U.S. patent number 10,385,380 [Application Number 14/874,297] was granted by the patent office on 2019-08-20 for personalized protease assay to measure protease activity in neoplasms.
This patent grant is currently assigned to The Regents of the University of California. The grantee listed for this patent is The Regents of the University of California. Invention is credited to Quyen T. Nguyen, Roger Y. Tsien, Mike Whitney.
View All Diagrams
United States Patent |
10,385,380 |
Whitney , et al. |
August 20, 2019 |
Personalized protease assay to measure protease activity in
neoplasms
Abstract
Disclosed herein, the invention pertains to methods and
compositions that find use in diagnostic, prognostic and
characterization of neoplasia samples based on the ability of a
neoplasia sample to cleave a MTS molecule of the present invention.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B-C), wherein A is a peptide with a sequence
comprising 5 to 9 consecutive acidic amino acids, wherein the amino
acids are selected from: aspartates and glutamates; B is a peptide
with a sequence comprising 5 to 20 consecutive basic amino acids; X
is a linker; and C is a detectable moiety.
Inventors: |
Whitney; Mike (San Diego,
CA), Nguyen; Quyen T. (La Jolla, CA), Tsien; Roger Y.
(La Jolla, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
56093766 |
Appl.
No.: |
14/874,297 |
Filed: |
October 2, 2015 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20160160263 A1 |
Jun 9, 2016 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
62059081 |
Oct 2, 2014 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/37 (20130101); G01N 2800/7028 (20130101) |
Current International
Class: |
C12Q
1/37 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 01/00245 |
|
Jan 2001 |
|
WO |
|
WO 01/75067 |
|
Oct 2001 |
|
WO |
|
WO 2005/042034 |
|
May 2005 |
|
WO |
|
WO 2006/007398 |
|
Jan 2006 |
|
WO |
|
WO 2006/125134 |
|
Nov 2006 |
|
WO |
|
WO 2011/008992 |
|
Jan 2011 |
|
WO |
|
WO 2011/008996 |
|
Jan 2011 |
|
WO |
|
WO 2399939 |
|
Dec 2011 |
|
WO |
|
WO 2013/019681 |
|
Feb 2013 |
|
WO |
|
WO 2014/120837 |
|
Aug 2014 |
|
WO |
|
Other References
Abdollahi, A. et al., "Inhibition of .alpha.V 3 Integrin Survival
Signaling Enhances Antiangiogenic and Antitumor Effects of
Radiotherapy," Clin Cancer Res., Sep. 1, 2005, 11(17), pp.
6270-6279. cited by applicant .
Adams, S.R. et al., "Anti-tubulin drugs conjugated to anti-ErbB
antibodies selectively radiosensitize," Nature Communications, Oct.
4, 2016, 7:13019, pp. 1-11. cited by applicant .
Advani, S.J. et al., "Increased oncolytic efficacy for high-grade
gliomas by optimal integration of ionizing radiation into the
replicative cycle of HSV-1," Gene Therapy, 2011, vol. 18, pp.
1098-1102. cited by applicant .
Advani, S.J. et al., "Preferential Replication of Systemically
Delivered Oncolytic Vaccinia Virus in Focally Irradiated Giloma
Xenografts," Clin Cancer Res., 2012; 18(9), pp. 2579-2590. cited by
applicant .
Aguilera, T.A. et al., "Systemic in vivo distribution of
activatable cell penetrating peptides is superior to that of cell
penetrating peptides," Integr. Biol., vol. 1, pp. 371-381 (2009).
cited by applicant .
Akashi, Y. et al., "The novel microtubule-interfering agent
TZT-1027 enhances the anticancer effect of radiation in vitro and
in vivo," British Journal of Cancer, 2007, vol. 96, pp. 1532-1539.
cited by applicant .
Albright et al. "Matrix metalloproteinase-activated doxorubicin
prodrugs inhibit HT1080 xenograft growth better than doxorubicin
with less toxicity" Molecular cancer therapeutics, vol. 4, pp.
751-760 (2005). cited by applicant .
Al-Sarraf et al. "Chemoradiotherapy versus radiotherapy in patients
with advanced nasopharyngeal cancer: phase III randomized
Intergroup study 0099" J. Clin. Oncol. 16, 1310-1317 (1998). cited
by applicant .
Ang et al. "Randomized phase III trial of concurrent accelerated
radiation plus cisplatin with or without cetuximab for stage III to
IV head and neck carcinoma: RTOG 0522" J. Clin. Oncol. 20,
2940-2950 (2014). cited by applicant .
Arnold, D. et al., "Substrate specificity of cathepsins D and E
determined by N-terminal and C-terminal sequencing of peptide
pools," Eur. J. Biochem., 1997, vol. 249, pp. 171-179. cited by
applicant .
Atalay et al. "Novel therapeutic strategies targeting the epidermal
growth factor receptor (EGFR) family and its downstream effectors
in breast cancer" Annals of Oncology, vol. 14, pp. 1346-1363
(2003). cited by applicant .
Ayoub et al. "Correct primary structure assessment and extensive
glyco-profiling of cetuximab by a combination of intact, middle-up,
middle-down and bottom-up ESI and MALDI mass spectrometry
techniques" mAbs 5, 699-710 (2013). cited by applicant .
Bai, R. et al., "Dolastatin 10, a powerful cytostatic peptide
derived from a marine animal. Inhibition of tubulin polymerization
mediated through the vinca alkaloid binding domain," Biochem
Pharmacol., 1990; 39:1941-49. cited by applicant .
Bartles, J.R. et al., "Identification and charactzerization of
espin, an actin-binding protein localized to the F-actinOrich
junctionla plaques of Sertoli cell ectoplasmic specializations,"
Journal of Cell Science, 1996, vol. 109, No. 6, pp. 1229-1239.
cited by applicant .
Baselga et al. "Phase II study of weekly intravenous recombinant
humanized anti-p185HER2 monoclonal antibody in patients with
HER2/neu-overexpressing metastatic breast cancer" J. Clin. Oncol.,
vol. 14, pp. 737-744 (1996). cited by applicant .
Bauvois "New facets of matrix metalloproteinases MMP-2 and MMP-9 as
cell surface transducers: outside-in signaling and relationship to
tumor progression" Biochim Biophys Acta1825, pp. 29-36 (2012).
cited by applicant .
Bhorade, R. et al., "Macrocyclic Chelators with Paramagnetic
Cations Are Internalized into Mammalian Cells via a HIV-Tat Derived
Membrane Translocation Peptide," Bioconjugate Chemistry, May 1,
2000, vol. 11, No. 3, pp. 301-305. cited by applicant .
Blum, G. et al., "Noninvasive optical imaging of cysteine protease
activity using fluorescently quenched activity-based probes,"
Nature Chemical Biology, vol. 3, No. 10, pp. 668-677 (2007). cited
by applicant .
Bonner et al. "Radiotherapy plus cetuximab for locoregionally
advanced head and neck cancer: 5-year survival data from a phase 3
randomised trial, and relation between cetuximab-induced rash and
survival" Lancet Oncol. 11, 21-28 (2010). cited by applicant .
Bradley et al. "Standard-dose versus high-dose conformal
radiotherapy with concurrent and consolidation carboplatin plus
paclitaxel with or without cetuximab for patients with stage IIIA
or IIIB non-small-cell lung cancer (RTOG 0617): a randomised,
two-by-two factorial phase 3 study" Lancet Oncol. 16, 187-199
(2015). cited by applicant .
Brand et al. "AXL mediates resistance to cetuximab therapy" Cancer
Res. 74, 5152-5164 (2014). cited by applicant .
Breij, E.C.W. et al., "An Antibody-Drug Conjugate That Targets
Tissue Factor Exhibits Potent Therapeutic Activity against a Broad
Range of Solid Tumors," Cancer Res., Feb. 15, 2014,
74(4):1214-1226. cited by applicant .
Bremer, C. et al., "In vivo molecular target assessment of matrix
metalloproteinase inhibition," Nature Medicine, Jun. 2001, vol. 7,
No. 6, pp. 743-748. cited by applicant .
Bremer, C. et al., "Optical Imaging of Matrix Metalloproteinase-2
Activity in Tumors: Feasibility Study in a Mouse Model," Radiology,
2001, vol. 221, pp. 523-529. cited by applicant .
Bremer, C. et al., "Optical Imaging of Spontaneous Breast Tumors
Using Protease Sensing `Smart` Optical Probes," Invest Radiol.,
Jun. 6, 40(6):321-327 (2005). cited by applicant .
Buckel et al. "Tumor radiosensitization by monomethyl auristatin E:
mechanism of action and targeted delivery" Cancer Res. 75,
1376-1387 (2015). cited by applicant .
Buckel et al. "Tumor radiosensitization by monomethyl auristatin e:
mechanism of action and targeted delivery" Cancer research, vol.
75, pp. 1376-1387 (2015). cited by applicant .
Chaudhary et al. "Genetic polymorphisms of matrix
metalloproteinases and their inhibitors in potentially malignant
and malignant lesions of the head and neck" Journal of Biomedical
Science, vol. 17 (2010). cited by applicant .
Chaurand, P. et al., "Molecular imaging of thin mammalian tissue
sections by mass spectrometry," Curr Opinion Biotechnol., 2006;
17(4):431-436. cited by applicant .
Chen et al. "Targeted therapy for Hodgkin lymphoma and systemic
anaplastic large cell lymphoma-focus on brentuximab vedotin" Onco.
Targets Ther. 7, 45-56 (2013). cited by applicant .
Chen, B. et al., "Thrombin Activity Associated with Neuronal Damage
during Acute Focal Ischemia," The Journal of Neuroscience, May 30,
2012, vol. 32, No. 22, pp. 7622-7631. cited by applicant .
Chen, E.I. et al., "A Unique Substrate Recognition Profile for
Matrix Metalloprotinase-2," The Journal of Biological Chemistry,
Feb. 8, 2002, vol. 277, No. 6, pp. 4485-4491. cited by applicant
.
Chen, J. et al., "`Zipper` Molecular Beacons: A Generalized
Strategy to Optimize the Performance of Activatable Protease
Probes," Bioconjugate Chem., 2009, vol. 20, pp. 1836-1842. cited by
applicant .
Cho et al. "Structure of the extracellular region of HER2 alone and
in complex with the Herceptin Fab" Nature, vol. 421, pp. 756-760
(2003). cited by applicant .
Choi et al. "Protease-Activated Drug Development" Theranostics,
vol. 2, pp. 156-178 (2012). cited by applicant .
Cohen et al. "Controversies in the treatment of local and locally
advanced gastric and esophageal cancers" J. Clin. Oncol. 33,
1754-1759 (2015). cited by applicant .
Cooks, R.J. et al., "Ambient Mass Spectrometry," Science, 2006;
311(5767):1566-1570. cited by applicant .
Coussens et al. "Matrix metalloproteinase inhibitors and cancer:
trials and tribulations" Science, vol. 295, pp. 2387-2392 (2002).
cited by applicant .
Creedon et al. "Exploring mechanisms of acquired resistance to HER2
(human epidermal growth factor receptor 2)-targeted therapies in
breast cancer" Biochem. Soc. Trans. 42, 822-830 (2014). cited by
applicant .
Crisp et al. "Dual targeting of integrin alphavbeta3 and matrix
metalloproteinase-2 for optical imaging of tumors and
chemotherapeutic delivery" Molecular cancer therapeutics, vol. 13,
pp. 1514-1525 (2014). cited by applicant .
Damen et al. "Electrospray ionization quadrupole ion-mobility
time-of-flight mass spectrometry as a tool to distinguish the
lot-to-lot heterogeneity in N-glycosylation profile of the
therapeutic monoclonal antibody trastuzumab" J. Am. Soc. Mass
Spectrom. 20, 2021-2033 (2009). cited by applicant .
Derossi et al., "Trojan peptides: the penetratin system for
intracellular delivery," Trends in Cell Biology, vol. 8, pp. 84-87
(1998). cited by applicant .
Doronina, S.O. et al., "Development of potent monoclonal antibody
auristatin conjugates for cancer therapy," Nat Biotechnol., 2003;
21:778-84. cited by applicant .
Dotan et al. "Positive Surgical Margins in Soft Tissue Following
Radical Cystectomy for Bladder Cancer and Cancer Specific Survival"
The Journal of Urology, vol. 178, No. 6, pp. 2308-2313 (2007).
cited by applicant .
D'Souza et al. Case-control study of human papillomavirus and
oropharyngeal cancer N. Engl J Med., vol. 356, pp. 1944-1956
(2007). cited by applicant .
Dutta et al. "Cellular responses to EGFR inhibitors and their
relevance to cancer therapy" Cancer Lett. 254, 165-177 (2007).
cited by applicant .
Egami, T. et al., "Up-regulation of integrin 3 in radioresistant
pancreatic cancer impairs adenovirus-mediated gene therapy," Cancer
Science, Oct. 2009, vol. 100, No. 10, pp. 1902-1907. cited by
applicant .
Egeblad et al. "New functions for the matrix metalloproteinases in
cancer progression" Nature reviews--Cancer, vol. 2, pp. 161-174
(2002). cited by applicant .
Epidermoid anal cancer: results from the UKCCCR randomised trial of
radiotherapy alone versus radiotherapy, 5-fluorouracil, and
mitomycin. UKCCCR Anal Cancer Trial Working Party. UK Co-ordinating
Committee on Cancer Research. Lancet 348, 1049-1054 (1996). cited
by applicant .
Fawell et al. "Tat-Mediated Delivery of Heterologous Proteins into
Cells", PNAS, vol. 91, pp. 664-668 (1994). cited by applicant .
Franklin et al. "Insights into ErbB signaling from the structure of
the ErbB2-pertuzumab complex" Cancer Cell, vol. 5, pp. 317-328
(2004). cited by applicant .
Friess et al. "CombinationTreatment with Erlotinib and Pertuzumab
against HumanTumor Xenografts Is Superior to Monotherapy" Clin.
Cancer Res., vol. 11, pp. 5300-5309 (2005). cited by applicant
.
Fujita, M. et al., "X-ray irradiation and Rho-kinase inhibitor
additively induce invasiveness of the cells of the pancreatic
cancer line, MIAPaCa-2, which exhibits mesenchymal and amoeboid
motility," Cancer Sci., Apr. 2011, vol. 102, No. 4, pp. 792-798.
cited by applicant .
Futaki et al., "Stearylated Arginine-Rich Peptides: A New Class of
Transfection Systems," Bioconj. Chem., vol. 12, pp. 1005-1011
(2001). cited by applicant .
Gallwitz, M. et al., "The Extended Cleavage Specificity of Human
Thrombin," PLoS One, Feb. 2012, vol. 7, Issue 2, e31756, pp. 1-16.
cited by applicant .
Garrett et al. "The Crystal Structure of a Truncated ErbB2
Ectodomain Reveals an Active Conformation, Poised to Interact with
Other ErbB Receptors" Mollecular Cell, vol. 11, pp. 495-505 (2003).
cited by applicant .
Girish et al. "Clinical pharmacology of trastuzumab
emtansine(T-DM1): an antibody-drug conjugate in development for the
treatment of HER2-positive cancer" Cancer Chemother. Pharmacol. 69,
1229-1240 (2012). cited by applicant .
Giustini, A.J. et al., "Ionizing radiation increases systemic
nanoparticle tumor accumulation," Nanomedicine 2012;8:818-21. cited
by applicant .
Golub, T.R. et al., "Molecular Classification of Cancer: Class
Discovery and Class Prediction by Gene Expression Monitoring,"
Science, Oct. 15, 1999, vol. 286, pp. 531-537. cited by applicant
.
Gounaris, E. et al., "Live Imaging of Cysteine-Cathepsin Activity
Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp
Growth," PLoS One, vol. 3, No. 8, e2916, pp. 1-9 (2008). cited by
applicant .
Gross et al. "Multi-tiered genomic analysis of head and neck cancer
ties TP53 mutation to 3p loss" Nature genetics, vol. 46, pp.
939-943 (2014). cited by applicant .
Hallahan, D. et al., "Integrin-mediated targeting of drug delivery
to irradiated tumor blood vessels," Cancer Cell, Jan. 2003, vol. 3,
pp. 63-74. cited by applicant .
Hallahan, D.E. et al., "Radiation-mediated control of drug
delivery," Am J Clin Oncol., 2001; 24:473-80. cited by applicant
.
Hallahan, D.E. et al., et al., "Spatial and temporal control of
gene therapy using ionizing radiation," Nat Med., 1995;1:786-91.
cited by applicant .
Hallbrink, M. et al., "Cargo delivery kinetics of cell-penetrating
peptides," Biochimica et Biophysica Acta, vol. 1515, pp. 101-109
(2001). cited by applicant .
Hamano et al. "Physiological levels of tumstatin, a fragment of
collagen IV alpha3 chain, are generated by MMP-9 proteolysis and
suppress angiogenesis via alphaV beta3 integrin" Cancer Cell, vol.
3, pp. 589-601 (2003). cited by applicant .
Hamblett et al. "Effects of drug loading on the antitumor activity
of a monoclonal antibody drug conjugate" Clin. Cancer Res. 10,
7063-7070 (2004). cited by applicant .
Haque et al. "Sugical Margins and Survival After Head and Neck
Cancer Surgery" BMC Ear, Nose and Throad Disorders, vol. 6, No. 2
(2006). cited by applicant .
Harir, G. et al., "Radiation-Guided Drug Delivery to Mouse Models
of Lung Cancer," Clin Cancer Res., Oct. 15, 2010, 16(1); pp.
4968-4977. cited by applicant .
Hauff et al. "Matrix-metalloproteinases in head and neck
carcinoma-cancer genome atlas analysis and fluorescence imaging in
mice" Otolaryngology--head and neck surgery, vol. 151, pp. 612-618
(2014). cited by applicant .
Herbst et al. "Monoclonal Antibodies to Target Epidermal Growth
Factor Receptor-Positive Tumors" American Cancer Society, vol. 94,
pp. 1593-1611 (2002). cited by applicant .
Herskovic et al. "Combined chemotherapy and radiotherapy compared
with radiotherapy alone in patients with cancer of the esophagus"
N. Engl. J. Med. 326, 1593-1598 (1992). cited by applicant .
Hudziak et al. "p185HER2 Monoclonal Antibody Has Antiproliferative
Effects In Vitro and Sensitizes Human Breast Tumor Cells to Tumor
Necrosis Factor" Mol. Cell. Biol., vol. 9, No. 3, pp. 1165-1172
(1989). cited by applicant .
Hussain et al. "Surgical molecular navigation with a Ratiometric
Activatable Cell Penetrating Peptide improves intraoperative
identification and resection of small salivary gland cancers" Head
& neck, vol. 38, pp. 715-723 (2014). cited by applicant .
Hutteman, M. et al., "Optimization of Near-Infrared Fluorescent
Sentinel Lymph Node Mapping for Vulvar Cancer," Am J Obstet
Gynecol., Jan. 2012, vol. 206, No. 1, pp. 89.e1-89.e5. cited by
applicant .
Ifa, D.R. et al., "Ambient Ionization Mass Spectrometry for Cancer
Diagnosis and Surgical Margin Evaluation," Clinical Chemistry,
2016, 62:1, pp. 111-123. cited by applicant .
Jaffer, F.A. et al., "In Vivo Imaging of Thrombin Activity in
Experimental Thrombi With Thrombin-Sensitive Near-Infrared
Molecular Probe," Arterioscler Thromb Vasc Biol., 2002, vol. 22,
pp. 1929-1935. cited by applicant .
Jiang, T. et al., "Tumor imaging by means of proteolytic activation
of cell-penetrating peptides," PNAS, vol. 101, No. 51., pp.
17867-17872 (2004). cited by applicant .
Joh, D.Y. et al., "Selective Targeting of Brain Tumors with Gold
Nanoparticle-Induced Radiosensitization," PLoS One, Apr. 2013, vol.
8, No. 4, e62425, pp. 1-10. cited by applicant .
Kesari et al. "DNA damage response and repair: insights into
strategies for radiation sensitization of gliomas" Future Oncol. 7,
1335-1346 (2011). cited by applicant .
Kohrt et al. "Profile of immune cells in axillary lymph nodes
predicts disease-free survival in breast cancer" PLoS Med., vol. 2,
Issue 9, e284 (2005). cited by applicant .
Kumar, A. et al., "Increased tyoe-IV collagenase (MMP-2 and MMP-9)
activity following preoperative radiotherapy in rectal cancer,"
British Journal of Cancer, 2000, 82(4), pp. 960-965. cited by
applicant .
Kuniyasu et al. "Relative expression of type IV collagenase,
E-cadherin, and vascular endothelial growth factor/vascular
permeability factor in prostatectomy specimens distinguishes
organ-confined from pathologically advanced prostate cancers" Clin
Cancer Res, vol. 6, pp. 2295-2308 (2000). cited by applicant .
Kwaan et al. "The apparent uPA/PAI-1 paradox in cancer: more than
meets the eye" Semin Thromb Hemost., vol. 39, pp. 382-391 (2013).
cited by applicant .
Laine et al. "Radiation therapy as a backbone of treatment of
locally advanced non-small cell lung cancer" Semin. Oncol. 41,
57-68 (2014). cited by applicant .
Lanekoff, I. et al., "Automated Platform for High-Resolution Tissue
Imaging Using Nanospray Desorption Electrospray Ionization Mass
Spectrometry," Anal Chem., 2012; 84(19):8351-8356. cited by
applicant .
Laskin, J. et al., "Ambient Mass Spectrometry Imaging Using Direct
Liquid Extraction Techniques," Anal. Chem., 2016; 88(1):52-73.
cited by applicant .
Lavaud et al. "Strategies to overcome trastuzumab resistance in
HER2-overexpressing breast cancers: focus on new data from clinical
trials" BMC Med. 12, 132 (2014). cited by applicant .
Le et al. "Integrating biologically targeted therapy in head and
neck squamous cell carcinomas" Semin. Radiat. Oncol. 19, 1953-1962
(2009). cited by applicant .
Lee et al. "Loss of Fhit expression is a predictor of poor outcome
in tongue cancer" Cancer Res., vol. 61, pp. 837-841 (2001). cited
by applicant .
Levenson, R. et al., "Review Article: Modern Trends in Imaging X:
Spectral imaging in preclinical research and clinical pathology,"
Anal Cell Pathol, 2012, vol. 35, pp. 339-361. cited by applicant
.
Levi, J. et al., "Design, Synthesis and Imaging of an Activatable
Photoacoustic Probe," J Am Chem Soc., Aug. 18, 2010, vol. 132, No.
32, pp. 11264-11269. cited by applicant .
Ley et al. "Cisplatin versus cetuximab given concurrently with
definitive radiation therapy for locally advanced head and neck
squamous cell carcinoma" Oncology 85, 290-296 (2013). cited by
applicant .
Li, C. et al., "Tumor Irradiation Enhances the Tumor-specific
Distribution of Poly(L-glutamate acid)-conjugated Paclitaxel and
Its Antitumor Efficacy," Clinical Cancer Research, Jul. 2000, vol.
6, pp. 2829-2834. cited by applicant .
Liang et al. "Sensitization of breast cancer cells to radiation by
trastuzumab" Mol. Cancer Ther. 2, 1113-1120 (2003). cited by
applicant .
Liauw, S.L. et al., "New paradigms and future challenges in
radiation oncology: an update of biological targets and
technology," Sci Transl Med., 2013;5:173sr2. cited by applicant
.
Lin, S.H. et al., "Opportunities and Challenges in the Era of
Molecularly Targeted Agents and Radiation Therapy," J Natl Cancer
Inst., 2013, vol. 105, pp. 686-693. cited by applicant .
Linder, K.E. et al., "Synthesis, In Vitro Evaluation, and In Vivo
Metabolism of Fluor/Quencher Compounds Containing IRDye 800CW and
Black Hole Quencher-3 (BHQ-3)," Bioconjugate Chemistry, 2011, vol.
22, pp. 1287-1297. cited by applicant .
Liu et al. "Overexpression of MMP-2 in laryngeal squamous cell
carcinoma: A potential indicator for poor prognosis"
Otolaryngology--Head and Neck Surgery, vol. 132, Issue 3, pp.
395-400 (2005). cited by applicant .
Liu, F-F. et al., "Lessons Learned from Radiation Oncology Clinical
Trials," Clin Cancer Res., 2013, 19(22):6089-6100. cited by
applicant .
Lyon et al. "Self-hydrolyzing maleimides improve the stability and
pharmacological properties of antibody-drug conjugates" Nat.
Biotechnol. 32, 1059-1062 (2014). cited by applicant .
Ma, D. et al., "Potent Antitumor Activity of an
Auristatin-Conjugated, Fully Human Monoclonal Antibody to
Prostate-Specific Membrane Antigen," Clin Cancer Res., 2006,
12(8):2591-2596. cited by applicant .
MacDonald et al. "Chemoradiotherapy after surgery compared with
surgery alone for adenocarcinoma of the stomach or gastroesophageal
junction" N. Engl. J. Med. 345, 725-730 (2001). cited by applicant
.
Maitz, M.F. et al., "Bio-responsive polymer hydrogels
homeostatically regulate blood coagulation," Nature Communications,
2013, pp. 1-7. cited by applicant .
Marur et al. "Challenges of integrating chemotherapy and targeted
therapy with radiation in locally advanced head and neck squamous
cell cancer" Curr. Opin. Oncol. 222, 206-211 (2010). cited by
applicant .
Maruyama et al. "Human papillomavirus and p53 mutations in head and
neck squamous cell carcinoma among Japanese population" Cancer
Sci., vol. 105, pp. 409-417 (2014). cited by applicant .
Mendelsohn et al. "The EGF receptor family as targets for cancer
therapy" Oncogene, vol. 19, pp. 6550-6565 (2000). cited by
applicant .
Meric et al. "Positive Surgical Margins and Ipsilateral Breast
Tumor Recurrence Predict Disease-Specific Survival after
Breast-Conserving Therapy" Cancer, vol. 97, No. 4, pp. 926-933
(2003). cited by applicant .
Metildi et al. "Ratiometric activatable cell-penetrating peptides
label pancreatic cancer, enabling fluorescence-guided surgery,
which reduces metastases and recurrence in orthotopic mouse models"
Annals of surgical oncology, vol. 22, 2082-2087 (2015). cited by
applicant .
Miller, S.M. et al., "Nanomedicine in chemoradiation," Ther Deliv.,
2013;4: 239-50. cited by applicant .
Moding et al. "Strategies for optimizing the response of cancer and
normal tissues to radiation" Nat. Rev. Drug Discov. 12, 526-542
(2013). cited by applicant .
Moding, E.J. et al., "Strategies for optimizing the response of
cancer and normal tissues to radiation," Nat Rev Drug Discov.,
2013; 12:526-42. cited by applicant .
Modjtahedi et al. "Phase I trial and tumour localisation of the
anti-EGFR monoclonal antibody ICR62 in head and neck or lung
cancer" British Journal of Cancer, vol. 73, pp. 228-235 (1996).
cited by applicant .
Montel et al. "Altered metastatic behavior of human breast cancer
cells after experimental manipulation of matrix metalloproteinase 8
gene expression" Cancer research, vol. 64, pp. 1687-1694 (2004).
cited by applicant .
Morris et al. "Interaction of radiation therapy with molecular
targeted agents" J. Clin. Oncol. 32, 2886-2893 (2014). cited by
applicant .
Morris et al. "Pelvic radiation with concurrent chemotherapy
compared with pelvic and para-aortic radiation for high-risk
cervical cancer" N. Engl. J. Med. 340, 1137-1143 (1999). cited by
applicant .
Mubard et al. "Maturing antibody-drug conjugate pipeline hits 30"
Nat. Rev. Drug. Discov. 12, 329-332 (2013). cited by applicant
.
Mullard, A., "Maturing antibody-drug conjugate pipeline hits 30,"
Nat Rev Drug Discov., 2013;12:329-32. cited by applicant .
Nagtegaal et al. "What Is the Role for the Circumferential Margin
in the Modern Treatment of Rectal Cancer?" Journal of Clinical
Oncology, vol. 26, No. 2, pp. 303-312 (2008). cited by applicant
.
Nguyen et al., "Surgery with molecular fluorescence imaging using
activatable cell-penetrating peptides decreases residual cancer and
improves survival," PNAS, vol. 107, No. 9, pp. 4317-4322 (2010).
cited by applicant .
Nguyen, Q.T. et al., "Fluorescence-guided surgery with live
molecular navigation--a new cutting edge," Nature Reviews Cancer,
Sep. 2013, vol. 13, pp. 653-662. cited by applicant .
Nguyen, Q.T. et al., "Surgery with molecular fluorescence imaging
using activatable cell-penetrating peptides decreases residual
cancer and improves survival," PNAS, vol. 107, No. 9, pp. 4317-4322
(2010). cited by applicant .
No et al. "Targeting HER2 signaling pathway for radiosensitization:
alternative strategy for therapeutic resistance" Cancer Biol. Ther.
8, 2351-2361 (2009). cited by applicant .
O-Charoenrat et al. "Expression of Matrix Metalloproteinases and
Their Inhibitors Correlates With Invasion and Metastasis in
Squamous Cell Carcinoma of the Head and Neck" Arch Otolayrngol Head
Neck Surg., vol. 127, pp. 813-820 (2001). cited by applicant .
Ohta et al. "The FHIT gene, spanning the chromosome 3p14.2 fragile
site and renal carcinoma-associated t(3;8) breakpoint, is abnormal
in digestive tract cancers" Cell, vol. 84, pp. 587-597 (1996).
cited by applicant .
Okeley et al. "Advancing antibody drug conjugation: from the
laboratory to a clinically approved anticancer drug" Hematol.
Oncol. Clin. North Am. 28, 13-25 (2014). cited by applicant .
Olson et al. "In vivo fluorescence imaging of atherosclerotic
plaques with activatable cell-penetrating peptides targeting
thrombin activity" Integr Biol (Camb)., vol. 4, pp. 595-605 (2012).
cited by applicant .
Olson et al. "In vivo characterization of activatable cell
penetrating peptides for targeting protease activity in cancer,"
Integr. Biol., vol. 1, pp. 382-393 (2009). cited by applicant .
Olson, E.S. et al., "Activatable cell penetrating peptides linked
to nanoparticles as dual probes for in vivo fluorescence and MR
imaging of proteases," PNAS, vol. 107, No. 9, pp. 4311-4316 (2010).
cited by applicant .
Olson, E.S., "Activatable cell penetrating peptides for imaging
protease activity in vivo," Electronic Theses and Dissertations UC
San Diego, 2008, 152 pages. cited by applicant .
Oshima et al. "Suppressing TGFbeta Signaling in Regenerating
Epithelia in an Inflammatory Microenvironment Is Sufficient to
Cause Invasive Intestinal Cancer" Cancer Research, vol. 75, pp.
766-776 (2015). cited by applicant .
Passarella, R.J. et al., "Targeted Nanoparticles That Deliver a
Sustained, Specific Release of Paclitaxel to Irradiated Tumors,"
Cancer Res., Jun. 1, 2010, 70(11); pp. 4550-4559. cited by
applicant .
Perentes et al. "Cancer cell-associated MT1-MMP promotes blood
vessel invasion and distant metastasis in triple-negative mammary
tumors" Cancer research. vol. 71, pp. 4527-4538 (2011). cited by
applicant .
Plowman et al. "Ligand-specific activation of HER4/p180erbB4, a
fourth member of the epidermal growth factor receptor family" Proc.
Natl. Acad. Sci., vol. 90, pp. 1746-1750 (1993). cited by applicant
.
Poeta et al. "TP53 mutations and survival in squamous-cell
carcinoma of the head and neck" N Engl J Med, vol. 357, pp.
2552-2561 (2007). cited by applicant .
Pretz, J.L. et al., "Chemoradiationtherapy: localized esophageal,
gastric, and pancreatic cancer," Surg Oncol Clin N Am.,
2013;22:511-24. cited by applicant .
Proimmune, "think peptides.RTM. the source for all peptides for
your research," 2012, pp. 1-15. cited by applicant .
Raju et al. "Combined TP53 mutation/3p loss correlates with
decreased radiosensitivity and increased matrix-metalloproteinase
activity in head and neck carcinoma" Oral oncology, vol. 51, pp.
470-475 (2015). cited by applicant .
Raleigh, D.R. et al., "Molecular targets and mechanisms of
radiosensitization using DNA damage response pathways," Future
Oncol., 2013; 9:219-223. cited by applicant .
Ratnikov et al. "Basis for substrate recognition and distinction by
matrix metalloproteinases" PNAS, vol. 111, pp. E4148-E4155 (2014).
cited by applicant .
Richard et al. "Cell-penetrating Peptides--A Reevaluation of the
Mechanism of Cellular Uptake" The Journal of Biological Chemistry,
vol. 278, No. 1, pp. 585-590 (2003). cited by applicant .
Rieken, S. et al., "Targeting .alpha.V 3 and .alpha.V 5 inhibits
photon-induced hypermigration of malignant glioma cells," Radiation
Oncology, 2011, 6(132):pp. 1-7. cited by applicant .
Rothbard, J. B. et al., "Conjugation of arginine oligomers to
cyclosporin A facilitates topical delivery and inhibition of
inflammation," Nature Medicine, vol. 6, No. 11, pp. 1253-1257
(2000). cited by applicant .
Rothbard, J.B. et al., "Arginine-Rich Molecular Transporters for
Drug Delivery: Role of Backbone Spacing in Cellular Uptake," J.
Med. Chem., vol. 45, pp. 3612-3618 (2002). cited by applicant .
Ryppa, C. et al., "In Vitro and in Vivo Evaluation of Doxorubicin
Conjugates with the Divalent Peptide E-[c(RGDfK)2] that Targets
Integrin .alpha.v 3," Bioconjugate Chem., 2008, vol. 19, pp.
1414-1422. cited by applicant .
Saki et al. "Acquired resistance to cetuximab is associated with
the overexpression of Ras family members and the loss of
radio-sensitization in head and neck cancer cells" Radiother.
Oncol. 108, 473-478 (2013). cited by applicant .
Sanderson et al. "In vivo drug-linker stability of an anti-CD30
dipeptide-linked auristatin immunoconjugate" Clin Cancer Res.
11,843-852 (2005). cited by applicant .
Savariar et al. "Real-time In Vivo Molecular Detection of Primary
Tumors and Metastases with Ratiometric Activatable Cell-Penetrating
Peptides," Cancer Res., vol. 73, pp, 855-864 (2013). cited by
applicant .
Scherer, R.L. et al., "Optical imaging of matrix
metalloproteinase-7 activity in vivo using a proteolytic
nanobeacon," Mol Imaging, 2008, vol. 7, No. 3, pp. 118-131. cited
by applicant .
Sievers, E.L. et al., "Antibody-drug conjugates in cancer therapy,"
Annu Rev Med., 2013;64:15-29. cited by applicant .
Singletary et al. "Surgical margins in patients with early-stage
breast cancer treated with breast conservation therapy" vol. 184,
Issue 5, pp. 383-393 (2002). cited by applicant .
Sivars et al. "Human papillomavirus and p53 expression in cancer of
unknown primary in the head and neck region in relation to clinical
outcome" Cancer Med., vol. 3, pp. 376-384 (2014). cited by
applicant .
Slamon et al. "Human Breast Cancer: Correlation of Relapse and
Survival with Amplification of the HER-2/neu Oncogene" Science,
vol. 235, pp. 177-182 (1987). cited by applicant .
Snijder et al. "Survival in Resected Stage I Lung Cancer With
Residual Tumor at the Bronchial Resection Margin" Ann. Thoracic
Surg., vol. 65, pp. 212-216 (1998). cited by applicant .
Somiari et al. "Circulating MMP2 and MMP9 in breast
cancer--potential role in classification of patients into low risk,
high risk, benign disease and breast cancer categories" Int J
Cancer, vol. 119, pp. 1403-1411 (2006). cited by applicant .
Speake, W.J. et al., "Radiation induced MMP expression from rectal
cancer is short lived but contributes to in vitro invasion," Eur I
Surg Oncol., 2005;31:869-74. cited by applicant .
Sperling, C. et al., "Thrombin-responsive hydrogels with varied
cleavage kinetics," Society for Biomaterials, 2013, Abstract #208,
1 page. cited by applicant .
