, which allows the spe-
`cific imaging of FAP-α expressing tumors. The presently
`disclosed FAP-αtargeting moiety can be adapted for use
`with optical dyes and radioisotopes known in the art for
`imaging and therapeutic applications targeting FAP-α.
`More particularly, in some embodiments, the presently
`disclosed subject matter provides a compound of the general
`structure of Formula (I):
`
`B-L-A
`
`(I)
`
`wherein: A is a targeting moiety for FAP-α; B is any optical
`or radiolabeled functional group suitable for optical imag-
`ing, positron-emission tomography (PET) imaging, single-
`photon emission computed tomography (SPECT) imaging,
`or radiotherapy; and L is a linker having bi-functionalization
`adapted to form a chemical bond with B and A.
`Representative targeting moieties for FAP-α are disclosed
`in U.S. Patent Application Publication No. US2014/0357650
`for Novel FAP Inhibitors to Jansen et al., published Dec. 4,
`2014; U.S. Pat. No. 9,346,814 for Novel FAP Inhibitors to
`Jansen et al., issued May 24, 2016; and International PCT
`Patent Publication No. WO 2013/107820 for Novel FAP
`Inhibitors to Jansen et al., published Jul. 25, 2013, each of
`which are incorporate by reference in their entirety.
`More particularly, U.S. Pat. No. 9,346,814 to Jansen et al.,
`discloses FAP-α inhibitors of formula (X), or a stereoisomer,
`tautomer, racemate, salt, hydrate, or solvate thereof, which
`are suitable for use with the presently disclosed subject
`matter:
`
`(X)
`
`R1x
`
`R2x
`
`N
`
`R3x;
`
`O
`
`HN
`
`O
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`N
`
`CH2
`
`v
`
`R4x
`
`50
`
`R5x
`
`R7x
`
`R6x
`
`55
`
`60
`
`65
`
`wherein:
`R1x and R2x are each independently selected from the
`group consisting of H, OH, halogen, C1-6alkyl,
`—O—C1-6alkyl, and —S—C1-6alkyl;
`R3x is selected from the group consisting of H, —CN,
`—B(OH)2, —C(O)alkyl, —C(O)aryl-, —C~C—C(O)
`aryl, —C~C—S(O)2aryl, —CO2H, —SO3H,
`—SO2NH2, —PO3H2, and 5-tetrazolyl;
`R4x is H;
`R5x, R6x, and R7x are each independently selected from the
`group consisting of H, —OH, oxo, halogen,
`—C1-6alkyl, —O—C1-6alkyl, —S—C1-6alkyl,
`—NR8xR9x, —OR12x, —Het2 and —Ar2 each of C1-6al-
`kyl being optionally substituted with from 1 to 3
`substituents selected from —OH and halogen;
`
`7
`in cancer by modifying bioactive signaling peptides through
`this enzymatic activity (Kelly, et al., 2005; Edosada, et al.,
`2006). FAP-α 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-α is of clinical impor-
`tance.
`FAP-α exists as a homodimer to carry out its enzymatic
`function. Inhibitors selectively targeting FAP-α 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-α 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-α.
`including positron
`imaging,
`Radionuclide molecular
`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 β- 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
`β-particle emitters
`
`α-particle emitters
`Auger electron emitters
`
`90Y, 131I, 177Lu, 153Sm, 186Re,
`188Re, 67Cu, 212Pb
`225Ac, 213Bi, 212Bi, 211At, 212Pb
`125I, 123I, 67Ga, 111In
`
`The highly potent and specific binding moiety targeting
`FAP-α 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-α.
`Accordingly, in some embodiments, the presently dis-
`closed subject matter provides potent and selective low-
`molecular-weight (LMW) ligands of FAP-α, i.e., an FAP-α
`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-
`α. 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-α
`ligand with optical dyes and PET or SPECT isotopes,
`including, but not limited to, 68Ga, 64Cu, 18F, 86Y, 90Y, 89Zr,
`111In, 99mTc, 125I, 124I, for FAP-α related imaging applica-
`tions. Further, the presently disclosed approach allows for
`the radiolabeling of the FAP-α ligand with radiotherapeutic
`isotopes, including but not limited to, 90Y, 177Lu, 125I, 131I,
`211At, 111In, 153Sm, 186Re, 188Re, 67Cu, 212Pb, 225Ac, 213Bi,
`212Bi, 212Pb, and 67Ga, for FAP-α related radio-therapy.
`In a particular embodiment, an optical agent conjugated
`with IRDye-800CW (XY-FAP-01) was synthesized and
`
`Petitioner GE Healthcare – Ex. 1001, p. 15
`
`

`

`US 11,938,201 B2
`
`10
`-continued
`S
`*
`
`*
`
`N
`
`and
`
`*
`
`O
`
`N
`
`N
`
`N
`
`N
`
`N
`
`*;
`
`N
`
`NH
`
`wherein * indicates the point of attachment of the 5 to
`10-membered N-containing aromatic or non-aromatic
`mono- or bicyclic heterocycle to —(CH2)v—.
`Accordingly, in some embodime