Stary, H. et al., "A Definition of Advanced Type of Atherosclerotic
Lesions and a Histologicial Classification of Atherosclerosis: A
Report From the Committee on Vascular Lesions of the Council on
Arteriosclerosis, American Heart Association," Circulation, Sep.
1995, vol. 92, No. 5, pp. 355-374. cited by applicant .
Stone, G.W. et al., "A Prospective Natural-History Study of
Coronary Atherosclerosis," The New England Journal of Medicine,
Jan. 20, 2011, vol. 364, No. 3, pp. 226-235. cited by applicant
.
Sun et al. "Reduction-alkylation strategies for the modification of
specific monoclonal antibody disulfides" Bioconjug. Chem. 165,
1282-1290 (2005). cited by applicant .
Swanton et al. "HER2-targeted therapies in non-small cell lung
cancer" Clin. Cancer Res. 12, 4377s-4383s (2006). cited by
applicant .
Thou et al. "Effects of the EGFR/HER2 kinase inhibitor GW572016 on
EGFR- and HER2-overexpressing breast cancer cell line
proliferation, radiosensitization, and resistance" Int. J. Radiat.
Oncol. Biol. Phys. 58, 344-352 (2004). cited by applicant .
Tishler, R.B. et al., "Taxol: a novel radiation sensitizer," Int J
Radiat Oncol Biol Phys., 1992; 122:613-7. cited by applicant .
Toth et al. "Assessment of gelatinases (MMP-2 and MMP-9) by gelatin
zymography" Methods Mol Biol vol. 878, pp. 121-135 (2012). cited by
applicant .
Tseng, W.W. et al., "Development of an Orthotopic Model of Invasive
Pancreatic Cancer in an Immunocompetent Murine Host," Clinical
Cancer Research, Jul. 15, 2010, vol. 16, No. 14, pp. 3684-3695.
cited by applicant .
Tsien et al. "Practical design criteria for a dynamic ratio imaging
system" Cell Calcium, vol. 11, pp. 93-109 (1990). cited by
applicant .
Tsien, R.Y., "Indicators Based on Fluorescence Resonance Energy
Transfer (FRET)," Imaging in Neuroscience and Development, Jul.
2009, vol. 4, No. 7, pp. 1-7. cited by applicant .
Tung et al. "Arginine Containing Peptides as Delivery Vectors"
Advanced Drug Delivery Reviews, vol. 55, pp. 281-294 (2003). cited
by applicant .
Tung, C-H. et al., "A Novel Near-Infrared Fluorescence Sensor for
Detection of Thrombin Activation in Blood," ChemBioChem, 2002, vol.
3, pp. 207-211. cited by applicant .
Ullrich, K.J. et al., "Controluminal para-aminohippurate (PAH)
transport in the proximal tubule of the rat kidney," Pflugers
Arch., 1989, vol. 415, pp. 342-350. cited by applicant .
Uloza et al. "Expression of matrix metalloproteinases (MMP-2 and
MMP-9) in recurrent respiratory papillomas and laryngeal carcinoma:
clinical and morphological parallels" Eur Arch Otorhinolaryngol,
vol. 268, pp. 871-878 (2011). cited by applicant .
Van Berkel, S.S. et al., "Fluorogenic Peptide-Based Substrates for
Monitoring Thrombin Acitivity," ChemMedChem, 2012, vol. 7, pp.
606-617. cited by applicant .
Van Dam, G.M. et al., "Intraoperative tumor-specific fluorescence
imaging in ovarian cancer by folate receptor-.alpha.targeting:
first in-human results," Nature Medicine, 2011, vol. 17, pp.
1315-1319. cited by applicant .
Van Duijnhoven, S.M.J. et al., "Tumor Targeting of MMP-2/9
Activatable Cell-Penetrating Imaging Probes Is Caused by
Tumor-Independent Activation," J Nucl Med, 2011, vol. 52, pp.
279-286. cited by applicant .
Van Vlerken, L.E. et al., "Poly(ethylene glycol)-modified
Nanocarriers for Tumor-targeted and Intracellular Delivery,"
Pharmaceutical Research, Aug. 2007, vol. 24, No. 8, pp. 1404-1414.
cited by applicant .
Vartak, D.G. et al., "In vitro evaluation of functional interaction
of integrin .alpha.v 3 and matrix metalloprotease-2," Mol Pharm.,
2009, vol. 6, No. 6, pp. 1856-1867. cited by applicant .
Verna et al. "Trastuzumab emtansine for HER2-positive advanced
breast cancer" N. Engl. J. Med. 367, 1783-1791 (2013). cited by
applicant .
Visse et al. "Matrix Metalloproteinases and Tissue Inhibitors of
Metalloproteinases" Circulation Research, vol. 92, pp. 827-839
(2003). cited by applicant .
Wadia et al., "Protein transduction technology," Curr. Opinion.
Biotech., vol. 13, pp. 52-56 (2002). cited by applicant .
Wang, Y. et al., "Efficacy and safety of dendrimer nanoparticles
with coexpression of tumor necrosis factor-.alpha.and herpes
simplex virus thymidine kinase in gene radiotherapy of the human
uveal melanoma OCM-1 cell line," International Journal of
Nanomedicine, 2013, vol. 8, pp. 3805-3816. cited by applicant .
Wang, Y. et al., "Visualizing the mechanical activation of Src,"
Nature, Apr. 21, 2005, pp. 1040-1045, vol. 434. cited by applicant
.
Wender et al., "The design, synthesis, and evaluation of molecules
that enable or enhance cellular uptake: Peptoid molcular
transporters," PNAS, vol. 97, No. 24, pp. 13003-13008 (2000). cited
by applicant .
Werner, M.E. et al., "Preclinical evaluation of Genexol-PM, a
nanoparticle formulation of paclitaxel, as a novel radiosensitizer
for the treatment of non-small cell lung cancer," Int J Radiat
Oncol Biol Phys., 2013;86:463-8. cited by applicant .
Wheeler et al. "Understanding resistance to EGFR inhibitors-impact
on future treatment strategies" Nat. Rev. Clin. Oncol. 7, 493-507
(2010). cited by applicant .
Whitney, M. et al., "Parallel in Vivo and in Vitro Selection Using
Phage Display Identifies Protease-dependent Tumor-targeting
Peptides," The Journal of Biological Chemistry, Jul. 16, 2010, vol.
285, No. 29, pp. 22532-22541. cited by applicant .
Wieder et al. "Incidence, etiology, location, prevention and
treatment of positive surgical margins after radical prostatectomy
for prostate cancer" The Journal of Urology, vol. 160, No. 2, pp.
299-315 (1998). cited by applicant .
Willis et al. "Extracellular matrix determinants and the regulation
of cancer cell invasion stratagems" Journal of Microscopy, vol.
251, pp. 250-260 (2013). cited by applicant .
Wittekindt et al. "Expression of matrix metalloproteinase-9 (MMP-9)
and blood vessel density in laryngeal squamous cell carcinomas"
Acta Oto-Laryngologica, vol. 131, Issue 1, pp. 101-106 (2011).
cited by applicant .
Xu, W. et al., "RGD-conjugated gold nanorods induce
Radiosensitization in melanoma cancer cells by down regulating
.alpha.v 3 expression," International Journal of Nanomedicine,
2012, vol. 7, pp. 915-924. cited by applicant .
Zanesi et al. "The tumor spectrum in FHIT-deficient mice" PNAS,
vol. 98, pp. 10250-10255 (2001). cited by applicant .
Zhang et al. "Preparation of Functionally Active Cell-Permeable
Peptides by Single-Step Ligation of Two Peptide Modules" PNAS, vol.
95, pp. 9184-9189 (1998). cited by applicant .
Zhou et al. "Immunoexpression of matrix metalloproteinase-2 and
matrix metalloproteinase-9 in the metastasis of squamous cell
carcinoma of the human tongue" Australian Dental Journal, vol. 55,
pp. 385-389 (2010). cited by applicant .
Zhu, L. et al., "Dual-Functional, Receptor-Targeted Fluorogenic
Probe for In Vivo Imaging of Extracellular Protease Expressions,"
Bioconjugate Chemistry, Jun. 15, 2011, vol. 22, No. 6, pp.
1001-1005. cited by applicant .
Znati, C. et al., "Effect of Radiation on Interstitual Fluid
Pressure and Oxygenation in a Human Tumor Xenograft," Cancer
Research, Mar. 1, 1996, vol. 56, pp. 964-968. cited by
applicant.
|
Primary Examiner: Lieb; Jeanette M
Attorney, Agent or Firm: Morgan Lewis & Bockius LLP
Government Interests
STATEMENT OF FEDERALLY-SPONSORED RESEARCH
This work was supported in part by grants from the Howard Hughes
Medical Institute, the Department of Defense (W81XWH-09-1-0699),
National Cancer Institute (CA158448-01 and P50 CA 097007-DRP),
NIBIB (K08 EB008122-01), and Burroughs Wellcome Fund. The
government has certain rights in this invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application
No. 62/059,081, filed on Oct. 2, 2014, and which is incorporated by
reference herein in its entirety for all purposes.
Claims
What is claimed:
1. An ex vivo method for detecting the presence of one or more
protease activities in a neoplasia sample from a subject with
cancer comprising: a) combining ex vivo a tissue sample from a
subject with cancer with a molecule of the structure A-X-B-C,
wherein B is a peptide portion of about 5 to about 20 basic amino
acid residues, which is suitable for cellular uptake, A is a
peptide portion of about 2 to about 20 acidic amino acid residues,
which when linked with portion B is effective to inhibit or prevent
cellular uptake of portion B, and X is a cleavable linker of about
2 to about 100 atoms joining A with B, where X is cleavable under
physiological conditions, and C is a detectable moiety; and b)
detecting cleavage of A-X-B-C by detecting a change in said
detectable moiety C, wherein said change in C is indicative of
cleavage, said cleavage is indicative of the presence of one or
more protease activities in said tissue sample, and the presence of
the protease activity is indicative that said tissue sample is a
neoplasia sample.
2. The method of claim 1, wherein the presence of the protease
activity is indicative of metastasis.
3. The method of claim 1, wherein C is a fluorescent detectable
moiety.
4. The method of claim 1, wherein C comprises a FRET pair.
5. The method of claim 1, said molecule further comprising a Q
moiety, wherein when said Q moiety is present, said molecule has
the structure Q-A-X-B-C.
6. The method of claim 5, wherein C and Q comprise a FRET pair.
7. The method of claim 6, wherein the FRET pair is selected from
the group consisting of CFP:YFP; Cy5:Cy7; FITC:TRITC; Cy3:Cy5;
EGFP:Cy3; EGFP:YFP; 6-FAM:LC Red 640 or Alexa Fluor 546;
fluorescein:tetramethylrhodamine; IAEDANS:fluorescein;
EDANS:Dabcyl; fluorescein:fluorescein; BODIPY FL:BODIPY FL; and
fluorescein:QSY 7 and QSY 9.
8. The method of claim 1, wherein cleavage of A-X-B-C is detected
by FRET.
9. The method of claim 6, wherein cleavage of Q-A-X-B-C is detected
by FRET.
10. The method of claim 1, wherein said peptide portion A comprises
about 5 to about 9 glutamates or aspartates.
11. The method of claim 1, wherein said peptide portion A comprises
about 5 to about 9 consecutive glutamates or aspartates.
12. The method of claim 1, wherein said peptide portion B comprises
about 9 to about 16 arginines.
13. The method of claim 1, wherein said peptide portion B comprises
about 9 to about 16 consecutive arginines.
14. The method of claim 1, wherein said peptide portion A comprises
D-amino acids.
15. The method of claim 1, wherein said peptide portion B comprises
D-amino acids.
16. The method of claim 1, wherein said peptide portion A consists
of D-amino acids.
17. The method of claim 1, wherein said peptide portion B consists
of D-amino acids.
18. The method of claim 1, wherein said peptide portions A and B
each consist of D-amino acids.
19. The method of claim 1, wherein cleavable linker X is a flexible
linker.
20. The method of claim 1, wherein cleavable linker X is a flexible
linker about 6 to about 30 atoms in length.
21. The method of claim 1, wherein cleavable linker X is cleavable
in an acidic environment.
22. The method of claim 1, wherein cleavable linker X comprises a
peptide linkage.
23. The method of claim 1, wherein cleavable linker X comprises
aminocaproic acid.
24. The method of claim 1, wherein cleavable linker X is configured
for cleavage exterior to a cell.
25. The method of claim 1, wherein cleavable linker X is configured
for cleavage by an enzyme.
26. The method of claim 25, wherein said enzyme is selected from
the group consisting of a matrix metalloprotease, elastase,
plasmin, thrombin, chymase, urokinase-type plasminogen activator
and tissue plasminogen activator.
27. The method of claim 1, wherein cleavable linker X comprises an
amino acid sequence selected from the group consisting of PLGLAG
(SEQ ID NO: 1), PLGC(met)AG (SEQ ID NO: 2), EDDDDKA (SEQ ID NO: 3),
RS-(Cit)-G-(homoF)-YLY (SEQ ID NO: 4), CRPAHLRDSG (SEQ ID NO: 5),
SLAYYTA (SEQ ID NO: 6), NISDLTAG (SEQ ID NO: 7), PPSSLRVT (SEQ ID
NO: 8), SGESLSNLTA (SEQ ID NO: 9), RIGFLR (SEQ ID NO: 10),
RLQLA(acetyl)L (SEQ ID NO: 11), RLQLKL (SEQ ID NO: 12), DPRSFL (SEQ
ID NO: 13), PPRSFL (SEQ ID NO: 14), Norleucine-TPRSFL (SEQ ID NO:
15), GVAY|SGA (SEQ ID NO: 16), YGRAAA (SEQ ID NO: 17), YGPRNR (SEQ
ID NO: 18), RSHP(Hfe)TLY (SEQ ID NO: 19), RSHG(Hfe)FLY (SEQ ID NO:
20), SNPYK-Y (SEQ ID NO: 21), SNPKG-Y (SEQ ID NO: 22), SNPYG-Y (SEQ
ID NO: 23), TLSE-LH (SEQ ID NO: 24), TIAHLA (SEQ ID NO: 25),
(RLQLK(acetyl)L (SEQ ID NO: 26), and KLRFSKQ (SEQ ID NO: 27).
28. The method of claim 1, wherein cleavable linker X comprises a
S-S linkage.
29. The method of claim 1, wherein cleavable linker X comprises a
transition metal complex, wherein said transition metal complex
linker is cleaved when the metal is reduced.
30. The method of claim 1, wherein in said method comprises
multiple molecules of the structure A-X-B-C and wherein the
cleavable linker X comprises a plurality of cleavable linkers
X.
31. The method of claim 30, wherein the plurality of cleavable
linkers X linking a portion A to a structure B-C are cleavable by a
single protease.
32. The method of claim 30, wherein the plurality of cleavable
linkers X linking a portion A to a structure B-C are cleavable by
more than one protease.
33. The method of claim 1, wherein cleavable linker X comprises an
amino acid sequence selected from the group consisting of
RSHP(Hfe)TLY (SEQ ID NO: 19), RSHG(Hfe)FLY (SEQ ID NO: 20), SNPYK-Y
(SEQ ID NO: 21), SNPKG-Y (SEQ ID NO: 22), SNPYG-Y (SEQ ID NO: 23),
TLSE-LH (SEQ ID NO: 24), TIAHLA (SEQ ID NO: 25), (RLQLK(acetyl)L
(SEQ ID NO: 26), and KLRFSKQ (SEQ ID NO: 27).
Description
FIELD OF THE INVENTION
This invention pertains to methods and composition that find use in
diagnostic, prognostic and characterization of neoplasia samples
based on the ability of a neoplasia sample to cleave a MTS molecule
of the present invention.
BACKGROUND OF THE INVENTION
Introduction
Cell membranes delimit the outer boundaries of cells, and regulate
transport into and out of the cell interior. Made primarily of
lipids and proteins, they provide a hydrophilic surface enclosing a
hydrophobic interior across which materials must pass before
entering a cell. Although many small, lipophilic compounds are able
to cross cell membranes passively, most compounds, particles and
materials must rely on active mechanisms in order to gain entry
into a living cell.
Transmembrane Transport
Regulation of transport into and out of a cell is vital for its
continued viability. For example, cell membranes contain ion
channels, pumps, and exchangers capable of facilitating the
transmembrane passage of many important substances. However,
transmembrane transport is selective: in addition to facilitating
the entry of desired substances into a cell, and facilitating the
exit of others, a major role of a cell membrane is to prevent
uncontrolled entry of substances into the cell interior. This
barrier function of the cell membrane makes difficult the delivery
of markers, drugs, nucleic acids, and other exogenous material into
cells.
Over the last decade, peptide sequences that can readily enter a
cell have been identified. For example, the Tat protein of the
human immunodeficiency virus 1 (HIV-1) is able to enter cells from
the extracellular environment (e.g., Fawell et al. P.N.A.S.
91:664-668 (1994)). Such uptake is reviewed in, for example,
Richard et al., J. Biol. Chem. 278(1):585-590 (2003).
Such molecules that are readily taken into cells may also be used
to carry other molecules into cells along with them. Molecules that
are capable of facilitating transport of substances into cells have
been termed "membrane translocation signals" (MTS) as described in
Tung et al., Advanced Drug Delivery Reviews 55:281-294 (2003). The
most important MTS are rich in amino acids such as arginine with
positively charged side chains. Molecules transported into cell by
such cationic peptides may be termed "cargo" and may be reversibly
or irreversibly linked to the cationic peptides. An example of a
reversible linkage is found in Zhang et al., P.N.A.S. 95:9184-9189
(1994)).
MTS molecules are discussed in, for example, Wender et al.,
P.N.A.S. 97:13003-13008 (2000); Hallbrink et al., Biochim. Biophys.
Acta 1515:101-109 (2001); Derossi et al., Trends in Cell Biology
8:84-87 (1998); Rothbard et al., J. Med. Chem. 45:3612-3618 (2002);
Rothbard et al., Nature Medicine 6(11):1253-1247 (2000); Wadia et
al., Curr. Opinion Biotech. 13:52-56 (2002); Futaki et al.;
Bioconj. Chem. 12:1005-1011 (2001); Rothbard et al., U.S. Pat. No.
6,306,993; Frankel et al., U.S. Pat. No. 6,316,003; Rothbard et
al., U.S. Pat. No. 6,495,663; Monahan et al., U.S. Pat. No.
6,630,351 and Jiang et al., WO 2005/042034.
Cancer Surgery
In cancer surgery, positive margins, defined as tumor cells present
at the cut edge of the surgical specimen, have been associated with
increased local recurrence and a poor prognosis (Hague R., et al.,
BMC Ear Nose Throat Disord. 16:2 (2006)). As in most solid tumors,
salvage surgery (i.e., re-excision of the positive margin) or
adjuvant chemotherapy and/or radiation not only cause extra trauma
and expense but also often fail to remediate the poor outcome
(Hague R., et al., BMC Ear Nose Throat Disord. 16:2 (2006);
Singletary S. Am. J. Surg. 184:383-393 (2002); Meric F., et al.,
Cancer 97:926-933 (2003); Snijder R., et al., Annals of Thoracic
Surg. 65 (1998); Nagtegaal I D, Quirke P., J. Clin. On. 26:303-312
(2008); Dotan Z, et al., J. Urol. 178:2308-2312 (2007); and Wieder
J. A., J. Urol. 160:299-315 (1998)).
The reason for this observation is likely multifactorial and
related in part to the difficulty in identifying the residual
cancer during repeat surgery. Therefore, development of more
sensitive imaging and diagnostic assays for more accurate detection
of positive surgical margins during the primary operation would be
one of the most effective means to minimize patient suffering and
expense and to improve survival.
Role of MMPs in Cancer
MMPs play crucial roles in cancer invasion and metastasis (Bauvois
B. et al. Biochim Biophys Acta. 1825:29-36 (2012)) are
overexpressed malignant tumors and their expression/activity is
associated with \poor patient prognosis. Increased MMP expression
has been shown to correlate with cancer grade (Wittekindt C., et
al. Acta Otolaryngol. 131:101-106 (2011)) and decreased survival
(Liu W. W., et al. Otolaryngol Head Neck Surg. 132:395-400 (2005)
and Mallis A., et al., Eur Arch Otorhinolaryngol. 269:639-642
(2012)). In carcinoma of the tongue, increased MMP expression has
been shown to correlate with incidence of lymph node metastases
(Zhou, C. X., et al., Aust Dent J. 55:385-389 (2010)).
Heterogeneity/Specificity
Although increased MMP expression has been shown to correlate with
increased cancer grade and stage and decreased survival (Wang W L,
et al., Mol Carcinog. 2012 and P. O. C., Arch Otolaryngol Head Neck
Surg. 127:813-820 (2001)), there is significant heterogeneity that
exists between patients in terms of absolute MMP levels (Wang W L,
et al., Mol Carcinog. 2012). This invention describes a method to
address this heterogeneity and evaluate the clinical utility of
ACPPs in cancers from multiple body sites using an ex-vivo
screening assay to determine MMP activity for individual human cell
line derived and surgical tumor samples. MMP activity can also be
elevated at some sites of nonmalignant inflammation, such as skin
lacerations and atherosclerotic plaques (Olson E. S., et al.,
Integr Biol (Camb). (2012)), but these are anatomically remote from
and easily distinguished intraoperatively from tumor margins and
potentially metastatic lymph nodes. In our experience, such other
sites of MMP activity are unlikely to confuse any experienced
clinician, just as the enormous .sup.18F signal in normal brain,
heart, and bladder during [.sup.18F]-FDG PET scans does not prevent
the usefulness of such imaging in locating tumors and metastases
with high glucose utilization. Because of the concern that MMP
expression is also increased in inflammation/wound healing, part of
the study is to evaluate the threshold of MTS (activatable cell
penetrating peptides; ACPP) uptake that can reliably distinguish
cancer from non-cancer tissue. Besides MMPs which were the focus of
initial studies, MTS have been developed the target other proteases
that have been proposed to be involved in cancer including,
elastases, thrombin, plasmin, legumain, cathepsins.
All patents and publications, both supra and infra, are hereby
incorporated by reference in their entirety.
As the field of molecularly targeting fluorescent markers for early
cancer detection and intraoperative margin evaluation progresses
and more enzymatically activatable probes (Jiang T., et al. Proc
Natl Acad Sci USA. 101:17867-17872 (2004); Aguilera T. A., et al.,
Integr. Biol. 1:371-381 (2009); Olson E. S., et al., Integr Biol
(Camb). 1:382-393 (2009); Olson E. S., et al., Proc Natl Acad Sci
USA. 107:4311-4316 (2010); Nguyen Q. T., Proc Natl Acad Sci USA.
107:4317-4322 (2010); Blum G., et al., Nat Chem Biol. 3:668-677
(2007); Gounaris E., et al., PLoS One. 3:e2916 (2008); Bremer C.,
et al., Invest Radiol. 40:321-327 (2005)) are becoming available
for clinical use, methods such as a personalized protease assay
(PePA) would be useful in a variety of diagnostic and prognostic
applications.
As such, there remains a need in the art for additional diagnosis,
prognosis and characterization, including development personalized
protease assays, useful in both in vivo and ex vivo applications.
Such methods would allow for the development of better and more
personalized treatment regimens. The present invention meets these
needs and provides methods for ex vivo diagnosis, prognosis and
characterization of tumors which can find use in a variety of
personalized medicine applications.
SUMMARY OF THE INVENTION
Methods of use and compositions comprising MTS molecules are
disclosed. MTS molecules having features of the invention include
peptide portions linked by a cleavable linker portion which may be
a peptide. The inventors have found that these MTS molecules can
find use in diagnostic, prognostic and characterization assays.
In some embodiments, the present invention provides an ex vivo
method for detecting the presence of one or more protease
activities in a neoplasia sample comprising a) combining ex vivo
said neoplasia sample from a subject with a molecule of the
structure A-X-B-C, wherein B is a peptide portion of about 5 to
about 20 basic amino acid residues, which is suitable for cellular
uptake, A is a peptide portion of about 2 to about 20 acidic amino
acid residues, which when linked with portion B is effective to
inhibit or prevent cellular uptake of portion B, and X is a
cleavable linker of about 2 to about 100 atoms joining A with B,
where X is cleavable under physiological conditions, and C is a
detectable moiety; and b) detecting cleavage of A-X-B-C by
detecting a change in said detectable moiety C, wherein said change
in C is indicative of cleavage and said cleavage is indicative of
the presence of one or more protease activities in said
neoplasia.
In some embodiments, the present invention provides an ex vivo
method of determining a treatment regimen based on the protease
profile of a neoplasia sample, comprising a) combining ex vivo said
neoplasia sample from a subject with a molecule of the structure
A-X-B-C, wherein B is a peptide portion of about 5 to about 20
basic amino acid residues, which is suitable for cellular uptake, A
is a peptide portion of about 2 to about 20 acidic amino acid
residues, which when linked with portion B is effective to inhibit
or prevent cellular uptake of portion B, and X is a cleavable
linker of about 2 to about 100 atoms joining A with B, where X is
cleavable under physiological conditions and C is a detectable
moiety; and b) detecting cleavage of A-X-B-C by detecting a change
in detectable moiety C, wherein said change in C is indicative of
cleavage and said cleavage is indicative of the presence of one or
more protease activities and wherein the presence and/or absence of
one or more protease activities allows for determining a medical
treatment regimen.
In some embodiments, the medical regimen is a surgical regimen.
In some embodiments, the protease activity is indicative of
neoplasia. In some embodiments, the protease activity is indicative
of metastasis.
In some embodiments, C is a fluorescent detectable moiety.
In some embodiments, C comprises a FRET pair.
In some embodiments, the molecule of the invention further
comprises a Q moiety, wherein when said Q moiety is present, said
molecule has the structure Q-A-X-B-C.
In some embodiments, the method of any of the preceding claims
wherein C and Q comprise a FRET pair. In some embodiments, the FRET
pair is selected from the group consisting of CFP:YFP; Cy5:Cy7;
FITC:TRITC; Cy3:Cy5; EGFP:Cy3; EGFP:YFP; 6-FAM:LC Red 640 or Alexa
Fluor 546; fluorescein:tetramethylrhodamine; IAEDANS:fluorescein;
EDANS:Dabcyl; fluorescein:fluorescein; BODIPY FL:BODIPY FL; and
fluorescein:QSY 7 and QSY 9.
In some embodiments, cleavage of A-X-B-C is detected by FRET.
In some embodiments, cleavage of Q-A-X-B-C is detected by FRET.
In some embodiments, the peptide portion A comprises about 5 to
about 9 glutamates or aspartates. In some embodiments, the peptide
portion A comprises about 5 to about 9 consecutive glutamates or
aspartates. In some embodiments, the peptide portion B comprises
about 9 to about 16 arginines. In some embodiments, the peptide
portion B comprises about 9 to about 16 consecutive arginines.
In some embodiments, the peptide portion A comprises D-amino acids.
In some embodiments, the peptide portion B comprises D-amino acids.
In some embodiments, the peptide portion A consists of D-amino
acids. In some embodiments, the peptide portion B consists of
D-amino acids. In some embodiments, the peptide portions A and B
consists of D-amino acids.
In some embodiments, the cleavable linker X is a flexible linker.
In some embodiments, the cleavable linker X is a flexible linker
about 6 to about 30 atoms in length.
In some embodiments, the cleavable linker X is cleavable in an
acidic environment.
In some embodiments, the cleavable linker X comprises a peptide
linkage.
In some embodiments, the cleavable linker X comprises aminocaproic
acid.
In some embodiments, the cleavable linker X is configured for
cleavage by an enzyme. In some embodiments, the enzyme is selected
from the group consisting of a matrix metalloprotease, elastase,
plasmin, thrombin, chymase, urokinase-type plasminogen activator
and tissue plasminogen activator. In some embodiments, the
cleavable linker X comprises an amino acid sequence selected from
the group consisting of PLGLAG (SEQ ID NO: 1), PLGC(met)AG (SEQ ID
NO: 2), EDDDDKA (SEQ ID NO: 3), RS-(Cit)-G-(homoF)-YLY (SEQ ID NO:
4), CRPAHLRDSG (SEQ ID NO: 5), SLAYYTA (SEQ ID NO: 6), NISDLTAG
(SEQ ID NO: 7), PPSSLRVT (SEQ ID NO: 8), SGESLSNLTA (SEQ ID NO: 9),
RIGFLR (SEQ ID NO: 10), RLQLA(acetyl)L (SEQ ID NO: 11), RLQLKL (SEQ
ID NO: 12), DPRSFL (SEQ ID NO: 13), PPRSFL (SEQ ID NO: 14),
Norleucine-TPRSFL (SEQ ID NO: 15), GVAY|SGA (SEQ ID NO: 16), YGRAAA
(SEQ ID NO: 17), YGPRNR (SEQ ID NO: 18), RSHP(Hfe)TLY (SEQ ID NO:
19), RSHG(Hfe)FLY (SEQ ID NO: 20), SNPYK-Y (SEQ ID NO: 21), SNPKG-Y
(SEQ ID NO: 22), SNPYG-Y (SEQ ID NO: 23), TLSE-LH (SEQ ID NO: 24),
TIAHLA (SEQ ID NO: 25), (RLQLK(acetyl)L (SEQ ID NO: 26), and
KLRFSKQ (SEQ ID NO: 27).
In some embodiments, the cleavable linker X comprises a S-S
linkage.
In some embodiments, the cleavable linker X comprises a transition
metal complex, wherein said transition metal complex linker is
cleaved when the metal is reduced.
In some embodiments, the method comprises multiple molecules of the
structure A-X-B-C and wherein the cleavable linker X comprises a
plurality of cleavable linkers X. In some embodiments, the
plurality of cleavable linkers X linking a portion A to a structure
B-C are cleavable by a single protease. In some embodiments, the
plurality of cleavable linkers X linking a portion A to a structure
B-C are cleavable by more than one protease.
In some embodiments, the method comprises an in vivo method of
determining a treatment regimen based on the protease profile of a
neoplasia, comprising a) providing to a subject a molecule of the
structure A-X-B-C, wherein B is a peptide portion of about 5 to
about 20 basic amino acid residues, which is suitable for cellular
uptake, A is a peptide portion of about 2 to about 20 acidic amino
acid residues, which when linked with portion B is effective to
inhibit or prevent cellular uptake of portion B, and X is a
cleavable linker of about 2 to about 100 atoms joining A with B,
where X is cleavable under physiological conditions and C is a
detectable moiety; and b) detecting cleavage of A-X-B-C by
detecting a change in detectable moiety C, wherein said change in C
is indicative of cleavage and said cleavage is indicative of the
presence of one or more protease activities and wherein the
presence and/or absence of one or more protease activities allows
for determining a medical treatment regimen.
In some embodiments, the medical treatment regimen is a surgical
regimen.
In some embodiments, the presence of the protease activity is
indicative of neoplasia.
In some embodiments, the cleavable linker X comprises an amino acid
sequence selected from the group consisting of PLGLAG (SEQ ID NO:
1), PLGC(met)AG (SEQ ID NO: 2), EDDDDKA (SEQ ID NO: 3),
RS-(Cit)-G-(homoF)-YLY (SEQ ID NO: 4), CRPAHLRDSG (SEQ ID NO: 5),
SLAYYTA (SEQ ID NO: 6), NISDLTAG (SEQ ID NO: 7), PPSSLRVT (SEQ ID
NO: 8), SGESLSNLTA (SEQ ID NO: 9), RIGFLR (SEQ ID NO: 10),
RLQLA(acetyl)L (SEQ ID NO: 11), RLQLKL (SEQ ID NO: 12), DPRSFL (SEQ
ID NO: 13), PPRSFL (SEQ ID NO: 14), Norleucine-TPRSFL (SEQ ID NO:
15), GVAY|SGA (SEQ ID NO: 16), YGRAAA (SEQ ID NO: 17), YGPRNR (SEQ
ID NO:18), RSHP(Hfe)TLY (SEQ ID NO: 19), RSHG(Hfe)FLY (SEQ ID NO:
20), SNPYK-Y (SEQ ID NO: 21), SNPKG-Y (SEQ ID NO: 22), SNPYG-Y (SEQ
ID NO: 23), TLSE-LH (SEQ ID NO: 24), TIAHLA (SEQ ID NO: 25),
(RLQLK(acetyl)L (SEQ ID NO: 26), and KLRFSKQ (SEQ ID NO: 27).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 describes that the ability to cleave RACPPs (radiometric
MTSs) to be assayed ex vivo on frozen tissue samples and may
differentiate normal from tumor tissues. Y axis indicates rates of
change of Cy5 fluorescence (arbitrary units) over time following
addition of MMP- and elastase-sensitive RACPPs to 100 mg
homogenized fatty tissue from mouse, normal human breast, two mouse
breast cancer grafts (4T1 and 8119), and several human head and
neck squamous cell carcinoma surgical specimens.
FIG. 2A describes Higher MMP expression in tumors versus normal
tissue in TCGA HNSCC. FIG. 2B HPV+ tumors have lower MMP expression
than HPV-tumors.
FIG. 2C and FIG. 2D Higher MMP-2/MMP-14 expression in HPV+ tumors
correlates with poorer prognosis. Abbreviations: MMP,
matrix-metalloproteinase; TCGA, the Cancer Genomic Atlas; HNSCC,
head and neck squamous cell carcinoma; HPV, human papilloma
virus.
FIG. 3 describes RACPP schematic showing (A) no tumor-contrast
immediately post-injection; (B) high tumor-contrast following
MMP-dependent cleavage, separating Cy5 from Cy7. (C) Application of
RACPP to HNSCC specimens produces faster Cy5/Cy7 ratio-change
compared to normal tissue. Abbreviations: RACPP, ratiometric
activatable cell-penetrating peptide; HNSCC, head and neck squamous
cell carcinoma.
FIG. 4 describes (A, B) RACPP injection produces greater
ratiometric fluorescent signal in HNSCC tumor versus normal tissue.
(C) Uncleavable-control does not produce tumor-specific contrast.
(D) RACPP is sensitive and specific for tumor detection.
Abbreviations: HNSCC, head and neck squamous cell carcinoma; RACPP,
ratiometric activatable cell-penetrating peptide.
FIG. 5 describes (A) Ratiometric images showing higher fluorescence
in tumor (white stippling). (B) Corresponding H&E images
confirming tumor burden (red stippling). (C) Ratiometric
activatable cell-penetrating peptide (RACPP) uptake correlates
directly with tumor burden.
FIG. 6 describes select ACPP substrates.
FIG. 7 describes selectivity for substrates for MMP2, 9 and 14 (1
uM peptide 20 nM enzyme). A) MT1 selective RSHPHfeTLY (SEQ ID NO:
19). B) MMP2 selective TIAHLA (SEQ ID NO: 25). C) MMP9 selective
SNPYKY (SEQ ID NO: 21).
FIG. 8A, FIG. 8B and FIG. 8C describe selectivity for substrate cut
with MMP-2, MMP9 and MMP-14. FIG. 8A, FIG. 8B and FIG. 8C describe
1 PLGmetCAG-MMP2 (SEQ ID NO: 28), 9,14, 2 TLSELH-MMP-2 selective
(SEQ ID NO: 24), 3 TIAHLA-MMP2 selective (SEQ ID NO: 25), 4
CATK-KLRFSKQ (SEQ ID NO: 29), 5 Cit-MMP14 selective, 6
RSHG(Hfe)FLY-MMP14 (SEQ ID NO: 20) selective, 7 RSHP(Hfe)TLY-MMP14
selective (SEQ ID NO: 19), 8 PLGLEEA-MMP12 (SEQ ID NO:30)
selective, and 9 SNPYKY-MMP-9 (SEQ ID NO: 21) selective.
FIG. 9 describes selectivity for substrates with A) Panc2
supernatant no radiation abd B) without MMP-2 substrates.
FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D describe testing of FRET
versions of newly optimized MMP-9 selective substrates. FIG. 10A
SNPYK-Y (SEQ ID NO: 21) substrate. FIG. 10B SNPKG-Y (SEQ ID NO: 22)
substrate. FIG. 10C SNPYG-Y (SEQ ID NO: 23) substrate. FIG. 10D
SNPFKY (SEQ ID NO: 31) substrate.
FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E and FIG. 11F
describe MMP2 FRET substrates based on rational substitution of
consensus/preferred residues. Peptides include:
MMP2-1=FAM-e9-dPEG(6)-SGTLAH-LHTA-r9-(D-cys)-NH2
MMP2-2=FAM-e9-dPEG(6)-SGTLSE-LHTA-r9-(D-cys)-NH2
MMP2-3=FAM-e9-dPEG(6)-SGTISH-LHTA-r9-(D-cys)-NH2
MMP2-4=FAM-e9-dPEG(6)-SGTLSH-LHTA-r9-(D-cys)-NH2
MMP2-5=FAM-e9-dPEG(6)-SGTIAH-FHTA-r9-(D-cys)-NH2
FIG. 12 describes design, synthesis and testing of new Cathepsin K
substrates. FAM-e9-dPEG(6)-XXXXXX-r9-(D-cys)-NH2-general
format.
FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, and FIG. 13E describe
design, synthesis and testing of new Cathepsin K substrates.
FAM/Cy5 FRET versions. FIG. 13A KPRGSKQ (SEQ ID NO: 32) substrate.
FIG. 13B KLRFSKQ (SEQ ID NO: 33) substrate. FIG. 13C KKPGSKQ (SEQ
ID NO: 34) substrate. FIG. 13D HPGGPQ (SEQ ID NO: 35) substrate.
FIG. 13E NleTLRSLQ (SEQ ID NO: 36) substrate.
FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D describe generation of
new FRET (FAM/Cy5) versions of MT1-MMP(MMP-14) selective ACPPs.
FIG. 14A O-RSHP(Hfe)TLY-(SEQ ID NO: 19) substrate. FIG. 14B
O-RSHG(Hfe)FLY (SEQ ID NO: 20) substrate. FIG. 14C
Original-R-S-cit-G-Hfe-YLY (SEQ ID NO: 38) substrate. FIG. 14D
dPEG6-SG-ARGIKL-TA (SEQ ID NO: 37) substrate.
FIG. 15 describes making ACPPs that have improved selectivity for
specific MMPs-Cy5/Cy7 FRET versions. A) PLGC(Me)AG (SEQ ID NO: 2)
substrate (MMP2/9)+. B) RS-cit-G-homoF-YLY (SEQ ID NO: 4) substrate
(MT1-MMP). C) Substrate diagram. D) Cy5/Cy7 tumor ratio.
FIG. 16 describes skin off images for Cal-27 tumors 2 hrs post 10
nmole injection. PLG, MT1-MMP "Cit" O-R-S-cit-G-Hfe-YLY (SEQ ID NO:
38), MT1-"New" 0-RSHP(Hfe)TLY-(SEQ ID NO: 19) substrates.
FIG. 17A and FIG. 17B are diagrams of Cy5/Cy7 FRET probes.
FIG. 18A, FIG. 18B, FIG. 18C and FIG. 18D provide additional
comparison data for Cy5/Cy7 FRET probes with "branched old" versus
"backbone new" peg12. FIG. 18A PLGC(met)AG (SEQ ID NO: 2) (Branched
Peg12). FIG. 18B New PLGC(met)AG (SEQ ID NO: 2) (backbone peg12).
FIG. 18C Nle-TPRSFL (SEQ ID NO: 15) (Original branched). FIG. 18D
CatK (backbone peg6).
FIG. 19 describes a comparison of Nle-TPRSFL (SEQ ID NO: 15) branch
PEG with new CatK substrate with backbone Peg6. A) CatK (backbone
peg6). B) Nle-TPRSFL (SEQ ID NO: 15) (Original branched).
FIG. 20A, FIG. 20B and FIG. 20C describe Synthesis if MMP selective
ACPPs in Cy5Cy7 format. (Cy7)-NH2-e9-c(Peg12)-0-Substrate-r9-c
(Cy5)-CONH2. FIG. 20A Control. FIG. 20B MMP2 selective TIAHLA (SEQ
ID NO: 25). FIG. 20C MMP9 selective SNPYGY (SEQ ID NO: 23).
FIG. 21 describes cleavage of derivatives of RS-(Cit)-G-(homoF)-YLY
(SEQ ID NO: 4) cut with MMP-2, MMP-9 and MT1-MMP.
FIG. 22 describes cleavage of derivatives of RS-(Cit)-G-(homoF)-YLY
(SEQ ID NO: 4) cut with MMP-2, MMP-9 and MT1-MMP. Insertion of
Proline at P3/P4 site makes the substrate a good MMP2
substrate.
FIG. 23 describes cleavage of derivatives of RS-(Cit)-G-(homoF)-YLY
(SEQ ID NO: 4) cut with MT2-MMP, Only RS-Q-G-(homoF)-YLY (SEQ ID
NO: 71) shows significant cleavage by MT2-MMP.
FIG. 24 describes cleavage of derivatives of RS-(Cit)-G-(homoF)-YLY
(SEQ ID NO: 4) cut with MT2-MMP, Only RS-Q-G-(homoF)-YLY (SEQ ID
NO: 71) shows significant cleavage by MT2-MMP.
FIG. 25 provides a diagram regarding the substitution of consensus
amino acids to our current best optimal MMT1 cleavable
substrate.
FIG. 26 describes MMP2/9/14 cleavage of
FAM-e9-dPEG(6)-SG-XXXXXX-TA-r9-(D-cys)-NH2 peptides.
FIG. 27 describes digestion of new ACPP with MMP2, 9 and 14 from
Ratinakov et. al. (2 hours/2 uM peptide/50 nM enzyme.
FAM-e9-dPEG(6)-SG-XXXXXX-TA-r9-(D-cys)-NH2.
FIG. 28A, FIG. 28B, FIG. 28C and FIG. 28D describe generation of
new FRET (FAM/Cy5) versions of MT1-MMP(MMP-14) selective ACPPs.
FIG. 28A 0-RSHP(Hfe)TLY-(SEQ ID NO: 19) substrate. FIG. 28B
O-RSHG(Hfe)FLY (SEQ ID NO: 20) substrate. FIG. 28C
Original-R-S-cit-G-Hfe-YLY (SEQ ID NO: 38) substrate. FIG. 28D
dPEG6-SG-ARGIKL-TA (SEQ ID NO: 37) substrate.
FIG. 29A, FIG. 29B, FIG. 29C and FIG. 29D describe FIG. 29A Higher
MMP expression in tumors versus normal tissue in TCGA HNSCC. FIG.
29B HPV+ tumors have lower MMP expression than HPV-tumors. FIG. 29C
and FIG. 29D Higher MMP-2/MMP-14 expression in HPV+ tumors
correlates with poorer prognosis.
FIG. 30 describes RACPP schematic showing (A) no tumor-contrast
immediately post-injection; (B) high tumor-contrast following
MMP-dependent cleavage, separating Cy5 from Cy7. (C) Application of
RACPP to HNSCC specimens produces faster Cy5/Cy7 ratio-change
compared to normal tissue.
FIG. 31 describes (A,B) RACPP injection produces greater
ratiometric fluorescent signal in HNSCC tumor vs. normal tissue.
(C) Uncleavable-control does not produce tumor-specific contrast.
(D) RACPP is sensitive and specific for tumor detection. E)
Receiver operating characteristic analysis.
FIG. 32 describes (A)Ratiometric images showing higher fluorescence
in tumor (white stippling). (B) Corresponding H&E images
confirming tumor burden (red stippling). (C) RACPP uptake
correlates directly with tumor burden.
FIG. 33 describes TCGA data showing that for patients with HPV+
tumor, mRNA expression of MMP-2 and MMP-14 positively
correlate.
FIG. 34 describes mouse HNSCC tongue xenografts demonstrate greater
MMP2/9 activity compared to normal tongue tissue. Bar graphs
display MMP activity of samples as a percentage of activity of pure
MMP standard.
FIG. 35 describes the ratio of tumor:control tissue MMP expression
levels (red means high ratio, blue means low ratio) for multiple
cancers represented within TCGA. HNSCC is the first column.
FIG. 36 provides a graph comparing uPA(aka PLAU) mRNA expression
levels in TCGA specimens showing increased levels in tumor (red)
compared to paired normal tissue (blue) in multiple cancers
including HNSC (number of specimen pairs analyzed for a given tumor
site in parentheses, p<0.01 for all tumor types shown).
FIG. 37 describes a) Schematics of regular non-ratiometric ACPP
(Standard ACPP) and RACPP induced tumor contrast shown in top and
bottom panels respectively. Immediately after IV injection neither
configurations produce any tumor contrasts (left panels). Within
1-2 hr spectacular tumor contrast can be obtained with RACPP
(bottom middle pane). However poor pharmacokinetic washout of the
uncleaved probe with the standard ACPP results in modest tumor to
background contrast (top middle panel). Longer waiting time such as
24 hr after IV injections result in loss of tumor contrasts in
either configurations (right panels). b) Graph shows the emission
spectrum of RACPP1, measured in mouse plasma in a cuvet
spectrofluorometer, before (black solid curve) and after (red
dashed curve) treatment with MMP-9. The starting spectrum shows
considerable quenching of the Cy5 peak at 670 nm and re-emission
from Cy7 at 780 nm.
FIG. 38 provides a schematic of RACPPs demonstrating the modular
nature of the molecule which enables rational modification of the
cleavable site (green) as well as payloads (yellow circles).
FIG. 39 describes that a ACPP fluorescence can be used to guide ex
vivo examination of surgical specimens. Photomicrographs showing a
representative specimens from tumor bearing mice following IV
administration of ACPPD. (A) Low-power Cy5 fluorescence showing
positive ACPPD uptake (arrowheads). (B) The same section as in A
stained with H&E, confirming the presence of malignant cells in
regions that show increased fluorescence uptake (arrowheads). (C
and E) Enlarged fluorescence images from the boxed areas in A,
showing the demarcation between high (*) and low (arrows)
fluorescence uptake. (D and F) Histological (H and E) analysis of C
and E, showing that the areas of high fluorescence uptake
correspond to malignant cells (*). (Scale bar in A and B: 0.5 mm; C
and D: 0.1 mm; E and F: 0.25 mm.) Adapted from Nguyen et al
2010.
FIG. 40 shows application of PLGCMeAG-RACPP (SEQ ID NO: 2) to HNSCC
specimens produces faster Cy5/Cy7 ratio-change compared to normal
tissue. Adapted from Hauff et al 2014.
FIG. 41 shows an increase in Cy5/Cy7 signal ratio of substrates
YGRAAA (SEQ ID NO: 17) upone cleavage by uPA (light purple)
compared to MMPs. Whitney et al, manuscript in preparation.
FIG. 42 shows a ratiometric fluorescence RACPP uptake (A)
correlates with H&E evidence of tumor burden (B) from Hauff et
al, 2014).
FIG. 43 shows a ratiometric fluorescence RACPP uptake (A-C)
correlates with more aggressive tumor genotype (D) (from Raju et
al, 2015).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based in part on the discovery that ex
vivo cleavage of ratiometric MTSs (ACPPs) by tumor extract
correlates with in-vivo MTS (ACPP) fluorescence uptake and
increased emission ratio in cancer, particularly carcinoma. In some
embodiments, measuring the ability of individual tumors to cleave
MTSs (ACPPs) and assessing the percentage of enzymatically positive
tumors in a clinical population provides valuable data in that the
ex vivo cleavage data can be correlated with MTS (ACPP) performance
in vivo. In some embodiments, the ex vivo cleavage assay may be
further developed into a personalized screening assay to determine
eligibility to use MTSs (ACPPs) during a given patient procedure
such as for example surgery. In some embodiments, the present
invention provides methods for assessing the distribution of human
surgical specimens with respect to their ability to cleave the MTSs
(ACPPs) and the correlation of the MTS with clinical grade and
outcome. Methods and compositions useful in such methods are
provided below.
Certain Definitions
The following terms have the meanings ascribed to them unless
specified otherwise.
The terms cell penetrating peptide (CPP), activatable cell
penetrating peptide (ACPP), membrane translocating sequence (MTS)
and protein transduction domain are used interchangeably. As used
herein, the terms mean a peptide (polypeptide or protein) sequence
that is able to translocate across the plasma membrane of a cell.
In some embodiments, a CPP facilitates the translocation of an
extracellular molecule across the plasma membrane of a cell. In
some embodiments, the CPP translocates across the plasma membrane
by direct penetration of the plasma membrane, endocytosis-mediated
entry, or the formation of a transitory structure. In some
embodiments the MTS is not transported across the membrane of a
cell, but is employed in an ex vivo assay or application.
As used herein, the term "aptamer" refers to a DNA or RNA molecule
that has been selected from random pools based on their ability to
bind other molecules with high affinity specificity based on
non-Watson and Crick interactions with the target molecule (see,
e.g., Cox and Ellington, Bioorg. Med. Chem. 9:2525-2531 (2001); Lee
et al., Nuc. Acids Res. 32:D95-D100 (2004)). In some embodiments,
the aptamer binds nucleic acids, proteins, small organic compounds,
vitamins, inorganic compounds, cells, and even entire
organisms.
The terms "polypeptide," "peptide" and "protein" and derivatives
thereof as used herein, are used interchangeably herein to refer to
a polymer of amino acid residues. The terms apply to naturally
occurring amino acid polymers as well as amino acid polymers in
which one or more amino acid residues is a non-naturally occurring
amino acid (e.g., an amino acid analog). The terms encompass amino
acid chains of any length, including full length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds. As used herein, the terms "peptide" refers to a
polymer of amino acid residues typically ranging in length from 2
to about 50 residues. In certain embodiments the peptide ranges in
length from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 50,
45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments the
peptide ranges in length from about 8, 9, 10, 11, or 12 residues to
about 15, 20 or 25 residues. Where an amino acid sequence is
provided herein, L-, D-, or beta amino acid versions of the
sequence are also contemplated as well as retro, inversion, and
retro-inversion isoforms. Peptides also include amino acid polymers
in which one or more amino acid residues is an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers. In addition, the
term applies to amino acids joined by a peptide linkage or by other
modified linkages (e.g., where the peptide bond is replaced by an
.alpha.-ester, a .beta.-ester, a thioamide, phosphonamide,
carbamate, hydroxylate, and the like (see, e.g., Spatola, Chem.
Biochem. Amino Acids and Proteins 7: 267-357 (1983)), where the
amide is replaced with a saturated amine (see, e.g., Skiles et al.,
U.S. Pat. No. 4,496,542, which is incorporated herein by reference,
and Kaltenbronn et al., (1990) Pp. 969-970 in Proc. 11th American
Peptide Symposium, ESCOM Science Publishers, The Netherlands, and
the like)).
The term "amino acid" and derivatives thereof as used herein,
refers to naturally occurring and synthetic amino acids, as well as
amino acid analogs and amino acid mimetics that function in a
manner similar to the naturally occurring amino acids. Naturally
occurring amino acids are those encoded by the genetic code, as
well as those amino acids that are later modified, e.g.,
hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, i.e., an
.alpha. carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide. Such analogs have modified R groups (e.g.,
norleucine) or modified peptide backbones, but retain the same
basic chemical structure as a naturally occurring amino acid. Amino
acid mimetics refers to chemical compounds that have a structure
that is different from the general chemical structure of an amino
acid, but that functions in a manner similar to a naturally
occurring amino acid. Amino acids may be either D amino acids or L
amino acids. In peptide sequences throughout the specification,
lower case letters indicate the D isomer of the amino acid
(conversely, upper case letters indicate the L isomer of the amino
acid).
Amino acids may be referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
One of skill will recognize that individual substitutions,
deletions or additions to a peptide, polypeptide, or protein
sequence which alters, adds or deletes a single amino acid or a
small percentage of amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. Such conservatively
modified variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
As used herein, a "linker" is any molecule capable of binding
(e.g., covalently) portion A and portion B of a MTS molecule
disclosed herein. Linkers include, but are not limited to, straight
or branched chain carbon linkers, heterocyclic carbon linkers,
peptide linkers, and polyether linkers. For example, poly(ethylene
glycol) linkers are available from Quanta Biodesign, Powell, Ohio.
These linkers optionally have amide linkages, sulfhydryl linkages,
or heterofunctional linkages.
As used herein, the term "label" refers to any molecule that
facilitates the visualization and/or detection of a MTS molecule
disclosed herein. In some embodiments, the label is a fluorescent
moiety.
The term "carrier" means an inert molecule that increases (a)
plasma half-life and (b) solubility. In some embodiments, a carrier
increases plasma half-life and solubility by reducing glomerular
filtration. In some embodiments, a carrier increases tumor uptake
due to enhanced permeability and retention (EPR) of tumor
vasculature.
The term "thrombin" means an enzyme (EC 3.4.21.5) that cleaves
fibrinogen molecules into fibrin monomers. Thrombin, acting through
its G-protein coupled receptor PAR-I, is a key player in a wide
range of vascular and extravascular disease processes throughout
the body, including cancer, cardiovascular diseases, acute kidney
injury, and stroke. In certain instances, thrombin activity
increases over the course of atherosclerotic plaque development. In
some embodiments, thrombin activity is a biomarker for
atherosclerotic plaque development.
The terms "individual," "patient," or "subject" are used
interchangeably. As used herein, they mean any mammal (i.e. species
of any orders, families, and genus within the taxonomic
classification animalia: chordata: vertebrata: mammalia). In some
embodiments, the mammal is a human. None of the terms require or
are limited to situation characterized by the supervision (e g
constant or intermittent) of a health care worker (e g a doctor, a
registered nurse, a nurse practitioner, a physician's assistant, an
orderly, or a hospice worker).
As used herein, the term "medical professional" means any health
care worker. By way of non-limiting example, the health care worker
may be a doctor, a registered nurse, a nurse practitioner, a
physician's assistant, an orderly, or a hospice worker.
The terms "administer," "administering," "administration," and
derivatives thereof as used herein, refer to the methods that may
be used to enable delivery of agents or compositions to the desired
site of biological action These methods include, but are not
limited to parenteral injection (e g, intravenous, subcutaneous,
intraperitoneal, intramuscular, intravascular, intrathecal,
intravitreal, infusion, or local) Administration techniques that
are optionally employed with the agents and methods described
herein, include e g, as discussed in Goodman and Gilman, The
Pharmacological Basis of Therapeutics, current ed, Pergamon, and
Remington's, Pharmaceutical Sciences (current edition), Mack
Publishing Co, Easton, Pa.
The term "pharmaceutically acceptable" and derivatives thereof as
used herein, refers to a material that does not abrogate the
biological activity or properties of the agents described herein,
and is relatively nontoxic (i e, the toxicity of the material
significantly outweighs the benefit of the material) In some
instances, a pharmaceutically acceptable material may be
administered to an individual without causing significant
undesirable biological effects or significantly interacting in a
deleterious manner with any of the components of the composition in
which it is contained.
The term "surgery" and derivatives thereof as used herein, refers
to any methods for that may be used to manipulate, change, or cause
an effect by a physical intervention These methods include, but are
not limited to open surgery, endoscopic surgery, laparoscopic
surgery, minimally invasive surgery, and robotic surgery.
The terms "neoplasm" or "neoplasia" and derivatives thereof as used
herein, include any non-normal or non-standard cellular growth.
Neoplasms can include tumors and cancers of any variety of stages,
from benign to metastatic. Neoplasms can be primary or metastatic
growths and can occur anywhere in a subject. Neoplasms can include
neoplasms of the lung, skin, lymph, brain, nerves, muscle, breast,
prostate, testis, pancreases, liver, kidneys, stomach, muscle, bone
and blood. Neoplasms can be solid and non-solid tumors.
The terms "sample" or "samples" and derivatives thereof as used
herein, include any samples obtained from a subject with can be
employed with the methods described herein. Samples can include but
are not limited to urine, blood, lymph, tears, mucus, saliva,
biopsy or other sample tissue samples. Sample can be frozen,
refrigerated, previously frozen, and/or stored for minutes, hours,
days, weeks, months, years. Sampling techniques, handling and
storage are well known and any such techniques for obtaining
samples for use with the present invention are contemplated.
The following symbols, where used, are used with the indicated
meanings Fl=fluorescein, aca=ahx=X=ammohexanoyl linker
(--HN--(CH2)<rCO-)aminohexanoyl, C=L-cysteine, E=L-glutamate,
R=L-arginme, D=L-aspartate, K=L-lysine, A=L-alanine, r=D-arginine,
c=D-cysteine, e=D-glutamate, P=L-proline, L=L-leucine, G=glycine,
V=valine, I=isoleucine, M=methionine, F==phenylalanine, Y=tyrosine,
W=tryptophan, H=histidine, Q=glutamine, N=asparagine, S=serine,
T=threonine, o is 5-amino-3-oxapentanoyl linker, and C(me) is
S-methylcysteine.
Methods of Use
The MTS molecules find use in a variety of ex vivo applications as
described herein and such MTS molecules have been thoroughly
described (see, WO 2005/042034, WO/2006/125134, WO2011008992 and
WO2011008996; all of which are incorporated herein by reference in
their entireties). As such, according to disclosure contained
herein, this invention pertains to methods and compositions that
find use in diagnostic, prognostic (e.g., patient prognosis) and
characterization (e.g., histologic grade/stage) of neoplasm samples
based on the ability of a tumor sample to cleave a MTS molecule of
the present invention.
Methods of use and compositions comprising MTS molecules are
disclosed. Molecules having features of the invention include
peptide portions linked by a cleavable linker portion which may be
a peptide. The inventors have found that these MTS molecules can
find use in diagnostic, detection, screening, prognosis (e.g.,
patient prognosis) and characterization (e.g., histologic
grade/stage) assays.
According to the present invention, such methods are based in part
on cleavage of the MTS molecule and detection of that cleavage
event. The presence of one or more proteases in a sample from a
subject can be detected ex vivo based on cleavage of the peptide.
Such cleavage is detected by detecting a change in a detectable
label (detectable moiety) that is part of the MTS peptide. In some
embodiments, the MTS molecule contains a detectable moieties which
provide for an indication of a cleavage event. In some embodiments,
cleavage could be detected by size changes in the length of the
peptide (e.g., gel electrophoresis, size exclusion, column
chromatography, immunoflourescence, etc.) or other biochemical and
physical changes that occur to the MTS molecule. In some
embodiments, the MTS molecule comprises a label which facilitates
cleavage detection. In some embodiments, cleavage could be detected
using a FRET-based pair (a reporter dye and an acceptor dye that
are involved in fluorescence resonance energy transfer known as
FRET), where a change in fluorescence is indicative of a cleavage
event. See, for examples, Examples 1-3. Methods for detecting and
monitoring cleavage of proteins are well known and any such methods
could be employed in detecting cleavage of the MTS molecules of the
invention.
In some embodiments, the invention provides an ex vivo method for
detecting the presence of one or more protease activities in a
neoplasia sample comprising a) combining ex vivo said sample from a
subject with a molecule of the structure A-X-B-C, wherein cleavage
of said A-X-B-C is indicative of the presence of protease activity
and wherein B is a peptide portion of about 5 to about 20 basic
amino acid residues, which is suitable for cellular uptake, A is a
peptide portion of about 2 to about 20 acidic amino acid residues,
which when linked with portion B is effective to inhibit or prevent
cellular uptake of portion B, and X is a cleavable linker of about
2 to about 100 atoms joining A with B, where X is cleavable under
physiological conditions, and C is a detectable moiety; and b)
detecting cleavage of A-X-B-C by detecting a change in said
detectable moiety C, wherein said change in C is indicative of
cleavage and said cleavage is indicative of the presence of one or
more protease activities in said neoplasia. In some embodiments,
one protease activity can be detected. In some embodiments, 2, 3,
4, 5, 6, 7, 8, 9 or 10 protease activities can be detected. In some
embodiments, one or more protease activities can be detected.
In some embodiments, the invention provides an ex vivo method of
screening for the presence of one or more protease activities in a
neoplasia sample comprising combining ex vivo said neoplasia sample
from a subject with a molecule of the structure A-X-B-C, wherein B
is a peptide portion of about 5 to about 20 basic amino acid
residues, which is suitable for cellular uptake, A is a peptide
portion of about 2 to about 20 acidic amino acid residues, which
when linked with portion B is effective to inhibit or prevent
cellular uptake of portion B, and X is a cleavable linker of about
2 to about 100 atoms joining A with B, where X is cleavable under
physiological conditions, and C is a detectable moiety; and b)
detecting cleavage of A-X-B-C by detecting a change in said
detectable moiety C, wherein said change in C is indicative of
cleavage and said cleavage is indicative of the presence of one or
more protease activities in said neoplasia. In some embodiments the
MTS molecules can be used in screening assays to determine how many
proteases and/or which proteases are expressed by a sample. In some
embodiments, the screening is small scale, involving screening of
1, 5, 10, 20 or 30 samples. In some embodiments, screening is large
scale, and involves screening of 100, 500, 1000, 10000, 100 000,
500000 or more samples. In some embodiments, samples are screened
for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more protease activities using
MTS molecules of the invention. In some embodiments, screening
information can be employed to develop data bases and incorporated
with other bioinformatic information in order to develop protease
profiles of samples.
In some embodiments, the invention provides an ex vivo method of
determining the protease profile of a neoplasia sample, comprising
a) combining said sample from a subject with a molecule of the
structure A-X-B-C, wherein B is a peptide portion of about 5 to
about 20 basic amino acid residues, which is suitable for cellular
uptake, A is a peptide portion of about 2 to about 20 acidic amino
acid residues, which when linked with portion B is effective to
inhibit or prevent cellular uptake of portion B, and X is a
cleavable linker of about 2 to about 100 atoms joining A with B,
where X is cleavable under physiological conditions and C is a
detectable moiety; and b) detecting cleavage of A-X-B-C by
detecting a change in said detectable moiety C, wherein said change
in C is indicative of cleavage and said cleavage is indicative of
the presence of one or more protease activities in said neoplasia
and wherein the protease profile is developed based on the cleavage
detected. In some embodiments, the MTS molecules are employed to
develop a protease profile for one or more neoplasia samples.
Protease profiles can be employed to develop databases and can be
incorporated with other information, including for example
bioinformatic information, in order to develop protease profiles of
neoplasia samples and for protease profiles for patients with
neoplasia.
In some embodiments, the invention provides An ex vivo method of
determining a treatment regimen based on the protease profile of a
neoplasia sample, comprising a) combining ex vivo said neoplasia
sample from a subject with a molecule of the structure A-X-B-C,
wherein B is a peptide portion of about 5 to about 20 basic amino
acid residues, which is suitable for cellular uptake, A is a
peptide portion of about 2 to about 20 acidic amino acid residues,
which when linked with portion B is effective to inhibit or prevent
cellular uptake of portion B, and X is a cleavable linker of about
2 to about 100 atoms joining A with B, where X is cleavable under
physiological conditions and C is a detectable moiety; and b)
detecting cleavage of A-X-B-C by detecting a change in detectable
moiety C, wherein said change in C is indicative of cleavage and
said cleavage is indicative of the presence of one or more protease
activities and wherein the presence and/or absence of one or more
protease activities allows for determining a medical treatment
regimen. In some embodiments, the MTS molecules are employed to
determine a treatment regimen. Protease information and/or protease
profiles can employed to develop databases and can be incorporated
with other information, for example bioinformatic information, in
order to develop protease profiles of samples. In some embodiments,
such information can be combined with information regarding
treatment and surgical options know to those of skill in the
medical arts in order to determine and develop personalize
treatment regimens for individual subjects. In some embodiments,
the medical regimen is a surgical regimen. After detecting the
presence or absence of one or more proteases based on MTS molecule
cleavage, a determination of the usefulness of an MTS molecule in
surgical procedures can be determined. Detection of cleavage of the
MTS molecule would be indicative of the presence of one or more
proteases and such information would allow for a determination of
usefulness of the peptide in a surgical procedure in order to
detect tumor borders and assist with surgical removal as previously
described (See, e.g., see, WO 2005/042034, WO/2006/125134,
WO2011008992 and WO2011008996). Non-detection of cleavage of the
MTS molecule would be indicative of the absence of a protease and
the non-usefulness of the peptide in a surgical procedure.
In some embodiments, the invention provides an ex vivo method of
characterizing a neoplasia based on the protease profile of said
neoplasia, comprising a) combining a sample of said neoplasia from
a subject with a molecule of the structure A-X-B-C, wherein B is a
peptide portion of about 5 to about 20 basic amino acid residues,
which is suitable for cellular uptake, A is a peptide portion of
about 2 to about 20 acidic amino acid residues, which when linked
with portion B is effective to inhibit or prevent cellular uptake
of portion B, and X is a cleavable linker of about 2 to about 100
atoms joining A with B, where X is cleavable under physiological
conditions and C is a detectable moiety; and detecting cleavage of
A-X-B-C by detecting a change is said detectable moiety C, wherein
said change in C is indicative of cleavage and said cleavage is
indicative of the presence of more than one protease activities and
wherein the characterization of the neoplasia is based on the
cleavage detected. In some embodiments, the neoplasia is
characterized based on histology, stage, grade, location, type, or
any of a variety of characteristics known to those skilled in the
medical arts. In some embodiments, the protease profile is
correlated with histology, stage, grade, location, type, or any of
a variety of characteristics known to those skilled in the medical
arts in order to characterize the neoplasia. In some embodiments,
the presence of the protease activity is indicative of neoplasia.
In some embodiments, the presence of the protease activity is
indicative of metastasis.
In some embodiments, the present invention provides a diagnostic
composition for use in the methods of any of the preceding claims
comprising: a molecule of the structure A-X-B-C, wherein B is a
peptide portion of about 5 to about 20 basic amino acid residues,
which is suitable for cellular uptake, A is a peptide portion of
about 2 to about 20 acidic amino acid residues, which when linked
with portion B is effective to inhibit or prevent cellular uptake
of portion B, and X is a cleavable linker of about 3 to about 30
atoms joining A with B, where X is a cleavable under physiological
conditions and C is a detectable moiety; and a diagnostic buffering
agent. In some embodiments of the diagnostic composition, the
cleavable linker X is of between about 6 to about 30 atoms in
length, said portion A has between about 5 to about 9 acidic amino
acid residues, and said portion B has between about 9 to about 16
basic amino acid residues.
In some embodiments, the present invention provides an array
comprising: a plurality of molecules of the structure A-X-B-C,
wherein B is a peptide portion of about 5 to about 20 basic amino
acid residues, which is suitable for cellular uptake, A is a
peptide portion of about 2 to about 20 acidic amino acid residues,
which when linked with portion B is effective to inhibit or prevent
cellular uptake of portion B, and X is a cleavable linker of about
3 to about 30 atoms joining A with B, where X is a cleavable under
physiological conditions, and C is a detectable moiety. In some
embodiments of the array, the cleavable linker X is of between
about 6 to about 30 atoms in length, said portion A has between
about 5 to about 9 acidic amino acid residues, and said portion B
has between about 9 to about 16 basic amino acid residues. In some
embodiments, the array comprises a plurality of molecules of the
structure A-X-B and wherein the cleavable linker X comprises a
plurality of cleavable linkers X. In some embodiments of the array,
the plurality of cleavable linkers X linking a portion A to a
structure B-C are cleavable by a single protease. In some
embodiments of the array, the plurality of cleavable linkers X
linking a portion A to a structure B-C are cleavable by more than
one protease. In some embodiments, an array of the invention would
contain a plurality of one species (one type) of MTS molecules. In
some embodiments, an array of the invention would contain a
plurality of one species (one type) of MTS molecules and multiple
samples could be screened for one protease activity type. In some
embodiments, an array of the invention would contain a plurality of
a plurality of species (multiple types) of MTS molecules. In some
embodiments, an array of the invention would contain a plurality of
a plurality of species (multiple types) of MTS molecules and one or
more samples could be screened for one or more protease activity
types. An array can include but is not limited to any substrate to
which the MTS molecules can be bound, and can include for examples
solid substrates, micro-arrays and microchips. Methods for making
arrays are well known and can even be supplied by commercial
suppliers. Arrays can be manually processed and/or automated or a
combination thereof. Such arrays can be employed in low-throughput
as well as high-throughput applications and can analyze one or more
samples, one or more proteases or any combination thereof.
In some embodiments of the above described methods, ratiometric
analysis can be employed to determine the level of enzyme activity
and/or to assess the percentage of enzymatically positive tumors in
a population. Such ratiometric analyses can be based on the ratio
of cleaved to non-cleaved MTS molecules. In some embodiments,
ratiometric analysis can be employed to correlate ex vivo cleavage
with in vivo cleavage activities.
In some embodiments, the protease information can be correlated
with histology, grade, type, characterization, etc. in order to
better characterize neoplasias and to provide personalized
prognosis and treatment regimens. Such information can be provided
to those of skill in the medical arts and be employed to develop
personalized medical treatment regimens for individuals.
MTS Peptides
In one embodiment, a generic structure for peptides having features
of the invention is A-X-B, where peptide portion B includes between
about 5 to about 20 basic amino acids, X is a cleavable linker
portion, in some embodiments cleavable under physiological
conditions, and where peptide portion A includes between about 2 to
about 20 acidic amino acids. In some embodiments of molecules
having features of the invention, peptide portion B includes
between about 5 to about 20, or between about 9 to about 16 basic
amino acids, and may be a series of basic amino acids (e.g.,
arginines, histidines, lysines, or other basic amino acids). In
some embodiments of molecules having features of the invention,
peptide portion A includes between about 2 to about 20, or between
about 5 to about 20 acidic amino acids, and may be series of acidic
amino acids (e.g., glutamates and aspartates or other acidic amino
acids). A schematic representation of a MTS molecule having
features of the invention comprising a basic portion B, a linker
portion X, and an acidic portion A is presented in FIG. 1A of WO
2005/042034. In embodiments, MTS molecules having features of the
invention may be cyclic molecules, as schematically illustrated in
FIG. 1B of WO 2005/04203. Thus, MTS molecules having features of
the invention may be linear molecules, cyclic molecules, or may be
linear molecules including a cyclic portion.
In some embodiments, a MTS molecule disclosed herein has the
formula A-X-B-C, wherein C is a cargo moiety (including for example
a detectable moiety); A is a peptide with a sequence comprising 5
to 9 consecutive acidic amino acids, wherein the amino acids are
selected from: aspartates and glutamates; B is a peptide with a
sequence comprising 5 to 20 consecutive basic amino acids; and X is
a linker that is cleavable by protease.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B-Qn-M, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; and M is a macromolecular carrier.
Regulation of transport into and out of a cell is important for its
continued viability. For example, cell membranes contain ion
channels, pumps, and exchangers capable of facilitating the
transmembrane passage of many important substances. However,
transmembrane transport is selective in addition to facilitating
the entry of desired substances into a cell, and facilitating the
exit of others, a major role of a cell membrane is to prevent
uncontrolled entry of substances into the cell interior. This
barrier function of the cell membrane makes difficult the delivery
of markers, drugs, nucleic acids, and other exogenous material into
cells.
As discussed above, molecules including a multiple basic amino
acids, such as a series of basic amino acids, are often taken up by
cells. However, the present inventors have discovered that
molecules having structures including a basic portion B, a linker
portion X, and an acidic portion A are not taken up by cells. An
acidic portion A may include amino acids that are not acidic.
Acidic portion A may comprise other moieties, such as negatively
charged moieties. In embodiments of MTS molecules having features
of the invention, an acidic portion A may be a negatively charged
portion, in some embodiments having about 2 to about 20 negative
charges at physiological pH, that does not include an amino acid. A
basic portion B may include amino acids that are not basic. Basic
portion B may comprise other moieties, such as positively charged
moieties. In embodiments of MTS molecules having features of the
invention, a basic portion B may be a positively charged portion,
having between about 5 and about 20 positive charges at
physiological pH, that does not include an amino acid. Including an
acidic portion A is effective to inhibit or prevent the uptake of a
portion B into cells. Such a block of uptake that would otherwise
be effected by the basic amino acids of portion B may be termed a
"veto" of the uptake by the acidic portion A. The present inventors
have made the further surprising discovery that cleavage of linker
X, allowing the separation of portion A from portion B is effective
to allow the uptake of portion B into cells.
In a further embodiment, a generic structure for peptides having
features of the invention is A-X-B-C, where C is a cargo moiety, X
a linker, A an acidic portion, and B a basic portion. An acidic
portion A may include amino acids that are not acidic. Acidic
portion A may comprise other moieties, such as negatively charged
moieties. In embodiments of MTS molecules having features of the
invention, an acidic portion A may be a negatively charged portion,
having about 2 to about 20 negative charges at physiological pH,
that does not include an amino acid. A basic portion B may include
amino acids that are not basic. Basic portion B may comprise other
moieties, such as positively charged moieties. In embodiments of
MTS molecules having features of the invention, a basic portion B
may be a positively charged portion, having between about 5 and
about 20 positive charges at physiological pH, that does not
include an amino acid. In some embodiments, the amount of negative
charge in portion A is approximately the same as the amount of
positive charge in portion B.
A cargo moiety C may be, for example, a variety of detectable
agents, including for example any detectable moiety for detection
in an ex vivo assay, a contrast agent for diagnostic imaging, or a
chemotherapeutic drug or radiation-sensitizer for therapy. B may
be, for example, a peptide portion having between about 5 to about
20 basic amino acids, such as a series of basic amino acids
(arginines are can be employed, as well as histidines, lysines or
other basic amino acids). In some embodiments, X is a cleavable
linker that is cleavable under physiological conditions. A may be a
peptide portion having between about 2 to about 20 about 2 to about
20 acidic amino acids, such as a series of acidic amino acids. In
some embodiments of molecules having features of the invention,
glutamates and aspartates are employed as acidic amino acids for
peptide portion A.
The present inventors have made the surprising discovery that
including an acidic portion A is also effective to inhibit or
prevent the uptake into cells of molecules combining a portion B
and a portion C. The present inventors have made the further
discovery that cleavage of linker X, allowing the separation of
portion A from portion B is effective to allow the uptake of
portions B and C into cells. Thus, delivery of cargo C can be
controlled and enhanced by molecules having features of the
invention.
For example, when peptide portion A contains about 5 to about 9
consecutive glutamates or aspartates, and X is a flexible linker of
about 2 to about 100, or about 6 to about 30 atoms in length, the
normal ability of a peptide portion B (e.g., a sequence of nine
consecutive arginine residues) to cause uptake into cells is
blocked. Cleavage of linker X allows the separation of portion A
from portion B and portion C, alleviating the veto by portion A.
Thus, when separated from A, the normal ability of portion B to
effect the uptake of cargo C into cells is regained. Such cellular
uptake typically occurs near the location of the cleavage event.
Thus, design of cleavable linker X such that it is cleaved at or
near a target cell is effective to direct uptake of cargo C into
target cells. Extracellular cleavage of X allows separation of A
from the rest of the molecule to allow uptake into cells.
A MTS molecule having features of the invention may be of any
length. In embodiments of MTS molecules having features of the
invention, a MTS molecule may be about 7 to about 40 amino acids in
length, not including the length of a linker X and a cargo portion
C. In other embodiments, particularly where multiple non-acidic (in
portion A) or non-basic (in portion B) amino acids are included in
one or both of portions A and B, portions A and B of a MTS molecule
may together be about 50, or about 60, or about 70 amino acids in
length. A cyclic portion of an MTS may include about 12 to about 60
amino acids, not including the length of a linker X and a cargo
portion C. For example, a linear MTS molecule having features of
the invention may have a basic portion B having between about 5 to
about 20 basic amino acids (in some embodiments between about 9 to
about 16 basic amino acids) and an acidic portion A having between
about 2 to about 20 acidic amino acids (e.g., between about 5 to
about 20, between about 5 to about 9 acidic amino acids). In some
embodiments, a MTS molecule having features of the invention may
have a basic portion B having between about 9 to about 16 basic
amino acids and between about 5 to about 9 acidic amino acids.
Portions A and B may include either L-amino acids or D-amino acids.
In embodiments of the invention, D-amino acids are employed for the
A and B portions in order to minimize immunogenicity and
nonspecific cleavage by background peptidases or proteases.
Cellular uptake of oligo-D-arginine sequences is known to be as
good or better than that of oligo-L-arginines. The generic
structures A-X-B and -A-X-B C can be effective where A is at the
amino terminus and where A is at the carboxy terminus, i.e. either
orientation of the peptide bonds is permissible. However, in
embodiments where X is a peptide cleavable by a protease, it may be
beneficial to join the C-terminus of X to the N-terminus of B, so
that the new amino terminus created by cleavage of X contributes an
additional positive charge that adds to the positive charges
already present in B.
In some embodiments, a MTS molecule disclosed herein has the
formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with a
sequence comprising 5 to 9 consecutive acidic amino acids, wherein
the amino acids are selected from: aspartates and glutamates; B is
a peptide with a sequence comprising 5 to 20 consecutive basic
amino acids; and X is a linker that is cleavable by thrombin. In
some embodiments, the acid amino acids are consecutive. In some
embodiments, the acid amino acids are not consecutive.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B-C)n-M, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; M is a macromolecular carrier; and n is an integer
between 1 and 20.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B)n-D, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; D is a dendrimer; and n is an integer between 1 and 20.
In some embodiments, D comprises a cargo moiety.
In some embodiments of molecules having features of the invention,
peptide portion A includes between about 2 to about 20, or between
about 5 to about 20 acidic amino acids, and may be series of acidic
amino acids (e.g., glutamates and aspartates or other acidic amino
acids). In some embodiments, A has a sequence comprising 5 to 9
consecutive glutamates. In some embodiments, portion A comprises 8
consecutive glutamates (i.e., EEEEEEEE (SEQ ID NO: 96) or
eeeeeeee).
An acidic portion A may include amino acids that are not acidic.
Acidic portion A may comprise other moieties, such as negatively
charged moieties. In embodiments of a MTS molecule disclosed
herein, an acidic portion A may be a negatively charged portion, in
some embodiments having about 2 to about 20 negative charges at
physiological pH that does not include an amino acid. In some
embodiments, the amount of negative charge in portion A is
approximately the same as the amount of positive charge in portion
B.
Portion A is either L-amino acids or D-amino acids. In embodiments
of the invention, D-amino acids are can be employed in order to
minimize immunogenicity and nonspecific cleavage by background
peptidases or proteases. Cellular uptake of oligo-D-arginine
sequences is known to be as good as or better than that of
oligo-L-arginines.
It will be understood that portion A may include non-standard amino
acids, such as, for example, hydroxylysine, desmosine,
isodesmosine, or other non-standard amino acids. Portion A may
include modified amino acids, including post-translationally
modified amino acids such as, for example, methylated amino acids
(e.g., methyl histidine, methylated forms of lysine, etc.),
acetylated amino acids, amidated amino acids, formylated amino
acids, hydroxylated amino acids, phosphorylated amino acids, or
other modified amino acids. Portion A may also include peptide
mimetic moieties, including portions linked by non-peptide bonds
and amino acids linked by or to non-amino acid portions.
The generic structures A-X-B and -A-X-B-C is effective where A is
at the amino terminus or where A is at the carboxy terminus, i.e.,
either orientation of the peptide bonds is permissible.
In some embodiments, a MTS molecule disclosed herein has the
formula A-X-B-C, wherein C is a cargo moiety, A is a peptide with a
sequence comprising 5 to 9 consecutive acidic amino acids, wherein
the amino acids are selected from: aspartates and glutamates; B is
a peptide with a sequence comprising 5 to 20 consecutive basic
amino acids; and X is a linker that is cleavable by thrombin.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B-C)n-M, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; M is a macromolecular carrier; and n is an integer
between 1 and 20.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B)n-D, wherein C is a cargo moiety, A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; D is a dendrimer; and n is an integer between 1 and 20.
In some embodiments, D comprises a cargo moiety.
In some embodiments of molecules having features of the invention,
peptide portion B includes between about 5 to about 20, or between
about 9 to about 16 basic amino acids, and may be a series of basic
amino acids (e.g., arginines, histidines, lysines, or other basic
amino acids). In some embodiments, portion B comprises 9
consecutive arginines (i.e., RRRRRRRRR (SEQ ID NO: 97) or
rrrrrrrrr). In some embodiments, the basic amino acids are
consecutive. In some embodiments, the basic amino acids are not
consecutive.
A basic portion B may include amino acids that are not basic. Basic
portion B may comprise other moieties, such as positively charged
moieties. In embodiments, a basic portion B may be a positively
charged portion, having between about 5 and about 20 positive
charges at physiological pH, that does not include an amino acid.
In some embodiments, the amount of negative charge in portion A is
approximately the same as the amount of positive charge in portion
B.
Portion B is either L-amino acids or D-amino acids. In embodiments
of the invention, D-amino acids are employed in order to minimize
immunogenicity and nonspecific cleavage by background peptidases or
proteases. Cellular uptake of oligo-D-arginine sequences is known
to be as good as or better than that of oligo-L-arginines.
It will be understood that portion B may include non-standard amino
acids, such as, for example, hydroxylysine, desmosine,
isodesmosine, or other non-standard amino acids. Portion B may
include modified amino acids, including post-translationally
modified amino acids such as, for example, methylated amino acids
(e.g., methyl histidine, methylated forms of lysine, etc.),
acetylated amino acids, amidated amino acids, formylated amino
acids, hydroxylated amino acids, phosphorylated amino acids, or
other modified amino acids. Portion B may also include peptide
mimetic moieties, including portions linked by non-peptide bonds
and amino acids linked by or to non-amino acid portions.
In embodiments where X is a peptide cleavable by a protease, it may
be beneficial to join the C-terminus of X to the N-terminus of B,
so that the new amino terminus created by cleavage of X contributes
an additional positive charge that adds to the positive charges
already present in B.
Cargo portion C may be attached to B in any location or
orientation. A cargo portion C need not be located at an opposite
end of portion B than a linker X. Any location of attachment of C
to B is acceptable as long as that attachment remains after X is
cleaved. For example, a cargo portion C may be attached to or near
to an end of portion B with linker X attached to an opposite end of
portion B. A cargo portion C may also be attached to or near to an
end of portion B with linker X attached to or near to the same end
of portion B. In some embodiments of the invention, a linker X may
link to a cargo portion C which is linked to a basic portion B
where a MTS molecule having features of the invention comprising a
cargo portion C linked to multiple basic portions B, each of which
basic portions B are linked to a linker portion X, and via the
linker to an acidic portion A.
A linker X may be designed for cleavage in the presence of
particular conditions or in a particular environment. In some
embodiments, a linker X is cleavable under physiological
conditions. Cleavage of such a linker X may, for example, be
enhanced or may be effected by particular pathological signals or a
particular environment related to cells in which cargo delivery is
desired. The design of a linker X for cleavage by specific
conditions, such as by a specific enzyme, allows the targeting of
cellular uptake to a specific location where such conditions
obtain. Thus, one important way that MTS molecules having features
of the invention provide specific detection of specific proteases
presence is by the design of the linker portion X to be cleaved by
the protease. The linker portion X can be designed to be cleaved
only by specific proteases or to be selective for specific
proteases. After cleavage of a linker X, the portions B-C of the
molecule are then a simple conjugate of B and C, in some instances
retaining a relatively small, inert stub remaining from a residual
portion of linker X.
A linker portion X may be cleavable by conditions found in the
extracellular environment, such as acidic conditions which may be
found near cancerous cells and tissues or a reducing environment,
as may be found near hypoxic or ischemic cells and tissues; by
proteases or other enzymes found on the surface of cells or
released near cells having a condition to be treated, such as
diseased, apoptotic or necrotic cells and tissues; or by other
conditions or factors. An acid-labile linker may be, for example, a
cis-aconitic acid linker. A linker portion X may also be cleaved
extracellularly in an ex vivo reaction. Other examples of
pH-sensitive linkages include acetals, ketals, activated amides
such as amides of 2,3-dimethylmaleamic acid, vinyl ether, other
activated ethers and esters such as enol or silyl ethers or esters,
imines, iminiums, enamines, carbamates, hydrazones, and other
linkages. A linker X may be an amino acid or a peptide. A peptide
linker may be of any suitable length, such as, for example, about 3
to about 30, or about 6 to about 24 atoms in sequence (e.g., a
linear peptide about 1 to 10 or about 2 to 8 amino acids long). A
cleavable peptide linker may include an amino acid sequence
recognized and cleaved by a protease, so that proteolytic action of
the protease cleaves the linker X.
In some embodiments, X is a cleavable linker. In some embodiments,
a linker X is designed for cleavage in the presence of particular
conditions or in a particular environment In some embodiments, a
linker X is cleavable under physiological conditions Cleavage of
such a linker X may, for example, be enhanced or may be affected by
particular pathological signals or a particular environment related
to cells in which cargo delivery is desired The design of a linker
X for cleavage by specific conditions, such as by a specific enzyme
(e g, thrombin), allows the targeting of cellular uptake to a
specific location where such conditions obtain Thus, one important
way that MTS molecules provide specific targeting of cellular
uptake to desired cells, tissues, or regions is by the design of
the linker portion X to be cleaved by conditions near such targeted
cells, tissues, or regions After cleavage of a linker X, the
portions B-C of the molecule are then a simple conjugate of B and
C, in some instances retaining a relatively small, inert stub
remaining from a residual portion of linker X.
In some embodiments, X is cleaved by thrombin. In some embodiments,
X is substantially specific for thrombin, MMPs or elastates. In
some embodiments, X is cleaved by or is substantially specific for
MMPs (PLGLAG (SEQ ID NO: 1) and PLGC(met)AG (SEQ ID NO: 2),
RSHP(Hfe)TLY (SEQ ID NO: 19), RSHG(Hfe)FLY (SEQ ID NO: 20), SNPYK-Y
(SEQ ID NO: 21), SNPKG-Y (SEQ ID NO: 22), SNPYG-Y (SEQ ID NO: 23),
TLSE-LH (SEQ ID NO: 24), TIAHLA (SEQ ID NO: 25)), elastases
(RLQLK(acetyl)L (SEQ ID NO: 26), plasmin and/or thrombin, cathepsin
K (KLRFSKQ (SEQ ID NO: 27)). In some embodiments, the MMP 2,9
cleavable or substantially specific sequence is PLGLAG and/or
PLGC(met)AG (SEQ ID NO: 2). In some embodiments, the MMP 14
cleavable or substantially specific sequences could include but are
not limited to RSHP(Hfe)TLY (SEQ ID NO: 19) or RSHG(Hfe)FLY (SEQ ID
NO: 20). In some embodiments, the MMP 9 cleavable or substantially
specific sequences could include but are not limited to SNPYK-Y
(SEQ ID NO: 21), SNPKG-Y (SEQ ID NO: 22), or SNPYG-Y (SEQ ID NO:
23). In some embodiments, the MMP 2 cleavable or substantially
specific sequences could include but are not limited to TLSE-LH
(SEQ ID NO: 24), TIAHLA (SEQ ID NO: 25). In some embodiments, the
cathepsin K cleavable or substantially specific sequences could
include but are not limited to KLRFSKQ (SEQ ID NO: 27). In some
embodiments, the MMP cleavable or substantially specific sequences
could include but are not limited to RS-(Cit)-G-(homoF)-YLY (SEQ ID
NO: 4), CRPAHLRDSG (SEQ ID NO: 5), SLAYYTA (SEQ ID NO: 6), NISDLTAG
(SEQ ID NO: 7), PPSSLRVT (SEQ ID NO; 8), SGESLSNLTA (SEQ ID NO: 9),
RIGFLR (SEQ ID NO: 10) elastase cleavable or substantially specific
sequence is RLQLA(acetyl)L (SEQ ID NO: 11). In some embodiments,
the plasmin cleavable or substantially specific sequence is RLQLKL
(SEQ ID NO: 12). Thrombin selective substrates DPRSFL (SEQ ID NO:
13), PPRSFL (SEQ ID NO: 14), Norleucine-TPRSFL (SEQ ID NO: 15). In
some embodiments, the chymase cleavable or substantially specific
sequence GVAY|SGA (SEQ ID NO: 16). Urokinase-type plasminogen
activator (uPA) and tissue plasminogen activator (tPA) cleavable or
substantially specific sequence is YGRAAA (SEQ ID NO: 17). In some
embodiments, the uPA cleavable or substantially specific sequence
is YGPRNR (SEQ ID NO: 18).
In some embodiments, a linker consisting of one or more amino acids
is used to join peptide sequence A (i.e., the sequence designed to
prevent uptake into cells) and peptide sequence B (i.e., the TS).
Generally the peptide linker will have no specific biological
activity other than to join the molecules or to preserve some
minimum distance or other spatial relationship between them.
However, the constituent amino acids of the linker may be selected
to influence some property of the molecule such as the folding, net
charge, or hydrophobicity.
In some embodiments, the linker is flexible. In some embodiments,
the linker is rigid.
In some embodiments, the linker comprises a linear structure. In
some embodiments, the linker comprises a non-linear structure. In
some embodiments, the linker comprises a branched structure. In
some embodiments, the linker comprises a cyclic structure.
In some embodiments, X is about 5 to about 30 atoms in length. In
some embodiments, X is about 6 atoms in length. In some
embodiments, X is about 8 atoms in length. In some embodiments, X
is about 10 atoms in length. In some embodiments, X is about 12
atoms in length. In some embodiments, X is about 14 atoms in
length. In some embodiments, X is about 16 atoms in length. In some
embodiments, X is about 18 atoms in length. In some embodiments, X
is about 20 atoms in length. In some embodiments, X is about 25
atoms in length. In some embodiments, X is about 30 atoms in
length.
In some embodiments, X is cleaved by thrombin. In some embodiments,
the linker is substantially specific for thrombin.
In some embodiments, the linker has a formula selected from: DPRSFL
(SEQ ID NO: 13), or PPRSFL (SEQ ID NO: 14).
In some embodiments, the linker binds peptide portion A (i.e., the
peptide sequence which prevents cellular uptake) to peptide portion
B (i.e., the MTS sequence) by a covalent linkage. In some
embodiments, the covalent linkage comprises an ether bond,
thioether bond, amine bond, amide bond, carbon-carbon bond,
carbon-nitrogen bond, carbon-oxygen bond, or carbon-sulfur
bond.
In some embodiments, X comprises a peptide linkage. The peptide
linkage comprises L-amino acids and/or D-amino acids. In
embodiments of the invention, D-amino acids are employed in order
to minimize immunogenicity and nonspecific cleavage by background
peptidases or proteases. Cellular uptake of oligo-D-arginine
sequences is known to be as good as or better than that of
oligo-L-arginines.
It will be understood that a linker disclosed herein may include
non-standard amino acids, such as, for example, hydroxylysine,
desmosine, isodesmosine, or other non-standard amino acids. A
linker disclosed herein may include modified amino acids, including
post-translationally modified amino acids such as, for example,
methylated amino acids (e.g., methyl histidine, methylated forms of
lysine, etc.), acetylated amino acids, amidated amino acids,
formylated amino acids, hydroxylated amino acids, phosphorylated
amino acids, or other modified amino acids. A linker disclosed
herein may also include peptide mimetic moieties, including
portions linked by non-peptide bonds and amino acids linked by or
to non-amino acid portions.
In some embodiments, a MTS molecule disclosed herein comprises a
single of linker Use of a single mechanism to mediate uptake of
both imaging and therapeutic cargoes is particularly valuable,
because imaging with noninjurous tracer quantities can be used to
test whether a subsequent therapeutic dose is likely to concentrate
correctly in the target tissue.
In some embodiments, a MTS molecule disclosed herein comprises a
plurality of linkers. Where a MTS molecule disclosed herein
includes multiple linkages X, separation of portion A from the
other portions of the molecule requires cleavage of all linkages X
Cleavage of multiple linkers X may be simultaneous or sequential
Multiple linkages X may include linkages X having different
specificities, so that separation of portion A from the other
portions of the molecule requires that more than one condition or
environment ("extracellular signals") be encountered by the
molecule Cleavage of multiple linkers X thus serves as a detector
of combinations of such extracellular signals For example, a MTS
molecule may include two linker portions Xa and Xb connecting basic
portion B with acidic portion A Both linkers Xa and Xb must be
cleaved before acidic portion A is separated from basic portion B
allowing entry of portion B and cargo moiety C (if any) to enter a
cell It will be understood that a linker region may link to either
a basic portion B or a cargo moiety C independently of another
linker that may be present, and that, where desired, more than two
linker regions X may be included
Combinations of two or more linkers X may be used to further
modulate the detection of multiple proteases with a single MTS
molecule, as well as targeting and delivery of molecules to desired
cells, tissue or regions. Combinations of extracellular signals are
used to widen or narrow the specificity of the cleavage of linkers
X if desired. Where multiple linkers X are linked in parallel, the
specificity of cleavage is narrowed, since each linker X must be
cleaved before portion. A may separate from the remainder of the
molecule. Where multiple linkers X are linked in series, the
specificity of cleavage is broadened, since cleavage on any one
linker X allows separation of portion A from the remainder of the
molecule For example, in order to detect either a protease OR
hypoxia (i. e., to cleave X in the presence of either protease or
hypoxia), a linker X is designed to place the protease-sensitive
and reduction-sensitive sites in tandem, so that cleavage of either
would suffice to allow separation of the acidic portion A
Alternatively, in order to detect the presence of both a protease
AND hypoxia (i e, to cleave X in the presence of both protease and
hypoxia but not in the presence of only one alone), a linker X is
designed to place the protease sensitive site between at least one
pair of cysteines that are disulfide-bonded to each other In that
case, both protease cleavage and disulfide reduction are required
in order to allow separation of portion A.
One important class of signals is the hydrolytic activity of matrix
metalloproteinases (MMPs), which are very important in the invasive
migration of metastatic tumor cells. MMPs are also believed to play
major roles in inflammation and stroke. MMPs are reviewed in Visse
et al., Circ. Res. 92:827-839 (2003). MMPs may be used to cleave a
linker X and so to allow separation of acidic portion A from
portions B and C, allowing cellular uptake of cargo C so that
cellular uptake of C is triggered by action of MMPs. Such uptake is
typically in the vicinity of the MMPs that trigger cleavage of X.
Thus, uptake of molecules having features of the invention are able
to direct cellular uptake of cargo C to specific cells, tissues, or
regions having active MMPs in the extracellular environment.
For example, a linker X that includes the amino-acid sequence
PLGLAG (SEQ ID NO: 1) may be cleaved by the metalloproteinase
enzyme MMP-2 (a major MMP in cancer and inflammation). Cleavage of
such a linker X occurs between the central G and L residues,
causing cell uptake to increase by 10 to 20-fold. A great deal is
known about the substrate preferences of different MMPs, so that
linkers X may be designed that are able to bias X to be
preferentially sensitive to particular subclasses of MMPs, or to
individual members of the large MMP family of proteinases. For
example, in some embodiments, linkers X designed to be cleaved by
membrane-anchored MMPs are particularly employed because their
activity remains localized to the outer surface of the expressing
cell. In alternative embodiments, linkers X designed to be cleaved
by a soluble secreted MMP are employed where diffusion of cargo C
away from the exact location of cleavage may be desired, thereby
increasing the spatial distribution of the cargo. Other linkers X
cleavable by other MMPs are discussed throughout the
application.
Hypoxia is an important pathological signal. For example, hypoxia
is thought to cause cancer cells to become more resistant to
radiation and chemotherapy, and also to initiate angiogenesis. A
linker X suitable for cleavage in or near tissues suffering from
hypoxia enables targeting of portion B and C to cancer cells and
cancerous tissues, infarct regions, and other hypoxic regions. For
example, a linker X that includes a disulfide bond is
preferentially cleaved in hypoxic regions and so targets cargo
delivery to cells in such a region. In a hypoxic environment in the
presence of, for example, leaky or necrotic cells, free thiols and
other reducing agents become available extracellularly, while the
0.sub.2 that normally keeps the extracellular environment oxidizing
is by definition depleted. This shift in the redox balance should
promote reduction and cleavage of a disulfide bond within a linker
X. In addition to disulfide linkages which take advantage of
thiol-disulfide equilibria, linkages including quinones that fall
apart when reduced to hydroquinones may be used in a linker X
designed to be cleaved in a hypoxic environment.
Necrosis often leads to release of enzymes or other cell contents
that may be used to trigger cleavage of a linker X. A linker X
designed for cleavage in regions of necrosis in the absence of
hypoxia, for example, may be one that is cleaved by calpains or
other proteases that may be released from necrotic cells. Such
cleavage of linkers X by calpains would release the connected
portions B-C from portion A, allowing cargo to be taken up by
diseased cells and by neighboring cells that had not yet become
fully leaky.
Acidosis is also commonly observed in sites of damaged or hypoxic
tissue, due to the Warburg shift from oxidative phosphorylation to
anaerobic glycolysis and lactic acid production. Such local acidity
could be sensed either by making an acid-labile linker X (e.g., by
including in X an acetal or vinyl ether linkage). Alternatively, or
in addition, acidosis may be used as a trigger of cargo uptake by
replacing some of the arginines within B by histidines, which only
become cationic below pH 7.
Molecules having features of the invention are suitable for
carrying different cargoes, including different types of cargoes
and different species of the same types of cargo, for uptake into
cells. For example, different types of cargo may include marker
cargoes (e.g., fluorescent or radioactive label moieties) and
therapeutic cargoes (e.g., chemotherapeutic molecules such as
methotrexate or doxorubicin), or other cargoes. Where destruction
of aberrant or diseased cells is therapeutically required, a
therapeutic cargo may include a "cytotoxic agent," i.e. a substance
that inhibits or prevents the function of cells and/or causes
destruction of cells. In some embodiments, a single molecule having
features of the invention may include more than one cargo portion C
so that a basic portion B may be linked to multiple cargoes C. Such
multiple cargoes C may include marker cargoes, therapeutic cargoes,
or other cargoes. Multiple cargo moieties may allow, for example,
delivery of both a radioactive marker and an ultrasound or contrast
agent, allowing imaging by different modalities. Alternatively, for
example, delivery of radioactive cargo along with an anti-cancer
agent, providing enhanced anticancer activity, or delivery of a
radioactive cargo with a fluorescent cargo, allowing multiple means
of localizing and identifying cells which have taken up cargo.
Delivery of cargo such as a fluorescent molecule may be used to
visualize cells having a certain condition or cells in a region
exhibiting a particular condition. For example, thrombosis (clot
formation) may be visualized by designing a linker X to be cleaved
by any of the many proteases in the blood clot formation cascade
for delivery of a cargo including a fluorescent or other marker to
the region. Similarly, complement activation may be visualized by
designing a linker X to be cleaved by any one or more of the
proteases in the complement activation cascades for delivery of a
fluorescent or other marker to the region. Thus, fluorescent
molecules are one example of a marker that may be delivered to
target cells and regions upon release of a portion A upon cleavage
of a linker X.
A molecule having features of the invention may include one or more
linkers X so that an acidic portion A may be linked to portions B
and C by one or more linkages. Such linkages connecting to portion
A may be to portion B, to portion C, or to both portions B and C.
Where a molecule having features of the invention includes multiple
linkages X, separation of portion A from the other portions of the
molecule requires cleavage of all linkages X. Cleavage of multiple
linkers X may be simultaneous or sequential. Multiple linkages X
may include linkages X having different specificities, so that
separation of portion A from the other portions of the molecule
requires that more than one condition or environment
("extracellular signals") be encountered by the molecule. Cleavage
of multiple linkers X thus serves as a detector of combinations of
such extracellular signals. In some embodiments, MTS molecule
having includes two linker portions Xa and Xb connecting basic
portion B with acidic portion A. In some embodiments, a cyclic MTS
molecule includes two linker regions Xa and Xb connecting basic
portion B with acidic portion A. In some embodiments, both linkers
Xa and Xb must be cleaved before acidic portion A is separated from
basic portion B allowing entry of portion B and cargo portion C (if
any) to enter a cell. It will be understood that a linker region
may link to either a basic portion B or a cargo portion C
independently of another linker that may be present, and that,
where desired, more than two linker regions X may be included.
Combinations of two or more linkers X may be used to further
modulate the targeting and delivery of molecules to desired cells,
tissue or regions. Boolean combinations of extracellular signals
can be detected to widen or narrow the specificity of the cleavage
of linkers X if desired. Where multiple linkers X are linked in
parallel, the specificity of cleavage is narrowed, since each
linker X must be cleaved before portion A may separate from the
remainder of the molecule. Where multiple linkers X are linked in
series, the specificity of cleavage is broadened, since cleavage on
any one linker X allows separation of portion A from the remainder
of the molecule. For example, in order to detect either a protease
OR hypoxia (i.e., to cleave X in the presence of either protease or
hypoxia), a linker X is designed to place the protease-sensitive
and reduction-sensitive sites in tandem, so that cleavage of either
would suffice to allow separation of the acidic portion A.
Alternatively, in order to detect the presence of both a protease
AND hypoxia (i.e., to cleave X in the presence of both protease and
hypoxia but not in the presence of only one alone), a linker X is
designed to place the protease sensitive site between at least one
pair of cysteines that are disulfide-bonded to each other. In that
case, both protease cleavage AND disulfide reduction are required
in order to allow separation of portion A.
D amino acids may be used in MTS molecules having features of the
invention. For example, some or all of the peptides of portions A
and B may be D-amino acids in some embodiments of the invention. In
an embodiment of the invention suitable for delivering a detectable
marker to a target cell, a MTS having features of the invention
includes a contrast agent as cargo C attached to a basic portion B
comprising 8 to 10 D-arginines. Acidic portion A may include
D-amino acids as well. Similarly, a drug may be delivered to a cell
by such molecules having a basic portion B including 8 to 10
D-arginines and an acidic portion A including acidic D-amino
acids.
It will be understood that a MTS molecule having features of the
invention may include non-standard amino acids, such as, for
example, hydroxylysine, desmosine, isodesmosine, or other
non-standard amino acids. A MTS molecule having features of the
invention may include modified amino acids, including
post-translationally modified amino acids such as, for example,
methylated amino acids (e.g., methyl histidine, methylated forms of
lysine, etc.), acetylated amino acids, amidated amino acids,
formylated amino acids, hydroxylated amino acids, phosphorylated
amino acids, or other modified amino acids. A MTS molecule having
features of the invention may also include peptide mimetic
moieties, including portions linked by non-peptide bonds and amino
acids linked by or to non-amino acid portions. For example, a MTS
molecule having features of the invention may include peptoids,
carbamates, vinyl polymers, or other molecules having non-peptide
linkages but having an acidic portion cleavably linked to a basic
portion having a cargo moiety.
The linker portion X may be designed so that it is cleaved, for
example, by proteolytic enzymes or reducing environment, as may be
found near cancerous cells. Such an environment, or such enzymes,
are typically not found near normal cells. In some embodiments, a
cleavable linker X is designed to be cleaved near cancerous cells.
In some embodiments, the cleavable linker is not cleaved near
normal tissue. A capable of vetoing cellular uptake of a portion B,
and of a portion B-C, blocking the entry of cargo into normal
tissue.
In some embodiments, the linker portion X may be cleaved, for
example, by proteolytic enzymes or reducing environment found near
cancerous cells to deliver a marker or a drug to cancerous cells.
In some embodiments, a MTS molecule with a cleavable linker X that
is cleaved by proteolytic enzymes or by the reducing environment
near cancer cells is able to facilitate cargo entry into diseased
tissue. Thus, the selective cleavage of the linker X and the
resulting separation of cargo C and basic portion B from acidic
portion A allows the targeted uptake of cargo into cells having
selected features (e.g., enzymes), or located near to, a particular
environment. Thus, molecules having features of the invention are
able to selectively deliver cargo to target cells without doing so
to normal or otherwise non-targeted cells.
In some embodiments, a MTS disclosed herein has the formula
(A-X-B)n-D, wherein C is a cargo moiety; A is a peptide with a
sequence comprising 5 to 9 consecutive acidic amino acids, wherein
the amino acids are selected from: aspartates and glutamates; B is
a peptide with a sequence comprising 5 to 20 consecutive basic
amino acids; X is a linker that is cleavable by thrombin; D is a
dendrimer; and n is an integer between 1 and 20. In some
embodiments, D comprises a cargo moiety.
In embodiments, a MTS molecule disclosed herein is a linear
molecule. In embodiments, a MTS molecule disclosed herein is a
cyclic molecule, as schematically illustrated in FIG. 1B of WO
2011/008996; incorporated herein by reference in its entirety. In
embodiments, a MTS molecule disclosed herein comprises a cyclic
portion and a linear portion.
A MTS disclosed herein may be of any length. In some embodiments, a
MTS molecule disclosed herein is about 7 to about 40 amino acids in
length, not including the length of a linker X and a cargo moiety
C. In other embodiments, particularly where multiple non-acidic (in
portion A) or non-basic (in portion B) amino acids are included in
one or both of portions A and B, portions A and B of a MTS molecule
disclosed herein may together be about 50, or about 60, or about 70
amino acids in length. A cyclic portion of a MTS molecule disclosed
herein may include about 12 to about 60 amino acids, not including
the length of a linker X and a cargo moiety C. For example, a
linear MTS molecule disclosed herein may have a basic portion B
having between about 5 to about 20 basic amino acids (between about
9 to about 16 basic amino acids) and an acidic portion A having
between about 2 to about 20 acidic amino acids (e.g., between about
5 to about 20, between about 5 to about 9 acidic amino acids). In
some particular embodiments, a MTS molecule disclosed herein may
have a basic portion B having between about 9 to about 16 basic
amino acids and between about 5 to about 9 acidic amino acids. In
some embodiments, A is consecutive glutamates (i.e., EEEEEEEE (SEQ
ID NO: 96), E9, eeeeeeee, or e9), B is nine consecutive arginines
(i.e., RRRRRRRRR (SEQ ID NO: 97), R9, rrrrrrrrr, or r9), and X is
PLGLAG (SEQ ID NO: 1).
In some embodiments, the MTS is selected from:
Suc-e9-XDPRSFL-r9-c(Cy5)-CONH2; Suc-e9-ODPRSFL-r9-c(Cy5)-CONH2; and
Suc-e9-Xdprsfl-r9-c(Cy5)-CONH2.
A MTS molecule disclosed herein may be of any length. In some
embodiments, a MTS molecule disclosed herein is about 7 to about 40
amino acids in length, not including the length of a linker X and a
cargo moiety C. In other embodiments, particularly where multiple
non-acidic (in portion A) or non-basic (in portion B) amino acids
are included in one or both of portions A and B, portions A and B
of a MTS molecule disclosed herein may together be about 50, or
about 60, or about 70 amino acids in length. A cyclic portion of a
MTS molecule disclosed herein may include about 12 to about 60
amino acids, not including the length of a linker X and a cargo
moiety.
For example, a linear MTS molecule disclosed herein may have a
basic portion B having between about 5 to about 20 basic amino
acids (in some embodiments between about 9 to about 16 basic amino
acids) and an acidic portion A having between about 2 to about 20
acidic amino acids (e.g., between about 5 to about 20, between
about 5 to about 9 acidic amino acids). In some embodiments, a MTS
molecule disclosed herein may have a basic portion B having between
about 9 to about 16 basic amino acids and between about 5 to about
9 acidic amino acids. In some embodiments, A is 9 consecutive
glutamates (i.e., EEEEEEEE (SEQ ID NO: 96), E9, eeeeeeee, or e9), B
is nine consecutive arginines (i.e., RRRRRRRRR (SEQ ID NO: 97), R9,
rrrrrrrrr, or r9), and X is PLGLAG (SEQ ID NO: 1).
In some embodiments, the MTS molecule has a formula given below. It
should be noted that in some instances the peptide sequence is
given by the amino acid symbol and a number indicating the number
of amino acids (for example, R9 translates to RRRRRRRRR (SEQ ID NO:
97) or nine consecutive L-argmines; and r9 translates to nine
consecutive D-argmines or rrrrrrrrr)
TABLE-US-00001 (SEQ ID NO: 39) EDDDDKA-aca-R9-aca-C(F1)-CONH2 (SEQ
ID NO: 40) Fl-aca-CRRRRRRRRR-aca-EEEEEEEEEC-CONH2 (SEQ ID NO: 41)
Fl-aca-CEEEE-aca-RRRRRRRRRC-CONH2 (SEQ ID NO: 42)
H2N-EEEEEDDDDKA-aca-RRRRRRRRR-aca-C(Fl)-CONH2 (SEQ ID NO: 43)
H2N-EDDDDKA-aca-RRRRRRRRR-aca-C(Fl)-CONH2 (SEQ ID NO: 44)
H2N-EEEEEDDDDK ARRRRRRRRR-aca-C(Fl)-CONH2 (SEQ ID NO: 45)
H2N-EEDDDDKA-aca-rarrarr-aca-C(Fl)-CONH2
H2N-DDDDDDKARRRRRRRRR-aca-C(Fl)-CONH2 (SEQ ID NO: 46)
H2N-EEDDDDKAR-aca-RR-aca-RR-aca-RR-aca-RR-aca- C(Fl)-CONH2 (SEQ ID
NO: 47) H2N-eeeeee-aca-PLGLAG-rrrrrrrrr-aca-c(Fl)-CONH2
EDA-aca-R,-aca-C(Fl)-CONH2 (SEQ ID NO: 48)
EDDDDKA-aca-R6-aca-C(DOX)-CONH2 (SEQ ID NO: 49)
EEEDDDEEEDA-aca-R9-aca-Y(12SI)-CONH2
ededdAAeeeDDDDKA-aca-R,,-aca-C(Fl)-CONH2
eddedededDDDDKA-aca-Rs-AGA-R6-aca-C(DOX)-CONH2
Ggedgddeeeeeeddeed-aca-PLGLAG-aca-R8-AAA-Ri2- aca-C(Fl)-CONH2
eeddeeddKA-aca-R7-aca-C(Fl)-CONH2
eDDDDKA-aca-RGRGRRR-aca-C(Fl)-CONH2
eddddeeeeeee-aca-PLGLAGKA-aca-R10-aca-C(Fl)-CONH2
eeeeeeeeeeeeeeee-aca-DDDDKA-aca-R20-aca-C(Fl)- CONH2
eeeeeeeeeddddd-aca-DDDDKA-aca-R, 7-aca-Y ('2<iI)-CONH2
dddddddddddddddd-aca-PLGLAG-aca-R, 4-aca-C(DOX)- CONH2
NH2-eeeeee-ahx-PLG LAG-rrrrrrrrr-ahx-c(Fl)-CONH2, where ''ahx''
indicates ammohexanoic acid (SEQ ID NO: 50)
EEEEEDDDDKAXRRRRRRRRRXC(FI) (SEQ ID NO: 51)
EEEEEDDDDKARRRRRRRRRXC(Fl) (SEQ ID NO: 52) EDDDDKAXRRRRRRRRRXC(Fl)
(SEQ ID NO: 53) EEDDDDKARXRRXRRXRRXRRXC(Fl) (SEQ ID NO: 54)
DDDDDDKARRRRRRRRRXC(Fl) EEDDDDKAXrrrrrrrrrXC(Fl)
eeeeeeXPLGLAGrrrrrrrrrXc(Fl) UeeeeeeeeXPLGLAGrrrrrrrrrXk(Fl)
eeeeeeXPLGLAGrrrrrrrrrXc(Cy5) UeeeeeeXPLGLAGrrrrrrrrrXc(Cy5)
UeeeeeeeeXPLGLACorturraXk(Cy5)
11-kDaPEG]XeeeeeeeeeXPLGLAGrarrarrXk(Cy5)
11-kDaPEG]XeeeeeeeeeXLALGPGrarrarrXk(Cy5)
Fl-XrarrarrXPLGLAGeeeeeeee-.beta.Ala
Fl-XrarrarrXSGRSAeeeeeeee-.beta.Ala eeeeeeXSGRSAXrrrrrrrrrXc(Cy5)
Fl-rrrrrrrrrc-SS-ceeeeee succinyl-e8-XPLGLAG-r9-Xk, where X denotes
6- aminohexanoyI [11 kDa PEG]-X-e9-XPLGLAG-r9 [11-kDa
PEG]-X-e9-XPLGLAG-r9-Xk(Cy5) H2N-e6-XPLGLAG-r9-Xc(Cy5)-CONH2, where
X .ident. aminohexanoic acid H2N-eeeeee-(ahx)-PLG
LAG-rrrrrrrrr-(ahx)-c (Fluor)-CONH2 XeeeeeeeeeXPLGLAGrrrrrrrrXk
eeeeeeeeeXLALGPG-rrrrrrrrrXk(Cy5) mPEG(11
kd)-S-CH2-CONH-ahx-e9-ahx-PLGLAG-r9-ahx- k-CONH2
mPEG-S-CH2CONH-e9-ahx-PLGLAG-r9- K[DOTA(Gd)]-CONH2 (11
KDa-mPEG)-e9-XPLGLAG-r9-[DPK-99mTc(CO)3] (70
KDa-dextran)-e9-XPLGLAG-r9-[DPK-99mTc(CO)3] murine serum
albumin)-e9-XPLGLAG-r9-[DPK- 99mTc(CO)3] (PAMAM generation 5
dendrimer)-e9-XPLGLAX-r9- [DPK-''mTc(CO)3] (70 KDa
dextran)-e9-XPLGLAX-r9-(DOTA-'11In)
(11-KDa-mPEG)-e9-XPLGLAG-r9-K(DOTA-Gd) Suc9-(70 KDa
dextran)-e9-XPLGLAG-r9-K(DOT A-Gd) Suc9-(70 KDa
dextran)-e9-XPLGLAX-r9-K(DOT A-Gd) Suc9-(70 KDa
dextran)-e9-XPLGLAG-r9-K(DOT A-Gd) cyclic[succinoyl-PLGLAG-c(11
KDa-mPEG)-e9- XPLGLAG-r9-K]-k(Cy5) Cy5-X-e6-XPLGLAG-r9-Xk(Cy5)
Cy7-X-e6-XPLGLAG-r9-Xk(Cy5) 11 KDa mPEG-e9-PLGLAG-r9 Ac-r9-k-NH2
mPEG(11 kd)-e9-XPLGLAG-r9-Xk-NH2 e9-XPLGLAG-r9-Xk-NH2
FAM-e9-dPEG(6)-SGRFPKTVHTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGSNPFKYHTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGGPQGIAGTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGPLKITRTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGIPFFMTTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGMGPWFMHTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGSNPYK-YTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGSNPKG-YTA-r9-(D-cys)-NH2
FAM-e9-dPEG(6)-SGSNPYG-YTA-r9-(D-cys)-NH2 FAM-e9-dPEG(6)-SG-
XXXXXX-TA-r9-(D-cys)-NH2
TABLE-US-00002 TABLE 1 Cap Macromolecule Polyanion P4 P3 P2 P1 P1'
P2' P3' . . . Pn' Polycation Cargo C-term Suc9 Dextran e9 X P L G L
A G r9 K[DOTA(Gd)] NH.sub.2 (70 KDa) Suc -- e9 X P L G C(Me) A X r9
DPK NH.sub.2 Suc -- e9 X P ThienylAla G C(Me) A X r9 DPK NH.sub.2
Suc -- e9 X P F(4---Cl) G C(Me) A X r9 DPK NH.sub.2 Suc -- e8 X P L
G L A G r9 c[Cy5] NH.sub.2 Suc -- e8 X P F(4---Cl) G C(Me) M X r9
c[Cy5] NH.sub.2 Suc -- e8 X P F(4---Cl) G C(Me) Y X r9 c[Cy5]
NH.sub.2 Suc -- e8 X P F(4---Cl) G C(Me) R X r9 c[Cy5] NH.sub.2 Suc
-- e8 X P F(4---Cl) G C(Me) PhGly X r9 c[Cy5] NH.sub.2 Suc -- e8 X
P F(4---Cl) G C(Me) C(Me) X r9 c[Cy5] NH.sub.2 -- Albumin e9 X P L
G L A X r9 DPK NH.sub.2 Suc -- e8 X P C(Me) G C(Me) A X r9 c[Cy5]
NH.sub.2 Suc -- e8 X P ThienylAla G C(Me) A X r9 c[Cy5] NH.sub.2
Suc -- e8 X P F(4---Cl) G C(Me) A X r9 c[Cy5] NH.sub.2 Suc -- e8 X
P K(Dnp) G C(Me) A X r9 c[Cy5] NH.sub.2 -- Albumin e9 X P L G L A X
r9 DPK NH.sub.2 Suc -- e8 X P L G C(Me) M X r9 c[Cy5] NH.sub.2 Suc
-- e8 X P L G C(Me) Y X r9 c[Cy5] NH.sub.2 Suc127 PAMAM-Gen5 e9 X P
L G L A X r9 DPK NH.sub.2 Suc -- e8 X P L G C(Me) A X r9 c[Cy5]
NH.sub.2 Suc9 Dextran e9 X P L G L A G r9 K[DOTA(Gd)] NH.sub.2 (70
KDa) Suc127 PAMAM-Gen5 e9 X P L G L A X r9 k[Cy5] NH.sub.2 -- -- --
-- -- -- -- -- -- -- r9 Xc[Cy5] NH.sub.2 Ac127 PAMAM-Gen5 e9 X P L
G L A X r9 k[Cy5] NH.sub.2 Suc -- e8 X P L G L F(4--- A Xr9 k[Cy5]
NH.sub.2 NO2) Suc127 PAMAM-Gen5 e9 X P L G L A X r9 k[Cy5] NH.sub.2
Suc63 PAMAM-Gen4 e9 X P L G L A X r9 k[Cy5] NH.sub.2 -- Albumin e9
X P L G L A G r9 DPK NH.sub.2 Suc136 Dextran e9 X P L G L A X r9
k[Cy5] NH.sub.2 (86 KDa) Suc -- e8 X P L G L A X r9 k[Cy5] NH.sub.2
Suc9 Dextran e9 X P L G L A X r9 K[DOTA(Gd)] NH.sub.2 (70 KDa) Suc9
Dextran e9 X P L G L A X r9 K[DOTA(Gd)] NH.sub.2 (70 KDa) Suc9
Dextran e9 X P L G L A G r9 k[Cy5] NH.sub.2 (70 KDa) Suc9 Dextran
e9 X P L G L A G r9 DPK NH.sub.2 (70 KDa) Suc -- e8 X p l g l a g
r9 k[Cy5] NH.sub.2 -- Albumin e9 X p l g l a g r9 k[Cy5] NH.sub.2
Suc9 Dextran e9 X p l g l a g r9 k[Cy5] NH.sub.2 Suc97 Dextran e9 X
P L G L A G r9 k[Cy5] NH.sub.2 (500 KDa) -- Albumin e9 X P L G L A
G r9 k[Cy5] NH.sub.2 Suc9 Dextran e9 X P L G L A G r9 k[Cy5]
NH.sub.2 (70 KDa) -- Albumin e9 X P L G L A G r9 k[Cy5] NH.sub.2
Suc e8 X P L G L A X r9 k[Cy5] NH.sub.2 Suc9 Dextran e9 X P L G L A
G r9 k[Cy5] NH.sub.2 (70 KDa) -- Albumin e9 X P L G L A G r9 k[Cy5]
NH.sub.2 -- Albumin e9 X P L G L A G r9 k[Cy5] NH.sub.2 -- Albumin
e9 X P L G L A G r9 k[Cy5] NH.sub.2 Suc9 Dextran e9 X P L G L A G
r9 k[Cy5] NH.sub.2 (70 KDa) Suc nonconj. e8 X P L G L A G r9X
k[Cy5] NH.sub.2 Albumin Suc -- e8 X P L G L A G r9X k[Cy5] NH.sub.2
-- Albumin e9 X P L G L A G r9 k[Cy5] NH.sub.2 -- mPEG e9 x p l g l
a g r9X k[Cy5] NH.sub.2 (5 KDa) -- mPEG e9 X P L G L A G r9
K[DOTA(Gd)] NH.sub.2 (11 KDa) -- mPEG e10 X P L G F(4- A Q Xr9
k[Cy5] NH.sub.2 (11 KDa) NO.sub.2) -- mPEG e10 X P L G C(Me) W A
Qr9 k[Cy5] NH.sub.2 (11 KDa) -- mPEG e9 X P L G C(Me) W A Qr9
k[Cy5] NH.sub.2 (11 KDa)
In some embodiments, cargo C may be a fluorescent molecule such as
fluorescein. Fluorescent cargo moieties enable easy measurement by
fluorescence microscopy or flow cytometry in unfixed cultured
cells. However, oligoarginine sequences, such as make up portion B,
have been demonstrated to import a very wide varieties of cargoes
C, ranging from small polar molecules to nanoparticles and vesicles
(Tung & Weissleder, Advanced Drug Delivery Reviews 55: 281-294
(2003)). Thus, in embodiments of the invention, a cargo portion C
may be any suitable cargo moiety capable of being taken up by a
cell while connected to a basic portion B.
For example, for in vivo imaging purposes, C may be labeled with a
positron-emitting isotope (e.g. .sup.18F)) for positron emission
tomography (PET), gamma-ray isotope (e.g. .sup.99mTc) for single
photon emission computed tomography (SPECT), a paramagnetic
molecule or nanoparticle (e.g. Gd.sup.3+ chelate or coated
magnetite nanoparticle) for magnetic resonance imaging (MRI), a
near-infrared fluorophore for near-infra red (near-IR) imaging, a
luciferase (firefly, bacterial, or coelenterate) or other
luminescent molecule for bioluminescence imaging, or a
perfluorocarbon-filled vesicle for ultrasound. For therapeutic
purposes, for example, suitable classes of cargo include but are
not limited to: a) chemotherapeutic agents such as doxorubicin,
mitomycin, paclitaxel, nitrogen mustards, etoposide, camptothecin,
5-fluorouracil, etc.; b) radiation sensitizing agents such as
porphyrins for photodynamic therapy, or .sup.10I3 clusters or
.sup.157Gd for neutron capture therapy; or c) peptides or proteins
that modulate apoptosis, the cell cycle, or other crucial signaling
cascades. Existing chemotherapeutic drugs may be used, although
they may not be ideal, because they have already been selected for
some ability to enter cells on their own. In some embodiments of
the molecules of the invention, cargoes that are unable to enter or
leave cells without the help of the polybasic portion B may be
employed.
Cargo C may include a radioactive moiety, for example a radioactive
isotope such as .sup.211At, .sup.131I, .sup.125I, .sup.90Y,
.sup.186Re, .sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P,
radioactive isotopes of Lu, and others.
Cargo portion C may include a fluorescent moiety, such as a
fluorescent protein, peptide, or fluorescent dye molecule. Common
classes of fluorescent dyes include, but are not limited to,
xanthenes such as rhodamines, rhodols and fluoresceins, and their
derivatives; bimanes; coumarins and their derivatives such as
umbelliferone and aminomethyl coumarins; aromatic amines such as
dansyl; squarate dyes; benzofurans; fluorescent cyanines;
carbazoles; dicyanomethylene pyranes, polymethine, oxabenzanthrane,
xanthene, pyrylium, carbostyl, perylene, acridone, quinacridone,
rubrene, anthracene, coronene, phenanthrecene, pyrene, butadiene,
stilbene, lanthanide metal chelate complexes, rare-earth metal
chelate complexes, and derivatives of such dyes. Fluorescent dyes
are discussed, for example, in U.S. Pat. Nos. 4,452,720, 5,227,487,
and 5,543,295. Cargo can also include detection agents.
A cargo portion C may include a fluorescein dye. Typical
fluorescein dyes include, but are not limited to,
5-carboxyfluorescein, fluorescein-5-isothiocyanate and
6-carboxyfluorescein; examples of other fluorescein dyes can be
found, for example, in U.S. Pat. Nos. 6,008,379, 5,750,409,
5,066,580, and 4,439,356. A cargo portion C may include a rhodamine
dye, such as, for example, tetramethylrhodamine-6-isothiocyanate,
5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives,
tetramethyl and tetraethyl rhodamine, diphenyldimethyl and
diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101
sulfonyl chloride (sold under the tradename of TEXAS RED.RTM.), and
other rhodamine dyes. Other rhodamine dyes can be found, for
example, in U.S. Pat. Nos. 6,080,852, 6,025,505, 5,936,087,
5,750,409. A cargo portion C may include a cyanine dye, such as,
for example, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7.
Some of the above compounds or their derivatives will produce
phosphorescence in addition to fluorescence, or will only
phosphoresce. Some phosphorescent compounds include porphyrins,
phthalocyanines, polyaromatic compounds such as pyrenes,
anthracenes and acenaphthenes, and so forth, and may be, or may be
included in, a cargo portion C. A cargo portion C may also be or
include a fluorescence quencher, such as, for example, a
(4-dimethylamino-phenylazo)benzoic acid (DABCYL) group.
In some embodiments, a cargo moiety is all or part of a molecular
beacon. In some embodiments a cargo moeity is combined with a
quencher moiety Q to form all or part of a molecular beacon. As
used herein, "molecular beacon" means a pair of connected compounds
having complementary regions with a fluorophore and a fluorescent
quencher so that the fluorescence of the fluorophore is quenched by
the quencher. One or both of the complementary regions may be part
of the cargo moiety. Where only one of the complementary regions
(e.g., the fluorescent moiety) is part of the cargo moiety, and
where the quencher moiety is part of the linker X or the acidic
portion A, then cleavage of the linker X will allow fluorescence of
the fluorescent portion and detection of the cleavage. Upon
cellular uptake, the fluorescent portion of a molecular beacon will
allow detection of the cell. For example, a quencher Q may be
attached to an acidic portion A to form a MTS molecule having
features of the invention Q-A-X-B-C where cargo is fluorescent and
is quenched by Q The quenching of the cargo moiety by Q is relieved
upon cleavage of X, allowing fluorescent marking of a cell taking
up portion B-C The combination of fluorescence dequenching and
selective uptake should increase contrast between tissues able to
cleave X compared to those that cannot cleave X.
A pair of compounds may be connected to form a molecular beacon or
FRET pair, having complementary regions with a fluorophore and a
fluorescent quencher associated together so that the fluorescence
of the fluorophore is quenched by the quencher. Such pairs can be
useful as detection agents and any fluorescent pairs known or
described herein can be employed with the present invention. One or
both of the complementary regions may be part of the cargo portion
C. Where only one of the complementary regions (e.g., the
fluorescent moiety) is part of the cargo portion C, and where the
quencher moiety is part of the linker X or the acidic portion A,
then cleavage of the linker X will allow fluorescence of the
fluorescent portion and detection of the cleavage. Upon cellular
uptake, the fluorescent portion of a molecular beacon will allow
detection of the cell. For example, a quencher Q may be attached to
an acidic portion A to form a MTS molecule having features of the
invention Q-A-X-B C where cargo C is fluorescent and is quenched by
Q. The quenching of C by Q is relieved upon cleavage of X, allowing
fluorescent marking of a cell taking up portion B-C. The
combination of fluorescence dequenching and selective uptake should
increase contrast between tissues able to cleave X compared to
those that cannot cleave X.
In some embodiments, C and/or Q and C for all or part of a
donor:acceptor FRET pair or a BRET (bioluminescence resonance
energy transfer) pair. Donors can include any appropriate molecules
listed herein or known in the art and as such include but are not
limited to FITC; Cy3; EGFP; cyan fluorescent protein (CFP); EGFP;
6-FAM; fluorescein, IAEDANS, EDANS and BODIPY FL. Acceptors can
include any appropriate molecules listed herein or known in the art
and as such include but are not limited to TRITC; Cy5; Cy3; YFP;
6-FAM; LC Red 640; Alexa Fluor 546; fluorescein;
tetramethylrhodamine; Dabcyl (acceptor); BODIPY FL; QSY 7 and QSY 9
dyes. Exemplary FRET pairs can include but are not limited to
CFP:YFP; Cy5:Cy7; FITC:TRITC; Cy3:Cy5; EGFP:Cy3; EGFP:YFP; 6-FAM:LC
Red 640 or Alexa Fluor 546; fluorescein:tetramethylrhodamine;
IAEDANS:fluorescein; EDANS:Dabcyl; fluorescein:fluorescein; BODIPY
FL:BODIPY FL; and fluorescein:QSY 7 and QSY 9 dyes.
In some embodiments, the cargo moiety C and/or quencher moeity Q
are a fluorescent moiety including but not limited to a fluorescent
protein, peptide, or fluorescent dye molecule. Common classes of
fluorescent dyes include, but are not limited to, xanthenes such as
rhodamines, rhodols and fluoresceins, and their derivatives;
bimanes; coumarins and their derivatives such as umbelliferone and
aminomethyl coumarins; aromatic amines such as dansyl; squarate
dyes; benzofurans; fluorescent cyanines; carbazoles;
dicyanomethylene pyranes, polymethine, oxabenzanthrane, xanthene,
pyrylium, carbostyl, perylene, acridone, quinacridone, rubrene,
anthracene, coronene, phenanthrecene, pyrene, butadiene, stilbene,
lanthanide metal chelate complexes, rare-earth metal chelate
complexes, and derivatives of such dyes. Fluorescent dyes are
discussed, for example, in U.S. Pat. Nos. 4,452,720, 5,227,487, and
5,543,295.
In some embodiments, a cargo moiety C and/or quencher moeity Q are
fluorescein dyes. Typical fluorescein dyes include, but are not
limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate and
6-carboxyfluorescein; examples of other fluorescein dyes can be
found, for example, in U.S. Pat. Nos. 6,008,379, 5,750,409,
5,066,580, and 4,439,356. A cargo moiety C may include a rhodamine
dye, such as, for example, tetramethylrhodamine-6-isothiocyanate,
5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives,
tetramethyl and tetraethyl rhodamine, diphenyldimethyl and
diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101
sulfonyl chloride (sold under the tradename of TEXAS RED.RTM.), and
other rhodamine dyes. Other rhodamine dyes can be found, for
example, in U.S. Pat. Nos. 6,080,852, 6,025,505, 5,936,087,
5,750,409. A cargo moiety C may include a cyanine dye, such as, for
example, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7.
In some embodiments, cargo moiety C and/or quencher moeity Q are
fluorophores. Fluorophores are commercially available and any known
and/or commercially available fluorophore can be employed as the
cargo moiety C detectable entity for the present invention. In some
embodiments, the fluorophore exhibits green fluorescence (such as
for example 494 nm/519 nm), orange fluorescence (such as for
example 554 nm/570 nm), red fluorescence (such as for example 590
nm/617 nm), or far red fluorescence (such as for example 651 nm/672
nm) excitation/emission spectra. In some embodiments, the
fluorophore is a fluorophore with excitation and emission spectra
in the range of about 350 nm to about 775 nm. In some embodiments
the excitation and emission spectra are about 346 nm/446 nm, about
494 nm/519 nm, about 554 nm/570 nm, about 555 nm/572 nm, about 590
nm/617 nm, about 651 nm/672 nm, about 679 nm/702 nm or about 749
nm/775 nm. In some embodiments, the fluorophore can include but is
not limited to AlexaFluor 3, AlexaFluor 5, AlexaFluor 350,
AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500,
AlexaFluor 514, AlexaFluor 532, AlexaFluor 546, AlexaFluor 555,
AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633,
AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, and
AlexaFluor 750 (Molecular Probes AlexaFluor dyes, available from
Life Technologies, Inc. (USA)). In some embodiments, the
fluorophore can include but is not limited to Cy dyes, including
Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 and Cy7 (available from
Lumiprobes). In some embodiments the fluorophore can include but is
not limited to DyLight 350, DyLight 405, DyLight 488, DyLight 550,
DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 750 and
DyLight 800 (available from Thermo Scientific (USA)). In some
embodiments, the fluorophore can include but is not limited to a
FluoProbes 390, FluoProbes 488, FluoProbes 532, FluoProbes 547H,
FluoProbes 594, FluoProbes 647H, FluoProbes 682, FluoProbes 752 and
FluoProbes 782, AMCA, DEAC (7-Diethylaminocoumarin-3-carboxylic
acid); 7-Hydroxy-4-methylcoumarin-3; 7-Hydroxycoumarin-3; MCA
(7-Methoxycoumarin-4-acetic acid); 7-Methoxycoumarin-3; AMF
(4'-(Aminomethyl)fluorescein); 5-DTAF
(5-(4,6-Dichlorotriazinyl)aminofluorescein); 6-DTAF
(6-(4,6-Dichlorotriazinyl)aminofluorescein); 6-FAM
(6-Carboxyfluorescein), 5(6)-FAM cadaverine; 5-FAM cadaverine;
5(6)-FAM ethylenediamme; 5-FAM ethylenediamme; 5-FITC (FITC Isomer
I; fluorescein-5-isothiocyanate); 5-FITC cadaverin;
Fluorescein-5-maleimide; 5-IAF (5-Iodoacetamidofluorescein); 6-JOE
(6-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5-CR110
(5-Carboxyrhodamine 110); 6-CR110 (6-Carboxyrhodamine 110); 5-CR6G
(5-Carboxyrhodamine 6G); 6-CR6G (6-Carboxyrhodamine 6G);
5(6)-Caroxyrhodamine 6G cadaverine; 5(6)-Caroxyrhodamine 6G
ethylenediamme; 5-ROX (5-Carboxy-X-rhodamine); 6-ROX
(6-Carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);
6-TAMRA (6-Carboxytetramethylrhodamine); 5-TAMRA cadaverine;
6-TAMRA cadaverine; 5-TAMRA ethylenediamme; 6-TAMRA ethylenediamme;
5-TMR C6 malemide; 6-TMR C6 malemide; TR C2 malemide; TR
cadaverine; 5-TRITC; G isomer
(Tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer
(Tetramethylrhodamine-6-isothiocyanate); Dansyl cadaverine
(5-Dimethylaminonaphthalene-1-(N-(5-aminopentyl))sulfonamide);
EDANS C2 maleimide; fluorescamine; NBD; and pyrromethene and
derivatives thereof.
Some of the above compounds or their derivatives will produce
phosphorescence in addition to fluorescence, or will only
phosphoresce. Some phosphorescent compounds include porphyrins,
phthalocyanines, polyaromatic compounds such as pyrenes,
anthracenes and acenaphthenes, and so forth, and may be, or may be
included in, a cargo moiety. A cargo moiety may also be or include
a fluorescence quencher, such as, for example, a
(4-dimethylamino-phenylazo)benzoic acid (DABCYL) group.
In some embodiments, a cargo moiety is a fluorescentl label. In
some embodiments, a cargo moiety C and/or quencher moeity Q is
indocarbocyanine dye, Cy5, Cy5.5, Cy7, IR800CW, or a combination
thereof. In some embodiments, a cargo moiety is a MRI contrast
agent. In some embodiments, a cargo moiety is Gd complex of
[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.
Cargo C may include a chemotherapeutic moiety, such as a chemical
compound useful in the treatment of cancer, or other therapeutic
moiety, such as an agent useful in the treatment of ischemic
tissue, or of necrotic tissue, or other therapeutic agent.
Multiple membrane translocation signals (MTS) have been identified.
For example, the Tat protein of the human immunodeficiency virus 1
(HIV-1) is able to enter cells from the extracellular environment.
A domain from Antennapedia homeobox protein is also able to enter
cells.
Molecules comprising a MTS may also be used to carry other
molecules into cells along with them. The most important MTS are
rich in amino acids such as arginine with positively charged side
chains. Molecules transported into cell by such cationic peptides
may be termed "cargo" and may be reversibly or irreversibly linked
to the cationic peptides.
The uptake facilitated by molecules comprising a MTS is currently
without specificity, enhancing uptake into most or all cells. It is
desirable to have the ability to target the delivery of cargo to a
type of cell, or to a tissue, or to a location or region within the
body of an animal. Accordingly, a need for a MTS molecule with
increased in vivo circulation has been identified.
In some embodiments, a MTS molecule disclosed herein has the
formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with a
sequence comprising 5 to 9 consecutive acidic amino acids, wherein
the amino acids are selected from: aspartates and glutamates; B is
a peptide with a sequence comprising 5 to 20 consecutive basic
amino acids; and X is a linker that is cleavable by thrombin.
In some embodiments, a MTS molecule disclosed herein has the
formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with a
sequence comprising 5 to 9 consecutive acidic amino acids, wherein
the amino acids are selected from: aspartates and glutamates; B is
a peptide with a sequence comprising 5 to 20 consecutive basic
amino acids; and X is a linker that is cleavable by thrombin.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B-C)n-M, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; M is a macromolecular carrier; and n is an integer
between 1 and 20.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B)n-D, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; D is a dendrimer; and n is an integer between 1 and 20.
In some embodiments, D comprises a cargo moiety. See Example 1 for
methods of attaching a label to a MTS molecule.
MTS molecules disclosed herein are suitable for carrying different
cargoes, including different types of cargoes and different species
of the same types of cargo, for uptake into cells. For example,
different types of cargo may include marker cargoes (e.g.,
fluorescent or radioactive label moieties) and therapeutic cargoes
(e.g., chemotherapeutic molecules such as methotrexate or
doxorubicin), or other cargoes. Where destruction of aberrant or
diseased cells is therapeutically required, a therapeutic cargo may
include a "cytotoxic agent," i.e., a substance that inhibits or
prevents the function of cells and/or causes destruction of
cells.
Delivery of cargo such as a fluorescent molecule may be used to
visualize cells having a certain condition or cells in a region
exhibiting a particular condition For example, thrombosis (clot
formation) may be visualized by designing a linker X to be cleaved
by thrombin Thus, fluorescent molecules are one example of a marker
that may be delivered to target cells and regions upon release of a
portion A upon cleavage of a linker X
In some embodiments, the cargo moiety is selected from an imaging
agent, a therapeutic agent, a lipid, a detection agent or a
combination thereof.
In some embodiments, the cargo portion comprises at least two cargo
moieties In some embodiments, C comprises a marker cargo and a
therapeutic cargo Multiple cargo moieties may allow, for example,
delivery of both a radioactive marker and an ultrasound or contrast
agent, allowing imaging by different modalities Alternatively, for
example, delivery of radioactive cargo along with an anti cancer
agent, providing enhanced anticancer activity, or delivery of a
radioactive cargo with a fluorescent cargo, allowing multiple means
of localizing and identifying cells which have taken up cargo.
The cargo moiety is attached to B in any location or orientation
The cargo moiety need not be located at an opposite end of portion
B than a linker X Any location of attachment of the cargo moiety to
B is acceptable as long as that attachment remains after X is
cleaved For example, the cargo moiety may be attached to or near to
an end of portion B with linker X attached to an opposite end of
portion B as illustrated in FIGS. 2A and 2B The cargo moiety may
also be attached to or near to an end of portion B with linker X
attached to or near to the same end of portion B.
Wherein the molecule comprises a dendrimer, the cargo is attached
directly to D By way of non-limiting example, the cargo is attached
as follows (A-X-B)n-D-cargo.
In some embodiments, a cargo moiety is a fluorescent molecule such
as fluorescein Fluorescent cargo moieties enable easy measurement
by fluorescence microscopy or flow cytometry in unfixed cultured
cells.
In some embodiments, a cargo moiety is labeled with a
positron-emitting isotope (e g, 18F) for positron emission
tomography (PET), gamma-ray isotope (e g, 99 mTc) for single photon
emission computed tomography (SPECT), a paramagnetic molecule or
nanoparticle (e g, Gd3+ chelate or coated magnetite nanoparticle)
for magnetic resonance imaging (MRI), a near-infrared fluorophore
for near-infra red (near-IR) imaging, a luciferase (firefly,
bacterial, or coelenterate) or other luminescent molecule for
bioluminescence imaging, or a perfluorocarbon-filled vesicle for
ultrasound.
In some embodiments, a cargo moiety is a radioactive moiety, for
example a radioactive isotope such as 21 1At, .pi. iI, 12SI, 90Y,
186Re, 188Re, `"Sm, 212Bi, 12P, radioactive isotopes of Lu, and
others.
In some embodiments, a cargo moiety is a therapeutic agent, such as
a chemical compound useful in the treatment of cancer, ischemic
tissue, or necrotic tissue.
For therapeutic purposes, for example, suitable classes of cargo
include but are not limited to a) chemotherapeutic agents, b)
radiation sensitizing agents, or c) peptides or proteins that
modulate apoptosis, the cell cycle, or other crucial signaling
cascades.
In some embodiments, a cargo moiety is an agent that treats a
cardiovascular disorder In some embodiments, the cargo moiety is a
niacin, a fibrate, a statin, an Apo-Al mimetic peptide, an apoA-I
transciptional up-regulator, an ACAT inhibitor, a CETP modulator,
or a combination thereof, a Glycoprotein (GP) Ilb/IIIa receptor
antagonist, a P2Y12 receptor antagonist, a Lp-PLA2-inhibitor, a
leukotriene inhibitor, a MIF antagonist, or a combination thereof.
In some embodiments the cargo moiety is atorvastatin, cerivastatin,
fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,
rosuvastatin, simvastatin, simvastatin and ezetimibe, lovastatin
and niacin, extended-release, atorvastatin and amlodipine besylate,
simvastatin and niacin, extended-release, bezafibrate,
ciprofibrate, clofibrate, gemfibrozil, fenofibrate, DF4
(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (SEQ ID NO: 55)), DF5,
RVX-208 (Resverlogix), avasimibe, pactimibe sulfate (CS-505),
CI-1011 (2,6-diisopropylphenyl [(2,
4,6-triisopropylphenyl)acetyl]sulfamate), CI-976
(2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide), VULM1457
(1-(2,6-diisopropyl-phenyl)-3-[4-(4'-mtrophenylthio)phenyl]urea),
CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide),
E-5324
(n-butyl-N'-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphe-
nyl)urea), HL-004 (N-(2,6-diisopropylphenyl)
tetradecylthioacetamide), KY-455
(N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide),
FY-087
(N-[2-[N'-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]-(2-met-
hyl-1-naphthyl-thio)acetamide), MCC-147 (Mitsubishi Pharma), F 1251
1 ((S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthioacetanihde),
SMP-500 (Sumitomo Pharmaceuticals), CL 277082
(2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N-(hepthyl)u-
rea), F-1394 ((1s,2s)-2
[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-1-yl
3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate),
CP-113818
(N-(2,4-bis(methylthio)-6-methylpyridm-3-yl)-2-(hexylthio)decan-
oic acid amide), YM-750, torcetrapib, anacetrapid, JTT-705 (Japan
Tobacco/Roche), abciximab, eptifibatide, tirofiban, roxifiban,
variabihn, XV 459
(N(3)-(2-(3-(4-formamidinophenyl)isoxazolm-5-yl)acetyl)-N(2)-(1-bu-
tyloxycarbonyl)-2,3-diaminopropionate), SR 121566A
(3-[N-{4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl
J-N-(I-carboxymethylpipe.pi.d-4-yl) aminol propionic acid,
trihydrochloride), FK419
((S)-2-acetylamino-3-[(R)-[1-[3-(pipe.pi.din-4-yl)propionyl]pipe.pi.dm-3--
ylcarbonyl]ammo]propionic acid t.pi.hydrate), clopidogrel,
prasugrel, cangrelor, AZD6140 (AstraZeneca); MRS 2395
(2,2-Dimethyl-propionic acid
3-(2-chloro-6-methylaminopurin-9-y])-2-(2,2-dimethyl-propionyloxymethyl)--
propyl ester); BX 667 (Berlex Biosciences); BX 048 (Berlex
Biosciences); darapladib (SB 480848); SB-435495 (GlaxoSmithKline);
SB-222657 (GlaxoSmithKline); SB-253514 (GlaxoSmithKline); A-81834
(3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylp-
henyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic
acid; AMEI 03 (Amira); AME803 (Amira); atreleuton; BAY-x-1005
((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic
acid); CJ-13610
(4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrah-
ydro-pyran-4-carboxylic acid amide); DG-031 (DeCode); DG-051
(DeCode); MK886
(1-[(4-chlorophenyl)methyl]3-[(1,1-dirnethylethyl)thio]-.alpha.,.al-
pha.-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic acid, sodium
salt); MK591
(3-(1-4[(4-chlorophenyl)methyl]-3-[(t-butylthio)-5-((2-quinoly)meth-
oxy)-1H-indole-2]-, dimehtylpropanoic acid); RP64966
([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy]acetic acid); SA6541
((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropy-
l-L-cycteine); SC-56938
(ethyl-1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-piperidine-carboxylate);
VIA-2291 (Via Pharmaceuticals); WY-47,288
(2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138
(6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-]-meth-
yl-2(1H)-quinlolinone); or combinations thereof.
In some embodiments, the drug is an agent that modulates death
(e.g., via apoptosis or necrosis) of a cell. In some embodiments,
the drug is a cytotoxic agent. In some embodiments, the drug is
maytansine, methotrexate (RHEUMATREX.RTM., Amethopterin);
cyclophosphamide (CYTOXAN.RTM.); thalidomide (THALIDOMID.RTM.);
paclitaxel; pemetrexed; pentostatin; pipobroman; pixantrone;
plicamycin; procarbazine; proteasome inhibitors (e.g.; bortezomib);
raltitrexed; rebeccamycin; rubitecan; SN-38; salinosporamide A;
satraplatin; streptozotocin; swainsonine; tariquidar; taxane;
tegafur-uracil; temozolomide; testolactone; thioTEPA; tioguanine;
topotecan; trabectedin; tretinoin; triplatin tetranitrate;
tris(2-chloroethyl)amine; troxacitabine; uracil mustard;
valrubicin; vinblastine; vincristine; vinorelbine; vorinostat;
zosuquidar; or a combination thereof. In some embodiments, the drug
is a pro-apoptotic agent. In some embodiments, the drug is an
anti-apoptotic agent. In some embodiments, the drug is selected
from: minocycline; SB-203580
(4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)
1H-imidazole); PD 169316
(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole);
SB 202190
(4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole-
); RWJ 67657
(4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-y-
l]-3-butyn-1-ol); SB 220025
(5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinlyl)imidazole-
); D-JNKI-I ((D)-hJIP175-157-DPro-DPro-(D)-HIV-TAT57-48); AM-1 1 1
(Auris); SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one); JNK Inhibitor
I ((L)-HIV-T AT48-57-PP-JBD20); JNK Inhibitor III ((L)-HIV-T
AT47-57-gaba-c-Jun.delta.33-57); AS601245 (1,3-benzothiazol-2-yl
(2-[[2-(3-pyridinyl)ethyl]amino]-4 pyrimidinyl) acetonitrile); JNK
Inhibitor VI (H2N-RPKRPTTLNLF-NH2 (SEQ ID NO: 56)); JNK Inhibitor
VIII
(N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamid-
e); JNK Inhibitor IX
(N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-1-naphthamide);
dicumarol (3,3'-Methylenebis(4-hydroxycoumarin)); SC-236
(4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfon-
amide); CEP-1347 (Cephalon); CEP-11004 (Cephalon); an artificial
protein comprising at least a portion of a Bcl-2 polypeptide; a
recombinant FNK; V5 (also known as Bax inhibitor peptide V5); Bax
channel blocker
((.+-.)-1-(3,6-Dibromocarbazol-9-yl)-3-piperazin-1-yl-propan-2-ol);
Bax inhibiting peptide P5 (also known as Bax inhibitor peptide P5);
Kp7-6; FAIM(S) (Fas apoptosis inhibitory molecule-short); FAIM(L)
(Fas apoptosis inhibitory molecule-long); Fas:Fc; FAP-I; NOK2;
F2051; F1926; F2928; ZB4; Fas M3 mAb; EGF; 740 Y-P; SC 3036
(KKHTDDGYMPMSPGVA (SEQ ID NO: 57)); PI 3-kinase Activator (Santa
Cruz Biotechnology, Inc.); Pam3Cys
((S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)--
Lys4-OH, trihydrochloride); Actl (NF-.kappa.B activator 1); an
anti-I.kappa.B antibody; Acetyl-1 1-keto-b-Boswellic Acid;
Andrographolide; Caffeic Acid Phenethyl Ester (CAPE); Gliotoxin;
Isohelenin; NEMO-Binding Domain Binding Peptide
(DRQIKIWFQNRRMKWKKTALDWSWLQTE (SEQ ID NO: 58)); NF-.kappa.B
Activation Inhibitor
(6-Amino-4-(4-phenoxyphenylethylamino)quinazoline); NF-.kappa.B
Activation Inhibitor II
(4-Methyl-N1-(3-phenylpropyl)benzene-1,2-diamine); NF-.kappa.B
Activation Inhibitor III
(3-Chloro-4-nitro-N-(5-nitro-2-thiazolyl)-benzamide); NF-.kappa.B
Activation Inhibitor IV ((E)-2-Fluoro-4'-methoxystilbene);
NF-.kappa.B Activation Inhibitor V
(5-Hydroxy-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione);
NF-.kappa.B SN50 (AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO: 59));
Oridonin; Parthenolide; PPM-18 (2-Benzoylamino-1,4-naphthoquinone);
RoI 06-9920; Sulfasalazine; TIRAP Inhibitor Peptide
(RQIKIWFNRRMKWKKLQLRDAAPGGAIVS (SEQ ID NO: 60)); Withaferin A;
Wogonin; BAY 1 1-7082
((E)3-[(4-Methylphenyl)sulfonyl]-2-propenenitrile); BAY 1 1-7085
((E)3-[(4-t-Butylphenyl)sulfonyl]-2-propenenitrile); (E)-Capsaicin;
Aurothiomalate (ATM or AuTM); Evodiamine; Hypoestoxide; IKK
Inhibitor m (BMS-345541); IKK Inhibitor VII; IKK Inhibitor X; IKK
Inhibitor II; IKK-2 Inhibitor IV; IKK-2 Inhibitor V; IKK-2
Inhibitor VI; IKK-2 Inhibitor (SC-514); I.kappa.B Kinase Inhibitor
Peptide; IKK-3 Inhibitor IX; ARRY-797 (Array BioPharma); SB-220025
(5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinlyl)imidazole-
); SB-239063
(trans-4-[4-(4-Fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl-
]cyclohexanol); SB-202190
(4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole);
JX-401
(-[2-Methoxy-4-(methylthio)benzoyl]-4-(phenylmethyl)piperidine);
PD-169316
(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazol-
e); SKF-86002
(6-(4-Fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo[2,1-b]thiazole
dihydrochloride); SB-200646
(N-(I-Methyl-1H-indol-5-yl)-N'-3-pyridinylurea); CMPD-I
(2'-Fluoro-N-(4-hydroxyphenyl)-[1, 1'-biphenyl]-4-butanamide);
EO-1428
((2-Methylphenyl)-[4-[(2-amino-4-bromophenyl)amino]-2-chlorophenyl]methan-
one); SB-253080
(4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyri-
dine); SD-169 (1H-Indole-5-carboxamide); SB-203580
(4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)
1H-imidazole); TZP-101 (Tranzyme Pharma); TZP-102 (Tranzyme
Pharma); GHRP-6 (growth hormone-releasing peptide-6); GHRP-2
(growth hormone-releasing peptide-2); EX-1314 (Elixir
Pharmaceuticals); MK-677 (Merck); L-692,429 (Butanamide,
3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-l-((2'-(1H-tetrazol-5-yl)(1,
1'-biphenyl)-4-yl)methyl)-1 H-1-benzazepin-3-yl)-, (R)-); EP1 572
(Aib-DTrp-DgTrp-CHO); diltiazem; metabolites of diltiazem; BRE
(Brain and Reproductive organ-Expressed protein); verapamil;
nimodipine; diltiazem; omega-conotoxin; GVIA; amlodipine;
felodipine; lacidipine; mibefradil; NPPB
(5-Nitro-2-(3-phenylpropylamino)benzoic Acid); flunarizine;
erythropoietin; piperine; hemin; brazilin; z-V AD-FMK
(Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone); z-LEHD-FMK
(SEQ ID NO: 61)
(benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethylketone);
B-D-FMK (boc-aspartyl(Ome)-fluoromethylketone); Ac-LEHD-CHO (SEQ ID
NO: 62) (N-acetyl-Leu-Glu-His-Asp-CHO); Ac-IETD-CHO (SEQ ID NO: 63)
(N-acetyl-Ile-Glu-Thr-Asp-CHO); z-IETD-FMK (SEQ ID NO: 64)
(benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethy Iketone);
FAM-LEHD-FMK (SEQ ID NO: 65) (benzyloxycarbonyl
Leu-Glu-His-Asp-fluoromethyl ketone); FAM-LETD-FMK (SEQ ID NO: 66)
(benzyloxycarbonyl Leu-Glu-Thr-Asp-iluoromethyl ketone); Q-VD-OPH
(Quinoline-Val-Asp-CH2-O-Ph); XIAP; cIAP-1; cIAP-2; ML-IAP; ILP-2;
NAIP; Survivin; Bruce; IAPL-3; fortilin; leupeptine; PD-150606
(3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid); MDL-28170
(Z-Val-Phe-CHO); calpeptin; acetyl-calpastatin; MG 132
(N-t(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-l-
eucinamide); MYODUR; BN 82270 (Ipsen); BN 2204 (Ipsen); AHLi-11
(Quark Pharmaceuticals), an mdm2 protein, pifithrin-.alpha.
(1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)et-
hanone); trans-stilbene; cis-stilbene; resveratrol; piceatannol;
rhapontin; deoxyrhapontin; butein; chalcon; isoliquirtigen; butein;
4,2',4'-trihydroxychalcone; 3,4,2',4',6'-pentahydroxychalcone;
flavone; morin; fisetin; luteolin; quercetin; kaempferol; apigenin;
gossypetin; myricetin; 6-hydroxyapigenin; 5-hydroxyflavone;
5,7,3',4',5'-pentahydroxyflavone; 3,7,3',4',5'-pentahydroxyflavone;
3,6,3',4'-tetrahydroxyflavone; 7,3',4',5'-tetrahydroxyflavone;
3,6,2',4'-tetrahydroxyflavone; 7,4'-dihydroxyflavone;
7,8,3',4'-tetrahydroxyflavone; 3,6,2',3'-tetrahydroxyflavone;
4'-hydroxyflavone; 5-hydroxyflavone; 5,4'-dihydroxyflavone;
5,7-dihydroxyflavone; daidzein; genistein; naringenin; flavanone;
3,5,7,3',4'-pentahydroxyflavanone; pelargonidin chloride; cyanidin
chloride; delphinidin chloride; (-)-epicatechin (Hydroxy Sites:
3,5,7,3',4'); (-)-catechin (Hydroxy Sites: 3,5,7,3',4');
(-)-gallocatechin (Hydroxy Sites: 3,5,7,3',4',5') (+)-catechin
(Hydroxy Sites: 3,5,7,3',4'); (+)-epicatechin (Hydroxy Sites:
3,5,7,3',4'); Hinokitiol (b-Thujaplicin;
2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one);
L-(+)-Ergothioneine
((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-1H-imidazole4-ethanam-
inium inner salt); Caffeic Acid Phenyl Ester; MCI-186
(3-Methyl-1-phenyl-2-pyrazolin-5-one); HBED
(N,N'-Di-(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid*H2O);
Ambroxol (trans-4-(2-Amino-3,5-dibromobenzylamino)cyclohexane-HCl;
and U-83836E
((-)-2-((4-(2,6-di-1-Pyrrolidinyl-4-pyrimidinyl)-1-piperzainyl)m-
ethyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol*2HCl);
.beta.-1'-5-methyl-nicotinamide-2'-deoxyribose;
.beta.-D-r-5-methyl-nico-tinamide-2'-deoxyribofuranoside;
.beta.-1'-4,5-dimethyl-nicotinamide-2'-de-oxyribose;
.beta.-D-1'-4,5-dimethyl-nicotinamide-2'-deoxyribofuranoside;
1-Naphthyl PPI
(1-(1,1-Dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-4-amine); Lavendustin A
(5-[[(2,5-Dihydroxyphenyl)methyl][(2-hydroxyphenyl)methy
1]amino]-2-hydroxybenzoic acid); MNS
(3,4-Methylenedioxy-b-nitrostyrene), PPI
(1-(1,1-Dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,
4-d]py.pi.midin-4-amine), PP2 (3-(4-chlorophenyl) 1-(1,
1-dimethylethyl)-l H-pyrazolo[3,4-d]py.pi.midin-4-amine), KX-004
(Kinex), KX-005 (Kinex), KX-136 (Kinex), KX-174 (Kinex), KX-141
(Kinex), KX2-328 (Kinex), KX-306 (Kinex), KX-329 (Kinex), KX2-391
(Kinex), KX2-377 (Kinex), ZD4190 (Astra Zeneca,
N-(4-bromo-2-fluorophenyl)-6-methoxy-7-(2-(1H-1,2,3-triazol-1-yl)ethoxy)q-
uinazolin-4-amine), AP22408 (Airad Pharmaceuticals), AP23236
(A.pi.ad Pharmaceuticals), AP23451 (Atad Pharmaceuticals), AP23464
(Atad Pharmaceuticals), AZD0530 (Astra Zeneca), AZM475271 (M475271,
Astra Zeneca), Dasatmib
(N-(2-chloro-6-methylphneyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-m-
ethylpynmidin-4-yl)ammo) thiazole-5-carboxamide), GN963
(trans-4-(6,7-dimethoxyqmnoxalm-2ylamino)cyclohexanol sulfate);
Bosutimb
(4-((2,4-dichloro-5-methoxyphenyl)ammo)-6-methoxy-7-(3-(4-methyl-1-prpera-
zmyl)propoxy)-3-quinolinecarboni.pi.le), or combinations
thereof.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B)n-L, wherein L is a lipid, A is a peptide with a
sequence comprising 5 to 9 consecutive acidic ammo acids, wherein
the amino acids are selected from aspartates and glutamates, B is a
peptide with a sequence comprising 5 to 20 consecutive basic amino
acids, X is a linker, and n is an integer between 1 and 20, and
wherein L is bound to an (A-X-B) moiety by a bond with a B.
In some embodiments, the lipid entraps a hydrophobic molecule In
some embodiments, the lipid entraps at least one agent selected
from the group consisting of a therapeutic moiety or an imaging
moiety.
In some embodiments, the lipid is PEGylated In some embodiments,
the lipid is PEG(2K)-phosphatidylethanolamine.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B-C)n-M, wherein C is a cargo moiety; A is a peptide
with a sequence comprising 5 to 9 consecutive acidic amino acids,
wherein the amino acids are selected from: aspartates and
glutamates; B is a peptide with a sequence comprising 5 to 20
consecutive basic amino acids; X is a linker that is cleavable by
thrombin; M is a macromolecular carrier; and n is an integer
between 1 and 20.
Disclosed herein, in certain embodiments, is a MTS molecule with
increased in vivo circulation In some embodiments, a MTS molecule
disclosed herein has the formula (A-X-B-C)n-M, wherein C is a cargo
moiety, A is a peptide with a sequence comprising 5 to 9
consecutive acidic ammo acids, wherein the amino acids are selected
from aspartates and glutamates, B is a peptide with a sequence
comprising 5 to 20 consecutive basic amino acids; X is a linker; M
is a macromolecular carrier; and n is an integer between 1 and
20.
The term "carrier" indicates an inert molecule that increases (a)
plasma half-life and (b) solubility. In some embodiments, a carrier
decreases uptake of a MTS molecule into cartilage. In some
embodiments, a carrier decreases uptake of a MTS molecule into
joints. In some embodiments, a carrier decreases uptake of a MTS
molecule into the liver. In some embodiments, a carrier decreases
uptake of a MTS molecule into kidneys.
In some embodiments, a carrier increases plasma half-life and
solubility by reducing glomerular filtration. In some embodiments,
a carrier increases tumor uptake due to enhanced permeability and
retention (EPR) of tumor vasculature.
In some embodiments, M is bound to A. In some embodiments, M is
bound to A at the n-terminal poly glutamate. In some embodiments, M
is bound to A (or, the n-terminal poly glutamate) by a covalent
linkage. In some embodiments, the covalent linkage comprises an
ether bond, thioether bond, amine bond, amide bond, carbon-carbon
bond, carbon-nitrogen bond, carbon-oxygen bond, or carbon-sulfur
bond.
In some embodiments, M is bound to B. In some embodiments, M is
bound to B at the c-terminal polyarginine. In some embodiments, M
is bound to B (or, the c-terminal polyarginine) by a covalent
linkage. In some embodiments, the covalent linkage comprises an
ether bond, thioether bond, amine bond, amide bond, carbon-carbon
bond, carbon-nitrogen bond, carbon-oxygen bond, or carbon-sulfur
bond.
In some embodiments, M is selected from a protein, a synthetic or
natural polymer, or a dendrimer. In some embodiments, M is selected
from dextran, a PEG polymer (e.g., PEG 5 kDa and PEG 12 kDa),
albumin, or a combination thereof. In some embodiments, M is a PEG
polymer.
In some embodiments, the size of the carrier is between 50 kDa and
70 kDa. In some embodiments, small amounts of negative charge keep
peptides out of the liver while not causing synovial uptake.
In some embodiments, the MTS molecule is conjugated to albumin. In
certain instances, albumin is excluded from the glomerular filtrate
under normal physiological conditions. In some embodiments, the MTS
molecule comprises a reactive group such as maleimide that can form
a covalent conjugate with albumin. A MTS molecule comprising
albumin results in enhanced accumulation of cleaved MTS molecules
in tumors in a cleavage dependent manner. See, Example 2. In some
embodiments, albumin conjugates have good pharmacokinetic
properties but are difficult to work with synthetically.
In some embodiments, the MTS molecule is conjugated to a PEG
polymer. In some embodiments, the MTS molecule is conjugated to a
PEG 5 kDa polymer. In some embodiments, the MTS molecule is
conjugated to a PEG 12 kDa polymer. In some embodiments, 5 kD PEG
conjugates behaved similarly to free peptides. In some embodiments,
12 kD PEG conjugates had a longer halflife as compared to free
peptides.
In some embodiments, the MTS molecule is conjugated to a dextran.
In some embodiments, the MTS molecule is conjugated to a 70 kDa
dextran. In some embodiments, dextran conjugates, being a mixture
of molecular weights, are difficult to synthesize and purify
reproducibly.
In some embodiments, the MTS molecule is conjugated to
streptavidin.
In some embodiments, the MTS molecule is conjugated to a fifth
generation PAMAM dendrimer.
In some embodiments, a carrier is capped. See Example 1 for methods
of capping. In some embodiments, capping a carrier improves the
pharmacokinetics and reduces cytotoxicity of a carrier by adding
hydrophilicity. In some embodiments, the cap is selected from:
Acetyl, succinyl, 3-hydroxypropionyl, 2-sulfobenzoyl, glycidyl,
PEG-2, PEG-4, PEG-8 and PEG-12.
In some embodiments, a MTS molecule disclosed herein has the
formula (A-X-B)n-D, wherein D is a dendrimer; A is a peptide with a
sequence comprising 5 to 9 consecutive acidic amino acids, wherein
the amino acids are selected from: aspartates and glutamates; B is
a peptide with a sequence comprising 5 to 20 consecutive basic
amino acids; X is a linker; and n is an integer between 1 and 20;
and wherein D is bound to an (A-X-B) moiety by a bond with a B. In
some embodiments, D is bound to an (A-X-B) moiety by a bond with a
polyarginine terminus. In some embodiments, D comprises at least
one cargo moiety.
As used herein, "dendrimer" means a poly-functional (or,
poly-branched) molecule. In some embodiments, a denrimer is a
structure in which a central molecule branches repetitively and
repetitiously. In some embodiments, the dendrimer is a
nanoparticle.
In some embodiments, D is bound to B (or, the c-terminal
polyarginine) by a covalent linkage. In some embodiments, the
covalent linkage comprises an ether bond, thioether bond, amine
bond, amide bond, carbon-carbon bond, carbon-nitrogen bond,
carbon-oxygen bond, or carbon-sulfur bond.
In some embodiments, a plurality of (A-X-B) moieties are attached
to D. See, Example 3. In some embodiments, a plurality of cargo
moieties are attached to D. In some embodiments, (a) a plurality of
(A-X-B) moieties are attached to D; and (b) a plurality of cargo
moieties are attached to D.
In some embodiments, the dendrimer comprises a reactive group such
as maleimide that can form a covalent conjugate with albumin. In
some embodiments, a dendrimer is conjugated to a MTS molecule via a
maleimide linker at the C-terminal end of the MTS molecule.
In some embodiments, conjugating a MTS molecule to a dendrimer
increases plasma half-life as compared to an unconjugated (or,
free) MTS molecule. In some embodiments, a MTS molecule conjugated
to a dendrimer results in a decrease in acute toxicity as compared
to unconjugated MTS molecules. In some embodiments, a MTS molecule
conjugated to a dendrimer reduces uptake by synovium, cartilage and
kidney as compared to unconjugated MTS molecules.
In some embodiments, a MTS molecule conjugated to a dendrimeric
nanoparticle is used to target tumor associated macrophages. In
some embodiments, a MTS molecule conjugated to a dendrimeric
nanoparticle, wherein the nanoparticle further comprises Ricin A,
is used to poison subcutaneous macrophages.
MTS molecules having features of the invention may be synthesized
by standard synthetic techniques, such as, for example, solid phase
synthesis including solid phase peptide synthesis. An example of
peptide synthesis using Fmoc is given as Example 1 below in WO
2005/042034). For example, conventional solid phase methods for
synthesizing peptides may start with N-alpha-protected amino acid
anhydrides that are prepared in crystallized form or prepared
freshly in solution, and are used for successive amino acid
addition at the N-terminus. At each residue addition, the growing
peptide (on a solid support) is acid treated to remove the
N-alpha-protective group, washed several times to remove residual
acid and to promote accessibility of the peptide terminus to the
reaction medium. The peptide is then reacted with an activated
N-protected amino acid symmetrical anhydride, and the solid support
is washed. At each residue-addition step, the amino acid addition
reaction may be repeated for a total of two or three separate
addition reactions, to increase the percent of growing peptide
molecules which are reacted. Typically, 1 to 2 reaction cycles are
used for the first twelve residue additions, and 2 to 3 reaction
cycles for the remaining residues.
After completing the growing peptide chains, the protected peptide
resin is treated with a strong acid such as liquid hydrofluoric
acid or trifluoroacetic acid to deblock and release the peptides
from the support. For preparing an amidated peptide, the resin
support used in the synthesis is selected to supply a C-terminal
amide, after peptide cleavage from the resin. After removal of the
strong acid, the peptide may be extracted into 1M acetic acid
solution and lyophilized. The peptide can be isolated by an initial
separation by gel filtration, to remove peptide dimers and higher
molecular weight polymers, and also to remove undesired salts The
partially purified peptide may be further purified by preparative
HPLC chromatography, and the purity and identity of the peptide
confirmed by amino acid composition analysis, mass spectrometry and
by analytical HPLC (e.g., in two different solvent systems).
The invention also provides polynucleotides encoding MTS molecules
described herein. The term "polynucleotide" refers to a polymeric
form of nucleotides of at least 10 bases in length. The nucleotides
can be ribonucleotides, deoxynucleotides, or modified forms of
either type of nucleotide. The term includes single and double
stranded forms of DNA. The term therefore includes, for example, a
recombinant DNA which is incorporated into a vector, e.g., an
expression vector; into an autonomously replicating plasmid or
virus; or into the genomic DNA of a prokaryote or eukaryote, or
which exists as a separate molecule (e.g., a cDNA) independent of
other sequences.
These polynucleotides include DNA, cDNA, and RNA sequences which
encode MTS molecules having features of the invention, or portions
thereof. Peptide portions may be produced by recombinant means,
including synthesis by polynucleotides encoding the desired amino
acid sequence. Such polynucleotides may also include promoter and
other sequences, and may be incorporated into a vector for
transfection (which may be stable or transient) in a host cell.
The construction of expression vectors and the expression of genes
in transfected cells involves the use of molecular cloning
techniques that are well known in the art. See, for example,
Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) and
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
(Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., most recent
Supplement). Nucleic acids used to transfect cells with sequences
coding for expression of the polypeptide of interest generally will
be in the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used herein, "operatively linked"
refers to a juxtaposition wherein the components so described are
in a relationship permitting them to function in their intended
manner. A control sequence operatively linked to a coding sequence
is ligated such that expression of the coding sequence is achieved
under conditions compatible with the control sequences. "Control
sequence" refers to polynucleotide sequences which are necessary to
effect the expression of coding and non-coding sequences to which
they are ligated. Control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence. The
term "control sequences" is intended to include, at a minimum,
components whose presence can influence expression, and can also
include additional components whose presence is advantageous, for
example, leader sequences and fusion partner sequences. As used
herein, the term "nucleotide sequence coding for expression of a
polypeptide refers to a sequence that, upon transcription and
translation of mRNA, produces the polypeptide. This can include
sequences containing, e.g., introns. As used herein, the term
"expression control sequences" refers to nucleic acid sequences
that regulate the expression of a nucleic acid sequence to which it
is operatively linked. Expression control sequences are operatively
linked to a nucleic acid sequence when the expression control
sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus,
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signals for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of the mRNA, and stop codons.
Any suitable method is used to construct expression vectors
containing the fluorescent indicator coding sequence and
appropriate transcriptional/translational control signals. Any
methods which are well known to those skilled in the art can be
used to construct expression vectors containing the fluorescent
indicator coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. (See, for example,
the techniques described in Maniatis, et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N. Y., 1989).
Transformation of a host cell with recombinant DNA may be carried
out by conventional techniques as are well known to those skilled
in the art.
Where the host is prokaryotic, such as E. coli, competent cells
which are capable of DNA uptake can be prepared from cells
harvested after exponential growth phase and subsequently treated
by the CaCl.sub.2 method by procedures well known in the art.
Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can
also be performed after forming a protoplast of the host cell or by
electroporation.
When the host is a eukaryote, such methods of transfection of DNA
as calcium phosphate co-precipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransfected with DNA sequences
encoding the fusion polypeptide of the invention, and a second
foreign DNA molecule encoding a selectable phenotype, such as the
herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (Eukagotic Viral Vectors, Cold
Spring Harbor Laboratory, Gluzman ed., 1982). Techniques for the
isolation and purification of polypeptides of the invention
expressed in prokaryotes or eukaryotes may be by any conventional
means such as, for example, preparative chromatographic separations
and immunological separations such as those involving the use of
monoclonal or polyclonal antibodies or antigen.
It will be understood that the compounds of the present invention
can be formulated in pharmaceutically and or diagnostically useful
compositions. Such pharmaceutical and diagnositcally useful
compositions may be prepared according to well known methods. For
example, MTS compounds having features of the invention, and having
a cargo portion C that is, for example, a therapeutic moiety or a
detection moiety, may be combined in admixture with a
pharmaceutically acceptable carrier vehicle or a diagnostic
buffering agent. Suitable vehicles and agents and their
formulation, inclusive of other human proteins, e.g. human serum
albumin are described, for example, in Remington's Pharmaceutical
Sciences by E. W. Martin and, the techniques described in Maniatis,
et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory, N. Y., 1989-2013, which are hereby incorporated by
reference. Such compositions will contain an effective amount of
the compounds hereof together with a suitable amount of vehicle in
order to prepare pharmaceutically acceptable compositions suitable
for effective administration. Dosages and dosing regimens may be
determined for the indications and compounds by methods known in
the art, including determining (e.g., in experimental animals) the
effective dose which causes half of those treated to respond to the
treatment (ED.sub.50) by providing a range of doses to experimental
animals or subjects and noting the responses.
EXAMPLES
Example 1
Methods for Measuring Ex Vivo Cleavage of FRET-Based Enzymatically
Cleavable Peptide Probes by Tumor Extract:
The rationale is to identify enzymatically positive tumors in a
patient population that may benefit from the use of enzymatically
cleavable peptide probes for early tumor detection and
intraoperative margin evaluation. Experiments have been performed
using probes that detected either MMPs (PLGLAG (SEQ ID NO: 1) and
PLGC(met)AG (SEQ ID NO: 2) or elastases (RLQLK(acetyl)L (SEQ ID NO:
26). A panel of probes will be expanded to include other tumor
expressed proteases.
Xenograft Tumor Extracts:
Animals models of a variety of cancers cancer were generated as
previously described. Tumors were grown to 0.5 cm-1 cm and then
surgically excised. Following dissection, tissue was gently
homogenized in PBS, while kept cooled on ice to minimize the
release of intracellular and intraorganellar proteases. Homogenates
were microcentrifuged at 14,000.times.g for 1 minute, and
supernatants (extracts) were tested for ability to increase the Cy5
donor:Cy7 acceptor emission ratio of a FRET-based ACPP. The effect
of freezing the tumor specimen before homogenization was tested and
showed that the assay can be reliably performed on frozen material,
simplifying the logistics of collection. Furthermore, banked
clinical specimens may be retrospectively analyzable and able to be
compared with known outcomes.
Surgical Specimen Extracts:
Surgical specimens from patients undergoing excision of neoplasms
were obtained under University of California, San Diego (UCSD) IRB
approval. Following receipt of tumor specimens from the pathology
department, tissue was frozen in -80C for varying periods of time
from 1 day to 1 year. Frozen specimens are thawed and assayed as
described in the previous subsection (Xenograft tumor
extracts).
Peptide Synthesis:
FRET-based ACPPs were synthesized by attaching a fluorescent
acceptor, Cy7, to the polyanionic domain of the previously
described ACPP2, 3 so that the donor (Cy5) and acceptor (Cy7)
fluorophores in the uncleaved probe are sufficiently close to each
other for FRET to occur This FRET quenches the Cy5 emission
intensity and causes re-emission of Cy7. Upon cleavage of the ACPP
linker, FRET was disrupted and Cy5 emission is increased while Cy7
re-emission is eliminated. This loss of FRET, imaged in vivo by
multispectral imaging in animal tumor models, produced faster and
more intense tumor:background contrast than occurred with
previously published non-FRET ACPPs.
Ex Vivo Cleavage:
Ex-vivo cleavages were done with 5 .mu.M peptide in 20 .mu.l of PBS
containing 1 .mu.M ZnCl2 plus 2-20 .mu.l of 20% tissue extract (20
mg of tissue homogenate per 100 .mu.l), followed by incubation at
37.degree. C. Cleavage of probe will be determined as a ratio of
Cy5:Cy7 fluorescence, using either a microplate fluorometer (Tecan
M1000) or multispectral imaging (Maestro, CRI) at various time
points for different tumor extract concentrations. Cleavage rate
will be determined by plotting Cy5:Cy7 ratio as a function of time
following addition of tumor extract to the peptide.
In Vivo Imaging:
Human-derived tongue SCC cell lines (named above) or minced tumor
specimens from patients will be implanted subcutaneously into nude
animals. Tumors were grown to 0.5 cm-1 cm. Animals were injected
intravenously with 10 nmoles of FRET-ACPP. Multispectral imaging
was performed using Maestro (CRI), and ratio of Cy5:Cy7
fluorescence was measured at 2 hours. Ratio of Cy5:Cy7 fluorescence
were measured for tumor and adjacent non-tumor tissue.
ACPP Cleavage Vs. ACPP Fluorescence Uptake Comparison:
Ratio of Cy5:Cy7 fluorescence were measured for tumor and adjacent
non-tumor tissue for a given tumor specimen (SCC cell lines or
individual human surgical specimens) and were compared with the
ex-vivo cleavage rate.
Example 2
Generation of Panel of ACPP for Profiling Tumor Protease
Activity:
Multiple ACPPs with varied protease cleavage sequence will be used
to establish each specific tumors protease profile. Currently the
panel consist of ACPPs that are selective for MMPs, elastases,
plasmin, thrombin. MMP 2,9 cleavable sequence PLGLAG (SEQ ID NO:
1), PLGC(met)AG (SEQ ID NO: 2). Other MMP selective substrates
could include RS-(Cit)-G-(homoF)-YLY (SEQ ID NO: 4), CRPAHLRDSG
(SEQ ID NO: 5), SLAYYTA (SEQ ID NO: 6), NISDLTAG (SEQ ID NO: 7),
PPSSLRVT (SEQ ID NO: 8), SGESLSNLTA (SEQ ID NO: 9), RIGFLR (SEQ ID
NO: 10). Elastase cleavable sequence RLQLA(acetyl)L (SEQ ID NO:
11). Plasmin selective substrate RLQLKL (SEQ ID NO: 12). Thrombin
selective substrates DPRSFL (SEQ ID NO: 13), PPRSFL (SEQ ID NO:
14), Norleucine-TPRSFL (SEQ ID NO: 15). Chymase selective substrate
GVAY|SGA (SEQ ID NO: 16). Urokinase-type plasminogen activator
(uPA) and tissue plasminogen activator (tPA) selective substrate
YGRAAA (SEQ ID NO: 17). uPA selective sequence YGPRNR (SEQ ID NO:
18).
Example 3
Personalized Protease Assay:
A personalized protease (PePA) assay will be provided which will
find use in a variety of situations. A PePA assay will provide an
assay for use in understanding the heterogeneity in levels of tumor
specific enzymes between patients for a given cancer diagnosis. A
PePA assay will also provide an assay for use in correlating tumor
specific enzyme activity with known histologic grade/stage and
patient prognosis. A PePA assay will provide an assay for use in
identifying which patients may benefit from the use of individual
enzymatically activatable probes for staging, medical and surgical
management in a variety of cancers.
Example 4
Abstract:
Objective:
1. Obtain matrix-metalloproteinase(MMP) expression profiles for
head and neck squamous cell carcinoma(HNSCC) specimens from The
Cancer Genomic Atlas (TCGA) 2. Demonstrate HNSCC imaging using
MMP-cleavable, fluorescently-labeled ratiometric activatable
cell-penetrating peptide (RACPP). Study Design:
Retrospective human cohort study; prospective animal study
Setting:
Translational Research Laboratory
Subjects and Methods:
Patient clinical data and mRNA expression levels of MMP genes were
downloaded from TCGA data portal. RACPP provides complementary
ratiometric fluorescent contrast (increased Cy5 and decreased Cy7
intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were
imaged in-vivo after RACPP injection. Histology was evaluated by a
pathologist blinded to experimental conditions. Zymography
confirmed MMP-2/9 activity in xenografts. RACPP was applied to
homogenized human HNSCC specimens and ratiometric fluorescent
signal was measured on a microplate reader for ex-vivo
analysis.
Results:
Expression of multiple MMPs including MMP2/9 is greater in patient
HNSCC tumors than matched control tissue. In patients with human
papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14
expression correlates with worse 5-year survival. Orthotopic tongue
HNSCC xenografts showed excellent ratiometric fluorescent labeling
with MMP2/9-cleavable RACPP(sensitivity=95.4%, specificity=95.0%).
Fluorescence ratios were greater in areas of higher tumor
burden(p<0.03), which is useful for intraoperative margin
assessment. Ex-vivo, human HNSCC specimens showed greater cleavage
of RACPP when compared to control tissue(p=0.009).
Conclusions:
Human HNSCC tumors show increased mRNA expression of multiple MMPs
including MMP2/9. RACPP, a ratiometric fluorescence assay of MMP2/9
activity, was used to show improved occult tumor identification and
margin clearance. Ex-vivo assays using RACPP in biopsy specimens
may identify patients who will benefit from intraoperative RACPP
use.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most
common cancer worldwide with an estimated annual burden of 355,000
deaths and 633,000 incident cases.sup.1. Major risk factors include
smoking, alcohol abuse, and human papilloma virus (HPV).sup.2.
Surgical management is usually the primary therapy for this
disease, although radiation and chemotherapy also have prominent
roles.sup.3.
For HNSCC, MMP expression has been shown to have prognostic
value.sup.4-8. Of the various MMPs thought to be involved in
cancer, attention has focused on MMPs 2 and 9 because they are
overexpressed in a variety of malignant tumors and their expression
is often associated with tumor grade and poor patient prognosis.
Absolute levels of MMP2/9 have been used to differentiate between
benign papillomas and carcinoma of the larynx.sup.4. Increased
MMP2/9 expression has also been shown to correlate with cancer
grade.sup.5 and decreased survival.sup.6,7. In carcinoma of the
tongue, increased MMP2/9 expression has been shown to correlate
with an increased incidence of lymph node metastases.sup.8.
In this example, MMP mRNA levels in HNSCC were evaluated using The
Cancer Genomic Atlas (TCGA), the largest available collection of
HNSCC specimens. The prognostic value of MMP mRNA levels in
patients with HPV+ and HPV-HNSCC tumors was evaluated.
Although MMP expression (mRNA and protein) has been associated with
tumor grade and poor patient prognosis for a variety of cancers, at
the tissue level, MMP activity is regulated by a variety of factors
including activation from pro-enzyme form and presence or absence
of inhibitors.sup.9. Consequently MMP activity, rather than
expression, may have closer association with tumor biological
behavior and therefore greater prognostic value. Activatable cell
penetrating peptides (ACPPs), which rely on tumor-associated
proteases MMP2/9 to unmask the adhesiveness of CPPs have been
previously described.sup.10,11. A ratiometric version of ACPPs
(RACPPS) which employs Cy5 as a far-red fluorescent resonance
energy transfer (FRET) donor and Cy7 as near-infrared FRET acceptor
has been recently described. The Cy5 emission is absorbed by Cy7
and re-emitted as near-infrared fluorescence until the intervening
linker is cleaved by tumor-associated MMP2/9. This cleavage event
increases Cy5:Cy7 emission ratio up to 40-fold and enables tissue
retention of the Cy5 fragment.sup.12. ACPP was previously used to
improve tumor margin (defined as tumor cells present at the cut
edge of the surgical specimen) detection in animal model of
melanoma and breast cancer.sup.13.
In HNSCC, positive margins have been associated with increased
local recurrence and a poor prognosis.sup.14. For the majority of
solid tumors, salvage surgery or adjuvant therapy not only cause
extra trauma and expense but also often fail to remediate the poor
outcome.sup.14-20. The reason for this observation is likely
multifactorial and related in part to the difficulty in identifying
the residual cancer during repeat surgery. Therefore, development
of more sensitive imaging for accurate detection of positive
surgical margins during the primary operation would be one of the
most effective means to prevent positive margins, thereby
minimizing patient suffering and expense, while improving
outcomes.
Using RACPP, MMP2/9 activity levels were compared between patient
derived ex-vivo HNSCC specimens versus non-tumor tissue. The use of
intravenously applied RACPPs to distinguish between orthotopic
HNSCC xenografts from normal tissue and stratify tumor burden at
the surgical margin in mice was evaluated.
Methods
All animal studies were approved by the UCSD Institutional Animal
Care and Use Committee. All studies involving tumor samples
obtained from HNSCC patients were approved by the UCSD
Institutional Review Board.
The Cancer Genomic Atlas (TCGA)
All available clinical and RNA expression data were downloaded from
the TCGA data-portal on Dec. 15, 2013. HPV status was obtained from
the TCGA HNSCC working group. HPV status was extracted from
sequencing data or RNA data.sup.21. For tumor-normal comparison, 37
patients (out of 377 total) with matched tumor/normal tissue were
considered and paired tests were used.
Ex-Vivo Assay on HNSCC
Tumor samples were obtained from patients undergoing surgery for
mucosal head and neck squamous cell carcinoma and stored at
-80.degree. C. until analysis. Samples were homogenized using equal
quantities of beads and tissue and twice the volume of PBS. 150
nmol of RACPP was added to 100-175 .mu.l of PBS containing 25 .mu.l
of 10% tissue extract. Cleavage of the probe was determined by
capturing the Cy5/Cy7 fluorescence ratio every 15 minutes for 2
hours (excitation 630 nm/emission 680-780 nm) using Tecan Infinite
M100 pro plate reader (Tecan Laboratories, Switzerland).
Zymogram
Zymogram was prepared as previously described.sup.22. Briefly,
30-40 mg of tissue was homogenized in buffered solution and
centrifuged. Tissue samples, along with SeeBlue Plus 2 Protein
ladder and MMP standards were loaded on the gel and run at 120V for
two hours. Following renaturation, development and staining, gels
were imaged and analyzed with Image J. MMP activity of samples was
recorded as a percentage of MMP activity within the positive
control lane.
Peptide Synthesis
RACPP and uncleavable-control were synthesized as previously
described.sup.12. The RACPP contains a poly-cationic moiety linked
to a neutralizing poly-anionic arm via a linker that is cleavable
by MMP-2 and MMP-9. A Cy5 fluorophore is attached to the
polycationic portion while the Cy7 fluorescent molecule is attached
to the poly-anionic domain. Following cleavage by MMPs, the
polycationic portion conjugated to Cy5 is dequenched and becomes
trapped within nearby tissue. Uncleavable-control peptide lacks an
MMP cleavable linker.
Cell Culture and Mouse Tongue Xenografts
Human tongue squamous cell carcinoma lines SCC-4, SCC-9, SCC-15,
and SCC-25 (ATCC) were maintained in Dulbecco's modified Eagle's
medium with nutrient mixture F-12 (DMEM/F-12) containing 10% fetal
bovine serum (FBS) and supplemented with 400 ng/mL of
hydrocortisone. Human tongue squamous cell carcinoma line CAL-27
(ATCC) was maintained in DMEM containing 10% FBS. Cells were
incubated at 37.degree. C. in 5% CO.sub.2. Nu/nu mice (age, 3-6
months) were injected with cultured HNSCC cells (.about.10.sup.6
for CAL-27, .about.5.times.10.sup.6 for SCC-4, SCC-9, SCC-15,
SCC-25) into the tip of the tongue. One cell line was used in each
mouse for these experiments (n=22 total; CAL-27:n=5, SCC-25 n=4,
SCC-15:n=4, SCC-4:n=4, SCC-9:n=5).
In Vivo Imaging with RACPP
Mice were monitored for 20% weight loss or tumor size >4-5 mm.
Once these parameters were met, animals were anesthetized with
isoflurane and injected intravenously with RACPP or control
uncleavable peptide (0.4 nmol/g). Two hours after injection, mice
were re-anesthetized (100 mg/kg ketamine and 5 mg/kg midazolam) and
subcutaneous cervical tissue/anterior tongue exposed for imaging
(Maestro, CRI). After completion of whole body imaging, animals
were euthanized. The entirety of the tongue was immediately
extracted and imaged in the dorsal position (Maestro, CRI).
Spectral imaging was carried out by exciting Cy5 at 620 (.+-.10) nm
followed by step-wise emission measurements from 640 to 840 nm
through a tunable LCD emission filter. For ratio imaging, numerator
(Cy5) and denominator (Cy7) images were generated by integrating
spectral images over a defined range at 10 nm intervals (660-720 nm
for Cy5 and 760-830 nm for Cy7). Ratio images were generated and
color-encoded using custom software. The ratio for each pixel was
encoded as hue on a blue to red scale and brightness was based on
the original Cy5 images. The software also generated monochromatic
Cy5/Cy7 images for further processing (see Image analysis and
histologic correlation).
Histology
Immediately following imaging, tongue tissues were embedded in
cryopreservative and stored at -80.degree. C. Samples were
cryosectioned into 5-.mu.M sections in the same orientation as the
whole tongue molecular imaging and stained with hematoxylin and
eosin (H&E). The entirety of the tongue was included in the
slice, including both tumor and normal tissue. Samples were
evaluated by a pathologist blinded to experimental conditions.
Mapping Histology to Molecular Imaging
For histologic samples, a pathologist blinded to experimental
conditions used a stage micrometer to determine the tumor's linear
position and extent along the length of the tongue. This
information was mapped to spectral images of the tongue (FIG. 4). A
mean Cy5/Cy7 ratio was calculated for segments containing
histologically-confirmed tumor and, separately, tumor-free
segments.
Percent tumor involvement was approximated by the pathologist as
the density of cancerous tissue (vs. non-cancerous tissue) within
the tumor-containing segment of tongue (FIG. 4). For example, if
the tumor-bearing length of the sample contained only malignant
cells and no normal tissue, percent involvement was recorded as
100%. If only half of this region contained malignancy, percent
involvement was recorded as 50%. This method for calculating
percent tumor involvement has been utilized in other
studies.sup.23,24.
Image Processing and Ratio Calculations
Monochromatic Cy5 and Cy7 images were extracted from the spectral
image using custom software. Using the "Image Calculator" feature
on Image J, the Cy5 image was divided by the Cy7 image to produce a
new image, where Cy5/Cy7 ratios were encoded by pixel intensity.
Ratios were calculated separately for tumor and normal tongue,
which were distinguished based on the histologic map described
above. These ratios were each normalized to Cy5/Cy7 ratios of
background tissue (cervical soft tissue).
Statistics
Statistical analysis between experimental groups was conducted
using either the 2-tailed independent sample student t test or
one-way ANOVA w/post-hoc analysis. Graphical bar-plots were
produced using Microsoft Excel, while ROC curves were created with
Sigmaplot (12.3). Paired tests were used for TCGA analysis due to
matched expression data. For survival analysis, Cox proportional
hazards regression was employed using the R `survival` package.
Results
MMPs are Overexpressed in HNSCC
To evaluate MMP expression levels in HNSCC from the TCGA,
patient-derived tumor specimens were compared with matched normal
control tissue. HNSCC tumors showed increased expression of
multiple MMPs compared to matched control non-tumor tissue (FIG.
2A, all p values <0.01). Interestingly, MMP14 (also known as
MT1-MMP) was the protease with the highest total expression in
tumor tissue and had significantly higher expression in tumor
compared to matched control tissue (p<10.sup.-5). The second
highest expressing MMP in tumor tissue was MMP2. MMP2 and 9 share a
common cleavage sequence and they have been particularly well
characterized in prior studies in association with HNSCC.sup.25. It
was found that both MMP2 (p<10.sup.-10) and MMP9
(p<10.sup.-6) have significantly greater RNA expression in HNSCC
tumors compared to paired-control tissue (n=37, (34 HPV- and 3
HPV+), Wilcoxon signed-rank test).
MMP 2 and 14 Stratify Survival in HPV+HNSCC
Next, the difference in MMP expression between HPV+ and HPV- tumors
was evaluated. It has been found that HPV+ tumors had less overall
MMP expression compared to HPV- tumors (FIG. 2B, Kruskal-Wallis
test on pooled RNA levels, p<10.sup.-10). This is consistent
with the hypothesis that HPV+ tumors are less biologically
aggressive, and consequently, that these patients tend to have
improved survival compared to patients with HPV- tumors.
Interestingly, it was found that in patients with HPV+HNSCC,
increased expression levels of MMP2 and MMP14 correlated with worse
survival (FIG. 2C, 2D p<0.01). Patients with HPV+ tumors who
have the highest MMP2 and MMP14 expression (FIG. 2C, 2D red lines)
had significantly worse 5 year survival compared to patients with
the lowest expression levels of these proteases (FIG. 2C, 2D blue
lines). Additionally, for a given patient with HPV+ tumor, there is
a significant correlation between MMP2 and MMP14 expression
(Spearman Rho=0.56, p<10.sup.-4). Thus, poor prognosis HPV+
tumors stratified in the highest quartiles of MMP2 expression are
also likely to have higher expression of MMP14. The same
correlation in MMP expression with survival in patients with HPV-
tumors was not found. The cause of this is multifactorial and
likely related to the observation that HPV- tumors have more
genetic mutations compared HPV+ tumors.sup.26.
Zymography
To confirm MMP2/9 activity in mouse HNSCC xenografts, cleavage of
gelatin by tumor homogenates via zymography was measured. A
two-fold increase in MMP9 and a 13-fold increase in MMP2 activity
in HNSCC xenografts compared to normal mouse tongue tissue was
found.
RACPP in Ex-Vivo HNSCC
To evaluate ex-vivo MMP2/9 activity in human and mouse HNSCC
specimens, the maximum rate of Cy5/Cy7 ratio change over time in
homogenates following addition of RACPP (FIG. 3A, 3B) was measured.
It was found that patient derived HNSCC specimens show higher
MMP2/9 activity compared to non-tumor tissue (FIG. 3C) (ROC
Analysis: AUC=1.000, p=0.01). Similarly, mouse HNSCC xenografts
also show higher Cy5/Cy7 rate change, signifying higher MMP2/9
activity compared to non-tumor tissue (ROC Analysis: AUC=1.000,
p=0.03).
RACPP Improves Detection of HNSCC
To test tumor-dependent Cy5/Cy7 ratiometric change in living mice,
tongue tumor bearing nu/nu mice were intravenously injected with
RACPP (n=25). Multispectral imaging of these live, anesthetized
mice (ex 620, em 640-840 nm, Maestro, CRI at 2 hours after
injection) with both tongue and subcutaneous cervical tissue
exposed was conducted(FIG. 4A). The tongue was excised and
ultispectral imaging of the tongue performed. Histologic
information regarding tumor location and size was correlated and
mapped to ratiometric fluorescence image of the tongue. Sample
(tumor and non tumor tissue) Cy5/Cy7 ratios were divided by
"background" subcutaneous cervical tissue Cy5/Cy7 ratio to compute
a "normalized Cy5/Cy7 ratio".sup.12.
It was found that higher Cy5/Cy7 ratiometric fluorescence in tumor
(FIG. 4B, red color) compared to adjacent normal tongue (FIG. 4B,
tan color). Injections of our control (uncleavable) probe revealed
no ratiometric difference between tongue tumor and normal tongue
(FIG. 4C). Following intravenous administration of MMP2/9-cleavable
RACPP, mice showed greater normalized Cy5/Cy7 ratio in tumor
(1.61.+-.0.05, n=22) compared to normal tongue (1.11.+-.0.03, n=20,
p<10.sup.-8). This increase in ratiometric fluorescence in
orthotopic tumors was not seen following intravenous injection of
uncleavable control probe (tumor=1.01.+-.0.04, n=3; normal
tongue=1.07.+-.0.03, n=3, p=0.30) (FIG. 4D).
The receiver-operating curve (ROC) for cleavable RACPP revealed an
area under the curve (AUC) of 0.995.+-.0.006 (p<10.sup.-4) with
a peak sensitivity of 95% and peak specificity of 100% for a
normalized ratio cutoff of 1.345 (FIG. 4D insert). The two tumor
specimens not detected by this threshold-cutoff had relatively low
tumor burden (<60% involvement, see below).
RACPPs Enable Stratification of Tumor Burden
One critical component of intraoperative margin evaluation is
determining how much tumor burden is present at the edges of the
surgical field. To evaluate the ability of RACPP to stratify tissue
with variable tumor burden, percent involvement of cancer within
tumor-bearing portions of each sample was approximated by a
pathologist blinded to experimental conditions. It was found that
varying levels of tumor burden among the 22 samples ranging from
25-100% invasion (FIG. 5A). To evaluate the stratification of
ratiometric fluorescence values between different levels of tumor
burden, samples were statistically separated into the following
tertiles of cancer involvement: 25-60% (n=8), 61-80% (n=8), 81-100%
(n=6) (FIG. 5B). Adjusted Cy5/Cy7 ratios were computed for each
tertile and compared with normal tongue tissue (n=20).
It was found that all tertiles of varying tumor burden showed
significantly greater normalized Cy5/Cy7 ratio than normal tongue
tissue (FIG. 5C; lowest tertile of tumor involvement=1.46.+-.0.07,
p<10.sup.-5, middle tertile=1.67.+-.0.12, p<10.sup.-7,
highest tertile=1.72.+-.0.04, p<10.sup.-7). Additionally, tumors
with percent involvement in the highest and middle tertiles showed
significantly greater normalized ratios than the lowest tertile
(p=0.01 for highest vs. lowest tertile, p=0.03 for middle vs.
lowest tertile). Future experiments will focus on evaluating the
ability of RACPP to detect incrementally smaller levels of tumor
burden (i.e. from 1% to 25% involvement).
DISCUSSION
In this study, mRNA expression levels for MMPs in human HNSCC were
analyzed using The Cancer Genomic Atlas (TCGA), the largest
available collection of human HNSCC specimens. The prognostic value
of MMP overexpression in terms of survival in patients with HPV+
and HPV-HNSCC tumors was evaluated. It was found that many MMPs are
overexpressed in HNSCC tumors compared to paired control tissue.
However, patients with HPV+HNSCC tumors have significantly lower
overall MMP levels compared to patients with HPV-HNSCC tumors. This
finding is consistent with previous studies showing that patients
with HPV+HNSCC tumors have better overall survival compared to
patients with HPV-HNSCC tumors.sup.27.
Of the various MMPs thought to be involved in cancer, attention has
focused on MMP2/9 because they are overexpressed in a variety of
malignant tumors and their expression is often associated with
tumor grade and poor patient prognosis. Interestingly, it was found
that of the MMPs that are increased in tumor compared to control
tissue, MMP2 and MMP14 are expressed at higher levels compared to
all other MMPs, suggesting that these proteinases may be
particularly important in HNSCC. Furthermore, it was found that in
patients with HPV+HNSCC tumors, increased MMP2 and MMP14 expression
levels correlated with worse overall survival. If clinically
validated in a prospective trial, the increases in MMP2 and MMP14
represent two molecular biomarkers that can individualize
management of patients with HPV+ tumors.
Using MMP2/9 cleavable RACPP, it was found that ex-vivo human HNSCC
specimens show greater activity compared to normal tissue. This
finding correlates with previous studies demonstrating higher
MMP2/9 expression at the invasive edge of tumors.sup.28. The high
sensitivity and specificity of RACPPs to differentiate between
tumor and normal tissue suggests that ex-vivo measurements of
MMP2/9 activity in HNSCC specimens may complement MMP mRNA
expression studies in evaluating patient prognosis and in
determining which patients would benefit from RACPP guided
surgery.
In multiple human cell line models of HNSCC xenografts, it was
found that higher MMP2/9 activity as evidenced by gelatinase
zymography and higher ratiometric fluorescence signal following
systemically applied RACPP compared to non-tumor tissue. All ratios
were computed from histologically confirmed tumor or normal tissue,
eliminating verification bias. The ideal discrimination threshold
for detecting cancer versus normal tissue is 1.345, which is
consistent with previously reported ratiometric thresholds for this
probe.sup.12. Our study tested multiple tongue squamous cell
carcinoma cell lines from ATCC to highlight the RACPP's broad
applicability.
One critical component of intraoperative margin evaluation is
determining how much tumor burden is present at the edges of the
surgical field. It was found that within tumor bearing tissue, the
greater the tumor burden, the greater the ratiometric fluorescence
signal following intravenous RACPP administration. Percent tumor
involvement has been shown to be important for survival and
recurrence outcomes in prostate and breast cancer.sup.23,29. The
correlation between intraoperative ratiometric fluorescence level
and tumor burden suggests that RACPP can improve intraoperative
decision making by providing information regarding local level of
tumor involvement and consequently margin clearance.
REFERENCES FOR EXAMPLE 4
1. Ferlay J, Shin H R, Bray F, Forman D, Mathers C, Parkin D M.
Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int
J Cancer 2010; 127:2893-2917. 2. D'Souza G, Kreimer A R, Viscidi R
et al. Case-control study of human papillomavirus and oropharyngeal
cancer. N Engl J Med 2007; 356:1944-1956. 3. Shin D M, Khuri F R.
Advances in the management of recurrent or metastatic squamous cell
carcinoma of the head and neck. Head Neck 2013; 35:443-453. 4.
Uloza V, Liutkevicius V, Pangonyte D, Saferis V, Lesauskaite V.
Expression of matrix metalloproteinases (MMP-2 and MMP-9) in
recurrent respiratory papillomas and laryngeal carcinoma: clinical
and morphological parallels. Eur Arch Otorhinolaryngol 2011;
268:871-878. 5. Wittekindt C, Jovanovic N, Guntinas-Lichius O.
Expression of matrix metalloproteinase-9 (MMP-9) and blood vessel
density in laryngeal squamous cell carcinomas. Acta Otolaryngol
2011; 131:101-106. 6. Liu W W, Zeng Z Y, Wu Q L, Hou J H, Chen Y Y.
Overexpression of MMP-2 in laryngeal squamous cell carcinoma: a
potential indicator for poor prognosis. Otolaryngol Head Neck Surg
2005; 132:395-400. 7. Mallis A, Teymoortash A, Mastronikolis N S,
Werner J A, Papadas T A. MMP-2 expression in 102 patients with
glottic laryngeal cancer. Eur Arch Otorhinolaryngol 2012;
269:639-642. 8. Zhou C X, Gao Y, Johnson N W, Gao J.
Immunoexpression of matrix metalloproteinase-2 and matrix
metalloproteinase-9 in the metastasis of squamous cell carcinoma of
the human tongue. Aust Dent J 2010; 55:385-389. 9. Chaudhary A K,
Singh M, Bharti A C, et al. Genetic polymorphisms of matrix
metalloproteinases and their inhibitors in potentially malignant
and malignant lesions of the head and neck. J Biomed Sci 2010;
17:10. 10. Olson E S, Aguilera T A, Jiang T et al. In vivo
characterization of activatable cell penetrating peptides for
targeting protease activity in cancer. Integr Biol (Camb) 2009;
1:382-393. 11. Aguilera T A, Olson E S, Timmers M M, Jiang T, Tsien
R Y. Systemic in vivo distribution of activatable cell penetrating
peptides is superior to cell penetrating peptides. Integr Biol
2009; 1:371-381. 12. Savariar E N, Felsen C N, Nashi N et al.
Real-time in vivo molecular detection of primary tumors and
metastases with ratiometric activatable cell-penetrating peptides.
Cancer Res 2013; 73:855-864. 13. Savariar E N, Felsen C N, Nashi N
et al. Real-time in vivo molecular detection of primary tumors and
metastases with ratiometric activatable cell-penetrating peptides.
Cancer Res 2013; 73:855-864. 14. Hague R, Contreras R, McNicoll M
P, Eckberg E C, Petitti D B. Surgical margins and survival after
head and neck cancer surger. BMC Ear Nose Throat Disord 2006; 16:2.
15. Singletary S. Surgical margins in patients with early-stage
breast cancer treated with breast conservation therapy. Am J Surg
2002; 184:383-393. 16. Nagtegaal I D, Quirke P. What is the role
for the circumferential margin in the modern treatment of rectal
cancer? J Clin On 2008; 26:303-312. 17. Meric F, Mirza N, Vlastos G
et al. Positive surgical margins and ipsilateral breast tumor
recurrence predict disease-specific survival after
breast-conserving therapy. Cancer 2003; 97:926-933. 18. Snijder R,
de la Riviere A, Elbers H, van den Bosch J. Survival in resected
stage I lung cancer with residual tumor at the bronchial resection
margin. Annals of Thoracic Surg 1998; 65. 19. Dotan Z, Kavanagh K,
Yossepowitch 0 et al. Positive surgical margins in soft tissue
following radical cystectomy for bladder cancer and cancer specific
survival. J Urol 2007; 178:2308-2312. 20. Wieder J A, Soloway M S.
Incidence, etiology, location, prevention and treatment of positive
surgical margins after radical prostatectomy for prostate cancer. J
Urol 1998; 160:299-315. 21. Gross A, Orosco R, Shen J et al. A
prognostic model of head and neck cancer ties TP53 mutation to 3p
loss. Nature Genetics [Under Review]. 22. Toth M, Sohail A, Fridman
R. Assessment of gelatinases (MMP-2 and MMP-9) by gelatin
zymography. Methods Mol Biol 2012; 878:121-35. 23. Rampersaud E N,
Sun L, Moul J W, Madden J, Freedland S J. Percent tumor involvement
and risk of biochemical progression after radical prostatectomy. J
Urol 2008; 180:571-576; discussion 576. 24. Thompson I M, 3rd,
Salem S, Chang S S et al. Tumor volume as a predictor of adverse
pathologic features and biochemical recurrence (BCR) in radical
prostatectomy specimens: a tale of two methods. World J Urol 2011;
29:15-20. 25. Xia T, Akers K, Eisen A Z, Seltzer J L. Comparison of
cleavage site specificity of gelatinases A and B using collagenous
peptides. Biochim Biophys Acta 1996; 1293(2):259-266. 26. Maruyama
H, Yasui T, Ishikawa-Fujiwara T, et al. Human papillomavirus and
p53 mutations in head and neck squamous cell carcinoma among
Japanese population. Cancer Sci 2014. [In Press] 27. Sivars L,
Nasman A, Tertipis N et al. Human papillomavirus and p53 expression
in cancer of unknown primary in the head and neck region in
relation to clinical outcome. Cancer Med 2014; 3(2): 376-84. 28.
Kuniyasu H, Troncoso P, Johnston D et al. Relative expression of
type IV collagenase, E-cadherin, and vascular endothelial growth
factor/vascular permeability factor in prostatectomy specimens
distinguishes organ-confined from pathologically advanced prostate
cancers. Clin Cancer Res 2000; 6(6): 2295-308. 29. Kohrt H E, Nouri
N, Nowels K, Johnson D, Holmes S, Lee P P. Profile of immune cells
in axillary lymph nodes predicts disease-free survival in breast
cancer. PLoS Med 2005; 2:e284.
Example 5
Matrix-Metalloproteinases in Head and Neck Carcinoma-Cancer Genome
Atlas Analysis and Fluorescence Imaging in Mice
Abstract:
Objective:
Obtain matrix-metalloproteinase(MMP) expression profiles for head
and neck squamous cell carcinoma(HNSCC) specimens from The Cancer
Genomic Atlas (TCGA)
Demonstrate HNSCC imaging using MMP-cleavable,
fluorescently-labeled ratiometric activatable cell-penetrating
peptide(RACPP).
Study Design:
Retrospective human cohort study; prospective animal study
Setting:
Translational Research Laboratory
Subjects and Methods:
Patient clinical data and mRNA expression levels of MMP genes were
downloaded from TCGA data portal. RACPP provides complementary
ratiometric fluorescent contrast (increased Cy5 and decreased Cy7
intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were
imaged in-vivo after RACPP injection. Histology was evaluated by a
pathologist blinded to experimental conditions. Zymography
confirmed MMP-2/9 activity in xenografts. RACPP was applied to
homogenized human HNSCC specimens and ratiometric fluorescent
signal was measured on a microplate reader for ex-vivo
analysis.
Results:
Expression of multiple MMPs including MMP2/9 is greater in patient
HNSCC tumors than matched control tissue. In patients with human
papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14
expression correlates with worse 5-year survival. Orthotopic tongue
HNSCC xenografts showed excellent ratiometric fluorescent labeling
with MMP2/9-cleavable RACPP(sensitivity=95.4%, specificity=95.0%).
Fluorescence ratios were greater in areas of higher tumor
burden(p<0.03), which is useful for intraoperative margin
assessment. Ex-vivo, human HNSCC specimens showed greater cleavage
of RACPP when compared to control tissue(p=0.009).
Conclusions:
Human HNSCC tumors show increased mRNA expression of multiple MMPs
including MMP2/9. RACPP, a ratiometric fluorescence assay of MMP2/9
activity, was used to show improved occult tumor identification and
margin clearance. Ex-vivo assays using RACPP in biopsy specimens
may identify patients who will benefit from intraoperative RACPP
use.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most
common cancer worldwide with an estimated annual burden of 355,000
deaths and 633,000 incident cases.sup.1. Major risk factors include
smoking, alcohol abuse, and human papilloma virus (HPV).sup.2.
Surgical management is usually the primary therapy for this
disease, although radiation and chemotherapy also have prominent
roles.sup.3.
For HNSCC, MMP expression has been shown to have prognostic
value.sup.4-8. Of the various MMPs thought to be involved in
cancer, attention has focused on MMPs 2 and 9 because they are
overexpressed in a variety of malignant tumors and their expression
is often associated with tumor grade and poor patient prognosis.
Absolute levels of MMP2/9 have been used to differentiate between
benign papillomas and carcinoma of the larynx.sup.4. Increased
MMP2/9 expression has also been shown to correlate with cancer
grade.sup.5 and decreased survival.sup.6,7. In carcinoma of the
tongue, increased MMP2/9 expression has been shown to correlate
with an increased incidence of lymph node metastases.sup.8.
In this example, MMP mRNA levels were examined in HNSCC using The
Cancer Genomic Atlas (TCGA), the largest available collection of
HNSCC specimens. The prognostic value of MMP mRNA levels in
patients with HPV+ and HPV-HNSCC tumors was also evaluated.
Although MMP expression (mRNA and protein) has been associated with
tumor grade and poor patient prognosis for a variety of cancers, at
the tissue level, MMP activity is regulated by a variety of factors
including activation from pro-enzyme form and presence or absence
of inhibitors.sup.9. Consequently MMP activity, rather than
expression, may have closer association with tumor biological
behavior and therefore greater prognostic value. Activatable cell
penetrating peptides (ACPPs), which rely on tumor-associated
proteases MMP2/9 to unmask the adhesiveness of CPPs have been
previously described.sup.10,11. A ratiometric version of ACPPs
(RACPPS) which employs Cy5 as a far-red fluorescent resonance
energy transfer (FRET) donor and Cy7 as near-infrared FRET acceptor
has been recently described. The Cy5 emission is absorbed by Cy7
and re-emitted as near-infrared fluorescence until the intervening
linker is cleaved by tumor-associated MMP2/9. This cleavage event
increases Cy5:Cy7 emission ratio up to 40-fold and enables tissue
retention of the Cy5 fragment.sup.12. ACPP has been previously used
to improve tumor margin (defined as tumor cells present at the cut
edge of the surgical specimen) detection in animal model of
melanoma and breast cancer.sup.13.
In HNSCC, positive margins have been associated with increased
local recurrence and a poor prognosis.sup.14. For the majority of
solid tumors, salvage surgery or adjuvant therapy not only cause
extra trauma and expense but also often fail to remediate the poor
outcome.sup.14-20. The reason for this observation is likely
multifactorial and related in part to the difficulty in identifying
the residual cancer during repeat surgery. Therefore, development
of more sensitive imaging for accurate detection of positive
surgical margins during the primary operation would be one of the
most effective means to prevent positive margins, thereby
minimizing patient suffering and expense, while improving
outcomes.
Using RACPP, MMP2/9 activity levels were compared between patient
derived ex-vivo HNSCC specimens versus non-tumor tissue. The use of
intravenously applied RACPPs was also evaluated to distinguish
between orthotopic HNSCC xenografts from normal tissue and stratify
tumor burden at the surgical margin in mice.
Methods
All animal studies were approved by the UCSD Institutional Animal
Care and Use Committee. All studies involving tumor samples
obtained from HNSCC patients were approved by the UCSD
Institutional Review Board.
The Cancer Genomic Atlas (TCGA)
All available clinical and RNA expression data were downloaded from
the TCGA data-portal on Dec. 15, 2013. HPV status was obtained from
the TCGA HNSCC working group. HPV status was extracted from
sequencing data or RNA data.sup.21. For tumor-normal comparison, 37
patients (out of 377 total) with matched tumor/normal tissue were
considered and paired tests were used.
Ex-Vivo Assay on HNSCC
Tumor samples were obtained from patients undergoing surgery for
mucosal head and neck squamous cell carcinoma and stored at
-80.degree. C. until analysis. Samples were homogenized using equal
quantities of beads and tissue and twice the volume of PBS. 150
nmol of RACPP was added to 100-175 .mu.l of PBS containing 25 .mu.l
of 10% tissue extract. Cleavage of the probe was determined by
capturing the Cy5/Cy7 fluorescence ratio every 15 minutes for 2
hours (excitation 630 nm/emission 680-780 nm) using Tecan Infinite
M100 pro plate reader (Tecan Laboratories, Switzerland).
Zymogram
Zymogram was prepared as previously described.sup.22. Briefly,
30-40 mg of tissue was homogenized in buffered solution and
centrifuged. Tissue samples, along with SeeBlue Plus 2 Protein
ladder and MMP standards were loaded on the gel and run at 120V for
two hours. Following renaturation, development and staining, gels
were imaged and analyzed with Image J. MMP activity of samples was
recorded as a percentage of MMP activity within the positive
control lane.
Peptide Synthesis
RACPP and uncleavable-control were synthesized as previously
described.sup.12. The RACPP contains a poly-cationic moiety linked
to a neutralizing poly-anionic arm via a linker that is cleavable
by MMP-2 and MMP-9. A Cy5 fluorophore is attached to the
polycationic portion while the Cy7 fluorescent molecule is attached
to the poly-anionic domain. Following cleavage by MMPs, the
polycationic portion conjugated to Cy5 is dequenched and becomes
trapped within nearby tissue. Uncleavable-control peptide lacks an
MMP cleavable linker.
Cell Culture and Mouse Tongue Xenografts
Human tongue squamous cell carcinoma lines SCC-4, SCC-9, SCC-15,
and SCC-25 (ATCC) were maintained in Dulbecco's modified Eagle's
medium with nutrient mixture F-12 (DMEM/F-12) containing 10% fetal
bovine serum (FBS) and supplemented with 400 ng/mL of
hydrocortisone. Human tongue squamous cell carcinoma line CAL-27
(ATCC) was maintained in DMEM containing 10% FBS. Cells were
incubated at 37.degree. C. in 5% CO.sub.2. Nu/nu mice (age, 3-6
months) were injected with cultured HNSCC cells (.about.10.sup.6
for CAL-27, .about.5.times.10.sup.6 for SCC-4, SCC-9, SCC-15,
SCC-25) into the tip of the tongue. One cell line was used in each
mouse for these experiments (n=22 total; CAL-27:n=5, SCC-25 n=4,
SCC-15:n=4, SCC-4:n=4, SCC-9:n=5).
In Vivo Imaging with RACPP
Mice were monitored for 20% weight loss or tumor size >4-5 mm.
Once these parameters were met, animals were anesthetized with
isoflurane and injected intravenously with RACPP or control
uncleavable peptide (0.4 nmol/g). Two hours after injection, mice
were re-anesthetized (100 mg/kg ketamine and 5 mg/kg midazolam) and
subcutaneous cervical tissue/anterior tongue exposed for imaging
(Maestro, CRI). After completion of whole body imaging, animals
were euthanized. The entirety of the tongue was immediately
extracted and imaged in the dorsal position (Maestro, CRI).
Spectral imaging was carried out by exciting Cy5 at 620 (.+-.10) nm
followed by step-wise emission measurements from 640 to 840 nm
through a tunable LCD emission filter. For ratio imaging, numerator
(Cy5) and denominator (Cy7) images were generated by integrating
spectral images over a defined range at 10 nm intervals (660-720 nm
for Cy5 and 760-830 nm for Cy7). Ratio images were generated and
color-encoded using custom software. The ratio for each pixel was
encoded as hue on a blue to red scale and brightness was based on
the original Cy5 images. The software also generated monochromatic
Cy5/Cy7 images for further processing (see Image analysis and
histologic correlation).
Histology
Immediately following imaging, tongue tissues were embedded in
cryopreservative and stored at -80.degree. C. Samples were
cryosectioned into 5-.mu.M sections in the same orientation as the
whole tongue molecular imaging and stained with hematoxylin and
eosin (H&E). The entirety of the tongue was included in the
slice, including both tumor and normal tissue. Samples were
evaluated by a pathologist blinded to experimental conditions.
Mapping Histology to Molecular Imaging
For histologic samples, a pathologist blinded to experimental
conditions used a stage micrometer to determine the tumor's linear
position and extent along the length of the tongue. This
information was mapped to spectral images of the tongue. A mean
Cy5/Cy7 ratio was calculated for segments containing
histologically-confirmed tumor and, separately, tumor-free
segments.
Percent tumor involvement was approximated by the pathologist as
the density of cancerous tissue (vs. non-cancerous tissue) within
the tumor-containing segment of tongue. For example, if the
tumor-bearing length of the sample contained only malignant cells
and no normal tissue, percent involvement was recorded as 100%. If
only half of this region contained malignancy, percent involvement
was recorded as 50%. This method for calculating percent tumor
involvement has been utilized in other studies.sup.23,24.
Image Processing and Ratio Calculations
Monochromatic Cy5 and Cy7 images were extracted from the spectral
image using custom software. Using the "Image Calculator" feature
on Image J, the Cy5 image was divided by the Cy7 image to produce a
new image, where Cy5/Cy7 ratios were encoded by pixel intensity.
Ratios were calculated separately for tumor and normal tongue,
which were distinguished based on the histologic map described
above. These ratios were each normalized to Cy5/Cy7 ratios of
background tissue (cervical soft tissue).
Statistics
Statistical analysis between experimental groups was conducted
using either the 2-tailed independent sample student t test or
one-way ANOVA w/post-hoc analysis. Graphical bar-plots were
produced using Microsoft Excel, while ROC curves were created with
Sigmaplot (12.3). Paired tests were used for TCGA analysis due to
matched expression data. For survival analysis, Cox proportional
hazards regression was employed using the R `survival` package.
Results
MMPs are Overexpressed in HNSCC
To evaluate MMP expression levels in HNSCC from the TCGA,
patient-derived tumor specimens were compared with matched normal
control tissue. TCGA profiled matched normal tissue for
approximately 10% of the patients (37 of 377). Thus, this data was
used in our analysis. HNSCC tumors showed increased expression of
multiple MMPs compared to matched control non-tumor tissue (FIG.
29A, all p values <0.01). Interestingly, MMP14 (also known as
MT1-MMP) was the protease with the highest total expression in
tumor tissue and had significantly higher expression in tumor
compared to matched control tissue (p<10.sup.-5). The second
highest expressing MMP in tumor tissue was MMP2. MMP2 and 9 share a
common cleavage sequence and they have been particularly well
characterized in prior studies in association with HNSCC.sup.25. It
was found that both MMP2 (p<10.sup.-10) and MMP9
(p<10.sup.-6) have significantly greater RNA expression in HNSCC
tumors compared to paired-control tissue (n=37, (34 HPV- and 3
HPV+), Wilcoxon signed-rank test).
MMP 2 and 14 Stratify Survival in HPV+HNSCC
Next, the difference in MMP expression between HPV+ and HPV- tumors
was evaluated. It was found that HPV+ tumors had less overall MMP
expression compared to HPV- tumors (FIG. 29B, Kruskal-Wallis test
on pooled RNA levels, p<10.sup.-10). This is consistent with the
hypothesis that HPV+ tumors are less biologically aggressive, and
consequently, that these patients tend to have improved survival
compared to patients with HPV- tumors. Interestingly, it was found
that in patients with HPV+HNSCC, increased expression levels of
MMP2 and MMP14 correlated with worse survival (FIG. 29C, D
p<0.01). Patients with HPV+ tumors who have the highest MMP2 and
MMP14 expression (FIG. 31C, D red lines) had significantly worse 5
year survival compared to patients with the lowest expression
levels of these proteases (FIG. 29C, D blue lines). Additionally,
for a given patient with HPV+ tumor, there is a significant
correlation between MMP2 and MMP14 expression (FIG. 33, Spearman
Rho=0.56, p<10.sup.4). Thus, poor prognosis HPV+ tumors
stratified in the highest quartiles of MMP2 expression are also
likely to have higher expression of MMP14. The same correlation in
MMP expression with survival in patients with HPV- tumors was not
found. The cause of this is multifactorial and likely related to
the observation that HPV-tumors have more genetic mutations
compared HPV+ tumors.sup.26.
Zymography
To confirm MMP2/9 activity in mouse HNSCC xenografts, cleavage of
gelatin by tumor homogenates was measured via zymography. A
two-fold increase in MMP9 and a 13-fold increase in MMP2 activity
in HNSCC xenografts compared to normal mouse tongue tissue was
found (FIG. 34).
RACPP in Ex-Vivo HNSCC
To evaluate ex-vivo MMP2/9 activity in human and mouse HNSCC
specimens, the maximum rate of Cy5/Cy7 ratio change over time in
homogenates following addition of RACPP was measured (FIG. 30A, B).
It was found that patient derived HNSCC specimens show higher
MMP2/9 activity compared to non-tumor tissue (FIG. 30C) (ROC
Analysis: AUC=1.000, p=0.01). Similarly, mouse HNSCC xenografts
also show higher Cy5/Cy7 rate change, signifying higher MMP2/9
activity compared to non-tumor tissue (ROC Analysis: AUC=1.000,
p=0.03).
RACPP Improves Detection of HNSCC
To test tumor-dependent Cy5/Cy7 ratiometric change in living mice,
tongue tumor bearing nu/nu mice were intravenously injected with
RACPP (n=25). Multispectral imaging of these live, anesthetized
mice (ex 620, em 640-840 nm, Maestro, CRI at 2 hours after
injection) with both tongue and subcutaneous cervical tissue
exposed was conducted (FIG. 31A). The tongue was excised and
multispectral imaging of the tongue performed. Histologic
information regarding tumor location and size was correlated and
mapped to ratiometric fluorescence image of the tongue. Sample
(tumor and non tumor tissue) Cy5/Cy7 ratios were divided by
"background" subcutaneous cervical tissue Cy5/Cy7 ratio to compute
a "normalized Cy5/Cy7 ratio".sup.12.
It was found higher Cy5/Cy7 ratiometric fluorescence in tumor (FIG.
3B, red color) compared to adjacent normal tongue (FIG. 31B, tan
color). Injections of our control (uncleavable) probe revealed no
ratiometric difference between tongue tumor and normal tongue (FIG.
31C). Following intravenous administration of MMP2/9-cleavable
RACPP, mice showed greater normalized Cy5/Cy7 ratio in tumor
(1.61.+-.0.05, n=22) compared to normal tongue (1.11.+-.0.03, n=20,
p<10.sup.-8). This increase in ratiometric fluorescence in
orthotopic tumors was not seen following intravenous injection of
uncleavable control probe (tumor=1.01.+-.0.04, n=3; normal
tongue=1.07.+-.0.03, n=3, p=0.30) (FIG. 31D).
The receiver-operating curve (ROC) for cleavable RACPP revealed an
area under the curve (AUC) of 0.995.+-.0.006 (p<10.sup.-4) with
a peak sensitivity of 95% and peak specificity of 100% for a
normalized ratio cutoff of 1.345 (FIG. 31D insert). The two tumor
specimens not detected by this threshold-cutoff had relatively low
tumor burden (<60% involvement, see below).
RACPPs Enable Stratification of Tumor Burden
One critical component of intraoperative margin evaluation is
determining how much tumor burden is present at the edges of the
surgical field. To evaluate the ability of RACPP to stratify tissue
with variable tumor burden, percent involvement of cancer within
tumor-bearing portions of each sample was approximated by a
pathologist blinded to experimental conditions. It was found that
varying levels of tumor burden among the 22 samples ranging from
25-100% invasion (FIG. 32A). To evaluate the stratification of
ratiometric fluorescence values between different levels of tumor
burden, samples were statistically separated into the following
tertiles of cancer involvement: 25-60% (n=8), 61-80% (n=8), 81-100%
(n=6) (FIG. 32B). Adjusted Cy5/Cy7 ratios were computed for each
tertile and compared with normal tongue tissue (n=20).
It was found that all tertiles of varying tumor burden showed
significantly greater normalized Cy5/Cy7 ratio than normal tongue
tissue (FIG. 32C; lowest tertile of tumor involvement=1.46.+-.0.07,
p<10.sup.-5, middle tertile=1.67.+-.0.12, p<10.sup.-7,
highest tertile=1.72.+-.0.04, p<10.sup.-7). Additionally, tumors
with percent involvement in the highest and middle tertiles showed
significantly greater normalized ratios than the lowest tertile
(p=0.01 for highest vs. lowest tertile, p=0.03 for middle vs.
lowest tertile). Future experiments will focus on evaluating the
ability of RACPP to detect incrementally smaller levels of tumor
burden (i.e. from 1-25% involvement).
Discussion
In this example, mRNA expression levels for MMPs in human HNSCC
were analyzed using The Cancer Genomic Atlas (TCGA), the largest
available collection of human HNSCC specimens. The prognostic value
of MMP overexpression in terms of survival in patients with HPV+
and HPV- HNSCC tumors was also evaluated. It was found that many
MMPs are overexpressed in HNSCC tumors compared to paired control
tissue. However, patients with HPV+HNSCC tumors have significantly
lower overall MMP levels compared to patients with HPV- HNSCC
tumors. This finding is consistent with previous studies showing
that patients with HPV+HNSCC tumors have better overall survival
compared to patients with HPV- HNSCC tumors.sup.27.
Of the various MMPs thought to be involved in cancer, attention has
focused on MMP2/9 because they are overexpressed in a variety of
malignant tumors and their expression is often associated with
tumor grade and poor patient prognosis. Interestingly, it was found
that of the MMPs that are increased in tumor compared to control
tissue, MMP2 and MMP14 are expressed at higher levels compared to
all other MMPs, suggesting that these proteinases may be
particularly important in HNSCC. Furthermore, it was found that in
patients with HPV+HNSCC tumors, increased MMP2 and MMP14 expression
levels correlated with worse overall survival. If clinically
validated in a prospective trial, the increases in MMP2 and MMP14
represent two molecular biomarkers that can individualize
management of patients with HPV+ tumors.
Using MMP2/9 cleavable RACPP, It was found that ex-vivo human HNSCC
specimens show greater activity compared to normal tissue. This
finding correlates with previous studies demonstrating higher
MMP2/9 expression at the invasive edge of tumors.sup.28. The high
sensitivity and specificity of RACPPs to differentiate between
tumor and normal tissue suggests that ex-vivo measurements of
MMP2/9 activity in HNSCC specimens may complement MMP mRNA
expression studies in evaluating patient prognosis and in
determining which patients would benefit from RACPP guided
surgery.
In multiple human cell line models of HNSCC xenografts, it was
found that higher MMP2/9 activity as evidenced by gelatinase
zymography and higher ratiometric fluorescence signal following
systemically applied RACPP compared to non-tumor tissue. All ratios
were computed from histologically confirmed tumor or normal tissue,
eliminating verification bias. The ideal discrimination threshold
for detecting cancer versus normal tissue is 1.345, which is
consistent with previously reported ratiometric thresholds for this
probe.sup.12. Our study tested multiple tongue squamous cell
carcinoma cell lines from ATCC to highlight the RACPP's broad
applicability.
One critical component of intraoperative margin evaluation is
determining how much tumor burden is present at the edges of the
surgical field. It was found that within tumor bearing tissue, the
greater the tumor burden, the greater the ratiometric fluorescence
signal following intravenous RACPP administration. Percent tumor
involvement has been shown to be important for survival and
recurrence outcomes in prostate and breast cancer.sup.23'.sup.29.
The correlation between intraoperative ratiometric fluorescence
level and tumor burden suggests that RACPP can improve
intraoperative decision making by providing information regarding
local level of tumor involvement and consequently margin
clearance.
REFERENCES FOR EXAMPLE 5
1. Ferlay J, Shin H R, Bray F, Forman D, Mathers C, Parkin D M.
Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int
J Cancer 2010; 127:2893-2917. 2. D'Souza G, Kreimer A R, Viscidi R
et al. Case-control study of human papillomavirus and oropharyngeal
cancer. N Engl J Med 2007; 356:1944-1956. 3. Shin D M, Khuri F R.
Advances in the management of recurrent or metastatic squamous cell
carcinoma of the head and neck. Head Neck 2013; 35:443-453. 4.
Uloza V, Liutkevicius V, Pangonyte D, Saferis V, Lesauskaite V.
Expression of matrix metalloproteinases (MMP-2 and MMP-9) in
recurrent respiratory papillomas and laryngeal carcinoma: clinical
and morphological parallels. Eur Arch Otorhinolaryngol 2011;
268:871-878. 5. Wittekindt C, Jovanovic N, Guntinas-Lichius O.
Expression of matrix metalloproteinase-9 (MMP-9) and blood vessel
density in laryngeal squamous cell carcinomas. Acta Otolaryngol
2011; 131:101-106. 6. Liu W W, Zeng Z Y, Wu Q L, Hou J H, Chen Y Y.
Overexpression of MMP-2 in laryngeal squamous cell carcinoma: a
potential indicator for poor prognosis. Otolaryngol Head Neck Surg
2005; 132:395-400. 7. Mallis A, Teymoortash A, Mastronikolis N S,
Werner J A, Papadas T A. MMP-2 expression in 102 patients with
glottic laryngeal cancer. Eur Arch Otorhinolaryngol 2012;
269:639-642. 8. Zhou C X, Gao Y, Johnson N W, Gao J.
Immunoexpression of matrix metalloproteinase-2 and matrix
metalloproteinase-9 in the metastasis of squamous cell carcinoma of
the human tongue. Aust Dent J 2010; 55:385-389. 9. Chaudhary A K,
Singh M, Bharti A C, et al. Genetic polymorphisms of matrix
metalloproteinases and their inhibitors in potentially malignant
and malignant lesions of the head and neck. J Biomed Sci 2010;
17:10. 10. Olson E S, Aguilera T A, Jiang T et al. In vivo
characterization of activatable cell penetrating peptides for
targeting protease activity in cancer. Integr Biol (Camb) 2009;
1:382-393. 11. Aguilera T A, Olson E S, Timmers M M, Jiang T, Tsien
R Y. Systemic in vivo distribution of activatable cell penetrating
peptides is superior to cell penetrating peptides. Integr Biol
2009; 1:371-381. 12. Savariar E N, Felsen C N, Nashi N et al.
Real-time in vivo molecular detection of primary tumors and
metastases with ratiometric activatable cell-penetrating peptides.
Cancer Res 2013; 73:855-864. 13. Savariar E N, Felsen C N, Nashi N
et al. Real-time in vivo molecular detection of primary tumors and
metastases with ratiometric activatable cell-penetrating peptides.
Cancer Res 2013; 73:855-864. 14. Hague R, Contreras R, McNicoll M
P, Eckberg E C, Petitti D B. Surgical margins and survival after
head and neck cancer surger. BMC Ear Nose Throat Disord 2006; 16:2.
15. Singletary S. Surgical margins in patients with early-stage
breast cancer treated with breast conservation therapy. Am J Surg
2002; 184:383-393. 16. Nagtegaal I D, Quirke P. What is the role
for the circumferential margin in the modern treatment of rectal
cancer? J Clin On 2008; 26:303-312. 17. Meric F, Mirza N, Vlastos G
et. al. Positive surgical margins and ipsilateral breast tumor
recurrence predict disease-specific survival after
breast-conserving therapy. Cancer 2003; 97:926-933. 18. Snijder R,
de la Riviere A, Elbers H, van den Bosch J. Survival in resected
stage I lung cancer with residual tumor at the bronchial resection
margin. Annals of Thoracic Surg 1998; 65. 19. Dotan Z, Kavanagh K,
Yossepowitch 0 et al. Positive surgical margins in soft tissue
following radical cystectomy for bladder cancer and cancer specific
survival. J Urol 2007; 178:2308-2312. 20. Wieder J A, Soloway M S.
Incidence, etiology, location, prevention and treatment of positive
surgical margins after radical prostatectomy for prostate cancer. J
Urol 1998; 160:299-315. 21. Gross A, Orosco R, Shen J et. al. A
prognostic model of head and neck cancer ties TP53 mutation to 3p
loss. Nature Genetics [Under Review]. 22. Toth M, Sohail A, Fridman
R. Assessment of gelatinases (MMP-2 and MMP-9) by gelatin
zymography. Methods Mol Biol 2012; 878:121-35. 23. Rampersaud E N,
Sun L, Moul J W, Madden J, Freedland S J. Percent tumor involvement
and risk of biochemical progression after radical prostatectomy. J
Urol 2008; 180:571-576; discussion 576. 24. Thompson I M, 3rd,
Salem S, Chang S S et al. Tumor volume as a predictor of adverse
pathologic features and biochemical recurrence (BCR) in radical
prostatectomy specimens: a tale of two methods. World J Urol 2011;
29:15-20.
25. Xia T, Akers K, Eisen A Z, Seltzer J L. Comparison of cleavage
site specificity of gelatinases A and B using collagenous peptides.
Biochim Biophys Acta 1996; 1293(2):259-266. 26. Maruyama H, Yasui
T, Ishikawa-Fujiwara T, et al. Human papillomavirus and p53
mutations in head and neck squamous cell carcinoma among Japanese
population. Cancer Sci 2014. [In Press] 27. Sivars L, Nasman A,
Tertipis N et al. Human papillomavirus and p53 expression in cancer
of unknown primary in the head and neck region in relation to
clinical outcome. Cancer Med 2014; 3(2): 376-84. 28. Kuniyasu H,
Troncoso P, Johnston D et al. Relative expression of type IV
collagenase, E-cadherin, and vascular endothelial growth
factor/vascular permeability factor in prostatectomy specimens
distinguishes organ-confined from pathologically advanced prostate
cancers. Clin Cancer Res 2000; 6(6): 2295-308. 29. Kohrt H E, Noun
N, Nowels K, Johnson D, Holmes S, Lee P P. Profile of immune cells
in axillary lymph nodes predicts disease-free survival in breast
cancer. PLoS Med 2005; 2:e284.
Example 6
A. Significance:
A1.
Multiple proteases have been evaluated for their roles in cancer
growth, invasion and metastasis, including matrix
metalloproteinases (MMPs).sup.1 and urokinase plasminogen activator
(uPA), cathepsins, interstitial collagenase (aka MMP1), elastases,
.sup.2. MMPs are a class of endopeptidases that breakdown
extracellular matrix leading to localized inflammation and tissue
permeability both of which are associated with tumorigenesis and
metastasis. Broad inhibition of MMPs for the treatment of advanced
cancer has been unsuccessful in clinical trials.sup.3. It is now
recognized that MMPs can have both inhibitory and stimulatory
effects on tumor progression.sup.4,5, thus a better understanding
of the in vivo activity of specific MMPs in the context of cancer
is needed to develop effective therapies or imaging agents. MMP2
and 9 are two very well studied gelatinases that can degrade
collagen in the basement membrane which is postulated to be
necessary for angiogenesis and metastasis.sup.6. Also the
inflammatory microenvironment within tumors causes upregulation of
MMP2 and 9 via MMP14 activation leading to invasion in intestinal
cancer.sup.7. MMP14 (also known as MT1-MMP) is a membrane-tethered
active protein that accumulate in invadopodia-like structures on
the cell membrane to allow the cells to tunnel through the
surrounding matrix. Inhibition of MMP14 expression with RNA
interference had no effect on triple negative breast cancer cell
growth but significantly diminished the number of migrating tumor
cells and the incidence of lung metastasis.sup.9.
Although MMP2,9 are also increased in inflammation/wound healing,
absolute levels of these gelatinases in the head and neck have been
used to differentiate between benign papillomas versus carcinoma of
the larynx.sup.10 Increased MMP2,9 expression has been shown to
correlate with cancer grade.sup.11 and decreased
survival.sup.12,13. In carcinoma of the tongue, increased MMP2,9
expression has been shown to correlate with incidence of lymph node
metastases.sup.14. From our own TCGA data analysis, it was found
that all cancers, including HNSCCs have significantly higher
expression of many MMPs compared to match normal tissue
(particularly MMP14, MMP1, MMP2, MMP9; FIG. 36).sup.15. It was also
found that tumors from patients with HPV- HNSCC (which have poor
prognosis compared to HPV+HNSCC) have significantly higher MMP
expression levels compared to tumors from patients with
HPV+HNSCC.sup.15. Interestingly, it was also found that in patients
with HPV+HNSCC, increased MMP2 and MMP14 expression correlated with
worse survival.sup.15, again, consistent with the hypothesis and
previous reports of a correlation between increased MMP levels and
worse prognosis.
In addition to the well-studied role of MMPs, plasminogen
activation is also believed to be critical in the progression of
multiple human cancers by facilitating matrix degradation during
invasion and metastasis.sup.16. Urokinase plasminogen activator
(uPA) levels as measured by zymography has been shown be highly
increased in tumor compared to adjacent normal tissue.sup.2. From
our own TCGA data analysis, it was also found that uPA mRNA
expression is highly increased in tumor compared to paired normal
tissue for multiple cancers including HNC (FIG. 36, Gross et al,
unpublished data). In contrast, mRNA expression levels of tissue
plaminogen activator (tPA), elastases and cathepsins are not
significantly increased in the TCGA tumor specimens (data not
shown, Gross et al, unpublished).
A3. Ratiometric Cell Penetrating Peptides (RACPP)s
Ratiometric activable cell penetrating peptides (RACPP, FIG. 36)
which are protease sensitive molecules that undergo a change in the
Cy5-Cy7 fluorescence intensity ratio and localized retention upon
cleavage by MMPs that are upregulated on the surface of many tumor
tissues have been previously described.sup.17. In contrast to mRNA
expression or copy number detection methods, RACPPs detect in vivo
protease activity, thus bypassing the complicated interplay between
transcription/translation, pro- and active forms of the enzymes, as
well as the presence of inhibitors/activators etc. It was shown
that RACPP uptake correlates with tumor burden.sup.15 and with
tumor characteristics corresponding to poor survival.sup.18. This
class of molecule has been used intraoperatively to demonstrate
improved tumor free survival following surgery in breast cancer,
melanoma.sup.19 salivary gland cancer.sup.20 and pancreatic
adenocarcinoma.sup.21. It the also demonstrated decreased tumor
burden following delivery of targeted chemotherapeutics.sup.22 and
radiosensitizer.sup.23 in animal models. Although this first
generation molecule promises wide applicability for use in surgical
resection of multiple types of solid tumors and is being tested in
an FDA Phase 1b clinical trial, one disadvantage is that the
current cleavable component of the molecule (PLGC(Me)AG) (SEQ ID
NO: 2) is cut by multiple MMPs, limiting signal to noise resolution
and potentially limiting sensitivity/specificity of the probe to
precisely define tumor boundaries.
Literature review and our own recent analysis of TCGA data showed
that mRNA expression of several proteases are selectively increased
in tumor compared to normal tissue in patient-derived samples (FIG.
35, 36).sup.2,24. In this example, rational design will be used to
improve selectivity of the cleavable component by the tumor
selective proteases (MMP14, 1, 2, 9, 11 and uPA) in human HNC. The
sensitivity/specificity of novel individual protease-selective
RACPPs for HNC tumor detection and compare tumor free survival
following surgery with and without protease-cleavable RACPP
fluorescence guidance will be determined.
Radiotherapy is a mainstay treatment modality for HNC, as
definitive therapy or postoperative adjuvant.
Intensity-modulated-radiation-therapy (IMRT) has the benefit of
reducing morbidity through highly conformal ionizing radiation
delivery.sup.25. In the second part of this example, it will be
tested whether uptake of these novel protease-selective RACPPs can
be exploited to modulate tissue sensitivity to radiotherapy with
the intent to spare adjacent tissue injury without compromising and
potentially enhancing tumor control. The novel protease-selective
ACPPs will be linked with known radiosensitizers and evaluate tumor
control following dual action radiotherapy.
B. Innovation
There are four main areas of innovation in this proposal. The first
one is the use of TCGA for direct identification of clinically
relevant proteases for a specific type of cancer (in this case head
and neck cancer). Several candidate proteases which show the
highest mRNA expression (MMP14, aka MT1-MMP, MMP1, 2, 9, 11 and
uPA) in tumor specimens compared to paired normal tissue were
identified.sup.15. Rational strategies.sup.26 to design the
cleavable site to provide selectivity for our molecularly targeted
agents were used.
The second area of innovation is the use of TCGA to direct
preclinical tumor model classification for use in this study. Using
TCGA, it was found that patients whose tumors have combined TP53
mutation and 3p deletion (double-hit) have significantly poorer
clinical outcome compared to patients with either events alone
(single-hit).sup.24. Multiple readily available cell lines that
mirror these classifications have been identified.sup.18. This
double/single-hit classification for tumor xenografts and
spontaneous oral carcinogen-derived models will be used in this
example to evaluate whether or not differential protease activity
and sensitivity to ionizing radiation mechanistically contribute to
this difference in clinical outcome.
A third area of innovation is the use of ratiometric fluorescence
guided molecular imaging during surgery to evaluate protease
activity at the advancing tumor edge vs. metastatic lymph
nodes.sup.17. The advantages of ratiometric vs. single-intensity
measurements are well known.sup.27 in fluorescence microscopy and
flow cytometry and are leveraged here for intraoperative molecular
imaging. It is hypothesized that high levels of specificity and
sensitivity for a given protease-specific probe at tumor edges or
metastatic lymph node will result in high signal to noise ratio
required for potential clinical translation. In vivo fluorescence
imaging will be used with molecular navigation to guide surgical
resection of tumors and compare tumor free survival with surgery
using protease-selective cleavable probes vs surgery using white
light reflectance alone in HNC, building on our expertise gained
during our prior studies in multiple other solid tumors, including
breast, melanoma, salivary gland carcinoma.sup.20 and pancreatic
adenocarcinoma.sup.11.
Finally, a fourth area of innovation is to use molecular targeting
of protease activity for guided radiotherapy or to localize
radiation sensitizers to the cells/tumors that have the highest
protease levels.sup.23. Tumors with high protease levels (MMPs,
uPA) have previously been shown to correlate with highest stage,
grade and metastatic potential.sup.2,11-13. It is hypothesized that
protease-cleavable probes may be useful for targeting advanced
stage tumors and that this characteristic can be leveraged to
target the highest levels of localized radiation with or without
radiosensitizers to improve tumor control and decrease damage to
surrounding tissue.
B1. Innovation 1a--TCGA Directed Identification of Clinically
Relevant Proteases.
Although there has been significant literature documenting the
correlation of protease levels with stage, grade and metastatic
potential.sup.11,13,14 data from TCGA was used which represent the
largest collection of tumors with detailed multi omics
characterization to identify proteases which are most highly
expressed (mRNA) for multiple cancers. One limitation of mRNA
expression is that it correlates poorly with protein abundance,
which further correlates poorly with enzymatic activity. As
mentioned above, increased MMP14 and MMP2 mRNA did correlate with
worse overall survival for patients with HPV+ tumors however this
correlation was not found in patients with HPV- tumors, likely due
to increased heterogeneity in the HPV- population.sup.17. This
example will leverage TCGA mRNA data to develop protease sensitive
probes that will report enzymatic activity which potentially has
much better correlation with actual tumor biology and prognostic
utility. Although the focus of this proposal is proteases in
HNC.sup.15, proteases important in multiple other solid tumors that
will benefit from rational design of protease-selective probes were
identified.
Innovation 1b--Rational Design of Molecular Imaging Agents Specific
for Select MMPs and uPA
In contrast to expression detection methods for proteases, RACPPs
which are capable of monitoring enzyme activity in real time in
living animals have been previously described.sup.17. RACPPs are
molecular targeting probes that target selective proteases by
linking positively charged cell penetrating peptide (FIG. 38, blue
segment) to the neutralizing negatively charged segment (FIG. 38,
red segment) with a protease selective linker (FIG. 38, green
segment). The emission of the Cy5 (far red) fluorescent donor is
quenched in favor of Cy7 re-emission until the intervening linker
is cleaved by tumor-associated proteases, which ratiometrically
increases the Cy5:Cy7 emission ratio up to 40 fold and triggers
tissue retention of the Cy5-containing fragment. This large change
in ratio provides a wide dynamic range in which protease activity
in tumors and metastases can be quantitatively differentiated from
adjacent normal tissue.sup.17. Furthermore, it has been shown that
RACPPs can be used as a high throughput assay to interrogate
existing ex vivo patient tumor specimens to probe protease
activity.sup.15 and correlate with clinical stage and outcome. This
represents a significant advance compared to existing technologies
measuring mRNA or protein expression which correlates poorly with
enzymatic activity and limits correlation with clinical
outcomes.
To generate the RACPP cleavable sites (FIG. 38, green segment) that
are selective for given MMPs, previously identified substrates and
recent data by Ranikov et al.sup.26 who used a large set of phage
peptide substrates were leveraged to interrogate cleavage rates of
multiple MMPs to determine the relative impact of individual
specificity determining positions (SDP). These authors identified
specific substrates and consensus residues from P3 to P2' that were
highly selected and therefore likely preferred by a given
MMP.sup.26. In particular, there was a focus on identifying and
testing specific substrates that distinguish the two gelatinases
MMP2 and MMP9, and MMP14 which have similar substrate preference
and were highly upregulated in head and neck tumors and most other
cancers from TCGA (FIG. 35).
To generate novel RACPP species that are selective for uPA, a set
of protease substrates were screened that were diverse and likely
cleaved by serine protease in that they contain either a lysine or
arginine residue flanked by diverse amino acids. Upon testing, one
6 amino acid substrate with sequence YGRAAA (SEQ ID NO: 17) was
found to be efficiently cleaved by urokinase plasminogen activator
(uPA) and to a lesser extent by tPA. Several other substrates that
were reported to be cleaved by uPA were also tested but were not
cleaved possible because the constrained structure of the substrate
within the context of an ACPP.
B2. Innovation 2--TCGA directed tumor model classification.
In our recent analysis of TCGA HNSCC specimens, it was found that
although TP53 has previously been shown to be the most commonly
mutated, prognostic driver gene in HNSCC.sup.28, in patients with
HPV- tumors, the detrimental impact of TP53 mutation occurs) .sub.4
only in combination with loss of chromosome 3p.sup.24. The combined
TP53 mutation and 3p deletion (double-hit) led to a marked decrease
in median survival from >5 years for TP53 mutation only
(single-hit) to 1.7 years for both events. In the 3p region, it was
found that copy number variation for Fragile Histidine Triad
Protein (FHIT) correlates best with 3p deletion status. FHIT has
been shown to be a tumor suppressor.sup.29 and FHIT deficient mice
show both increased incidence of spontaneous tumor formation as
well as increased tumor formation in response to
carcinogens.sup.30. Loss of FHIT expression has been shown to
correlate with poor outcome in patients with tongue cancer.sup.31.
Using already available, well characterized HNC cell lines which
have either the "double-hit" (Cal 27, SCC15, SCC25) or "single-hit"
(SCC4) genotype, it was previously shown that "double-hit" tumors
have significantly higher generalized MMP activity compared to
"single-hit" tumors (using our first generation RACPP).sup.15. This
example aims to test the difference in specific protease activity
(i.e. MMP14, 1, 2, 9, 11 or uPA between the two tumor types (double
vs. single-hit). A parallel experiment will be to test the specific
protease activity levels (i.e. MMP14, 1, 2, 9, 11 or uPA) in
metastatic lymph nodes derived from these tumor types. Our
hypothesis is that RACPPs that are uniquely sensitive to cleavage
by single individual proteases will demonstrate improved
sensitivity/specificity for differentiation tumor vs. adjacent
normal tissue compared to our existing first generation PLGC(me)AG
(SEQ ID NO: 2) substsrate which is cleaved by multiple
proteases.
B3. Innovation 3-Molecular Imaging to Guide Surgery, Pathological
Examination of Surgical Specimens and Ex Vivo Examination of
Protease Activity of Banked Tissues for Correlation with
Outcome
ACPPs have previously been shown to improve tumor free survival
following fluorescence guided surgery for breast, melanoma,
pancreatic and salivary gland cancers in animal models. Extensive
experience in fluorescence guided surgery with molecular navigation
to evaluate tumor-free survival with and without the use of these
novel protease-selective probes in animal models of HNC will be
used. One important difference between our proposed use of
ratiometric fluorescence imaging with RACPP vs. other antibody or
ligand-fluorescence conjugate strategies is quick wash out period
of approximately 90 minutes for our agents.sup.17 compared to days
for antibody based technologies.sup.12,32. The quick wash out
period allows RACPP agents to be administered on the day of surgery
during preoperative preparations instead of bringing the patient in
several days prior to surgery solely to administer the targeting
agent, simplifying the clinical work flow and minimizing related
costs.
Furthermore, previous work that surgical specimens treated with
intravenous RACPPs retain ratiometric fluorescence in the frozen
blocks to focus ex vivo pathological examination of the tissue will
be expanded (FIG. 39.sup.19), potentially improving diagnostic
accuracy for eventual ex vivo clinical translation. Finally, this
example aims to use protease-selective RACPPS to develop an ex vivo
screening assay of protease activity in banked tissue (FIG.
40.sup.15) for correlation with stage and clinical outcome.
Although focusing on HNC animal models in this examiner, it has
been found that MMPs 14, 1, 2, 9 and uPA are highly overexpressed
in patient specimens from multiple cancer sites including ones with
highest overall incidence of positive margins (FIGS. 35, 36).
Consequently, novel protease-specific targeting molecules developed
within this proposal for surgical guidance and modulation of
ionizing radiation can potentially be also used to improve outcome
in these other cancer sites with high unmet clinical need.
C2. Experimental Design
C2.1--Specific Aim 1. Rational Design of Novel Protease-Selective
Substrates
a: Use rational design to develop new peptide substrates that are
specific for cleavage by MMP14, MMP2, MMP1, MMP11, MMP9 and uPA.
There are 5 promising peptide substrate candidates for 4 different
proteases with high specificity (MMP14, MMP2, MMP9 and uPA). This
example will focus on characterizing these initial promising
substrate candidates and extent our strategy to other
substrates.
b: Incorporate these peptide substrates into our FRET based RACPP
design and evaluate enzyme kinetics
Problem being Addressed:
RACPPs are capable of monitoring protease activity in real time
living animal models of cancer. Our first generation RACPP,
although good enough to have received FDA approval for ongoing
clinical testing, shows non-specific cleavage by multiple MMPs. To
tease out the involvement of specific individual proteases involved
in the various stages of cancer growth, invasion and metastasis, it
was sought to identify RACPP cleavable sites that are specific for
individual select proteases.
Experimental Details:
Using TCGA, extracellular proteases were classified by their
differential mRNA expression between tumor and paired normal
tissue.sup.15. Several MMPs were identified (14, 1, 2 and 9, 11
FIG. 35) and uPA (FIG. 36) as showing highest differential
expression between cancer and paired normal tissue for HNC and many
other cancers. Next, it was sought to identify amino acids
sequences that might be cleaved specifically by these
proteases.
To generate new substrates for MMPs, amino acids were substituted
at key position at and near the cleavage site based on the
following rule derived by recent work by Ratnikov et al.sup.26 that
the amino acid at a certain position must improve cleavage by the
specific enzyme and reduce cleavage by other related enzymes. For
increased specificity, an amino acid should be red for the enzyme
of interest compared green for the other enzymes at the same
substitution position (FIG. 36A). These substrates will be tested
by treating 5 .mu.M peptide with 20 nM of each enzyme for 2 hours
and analyse by gel electrophoresis.
To generate novel RACPP species that are specific for uPA, a set of
protease substrates were screened that were diverse and likely
cleaved by serine protease in that they contain either a lysine or
arginine residue flanked by diverse amino acids.
Preliminary Results:
MMP Selective Substrates:
Our current best MMP14-selective substrate is derived from a
previously reported substrate which was selective for MMP-14 over
MMP-2 and 9 but was inefficiently cleaved.sup.23. This novel
substrate, RSHG-(homoF)-FLY (SEQ ID NO: 70), was generated using
site specific substitution based on consensus cleavage sequences
reported by Ratnikov et al. and is highly selective and efficiently
cleaved by MMP-14 over other MMPs. Our current best MMP2 selective
substrate (TIAH/LH) is selectively cleaved by MMP-2 versus 9 and
14. There are two MMP9 selective substrates, SNPYK-Y (SEQ ID NO:
21) and SNPYG-Y (SEQ ID NO: 23). FAM/Cy5 ratiometric versions of
RACPPs with these sequences were made and tested to confirm
selectivity. The preferred form Cy5/Cy7 ratiometric versions for in
vivo testing with a pegylated carrier to help with solubility is
currently being synthesized. For MMP 11 a similar approach will be
adopted in finding selective substrates..sup.26.
uPA Selective Substrates:
A substrate with sequence YGRAAA (SEQ ID NO: 17) was found to be
efficiently cleaved by uPA and to a lesser extent by tPA. Although
this cleavage sequence demonstrates specificity for uPA compared to
MMPs (FIG. 41), a goal is to further optimize cleavage kinetics
with additional sequential peptide substitutions and testing
against an extended panel of cancer-associated proteases.
Potential Pitfall:
Designing a completely specific substrate can be very challenging
since enzymes are known to be promiscuous and the substrate may be
cleaved to a small extent by other enzymes. This is especially true
for highly related enzymes like MMP2 and 9.
To assure specificity, time course measurements will be performed
with the fluorescence plate reader and determine Michaelis Menton
kinetics rates. Our substrate candidates will proceed to in vivo
testing if they are cleaved by the intended MMP at a 10 fold higher
rate than the other related MMPs. To be certain that a given
protease is specifically cleaving a given substrate, RNA
interference will be used to knock down each protease or explore
using murine knockout of a given protease.
c2.2--Specific Aim 2. Determine Sensitivity/Specificity of Novel
Protease-Selective RACPPs for Tumor Detection During Surgery, Ex
Vivo Pathological Examination of Surgical Specimens, Ex Vivo
Screening Assay to Determine Individual Protease Activity for
Banked Tissues.
a)Evaluate specificity and sensitivity of MMP14, 2, 9, 1, 11 or
uPA-selective RACPPs for tumor margin and lymph node metastasis
detection in vivo
b) Evaluate specificity and sensitivity of MMP14, 2, 1, 9, 11 or
uPA-selective RACPPs for tumor margin and lymph node metastasis
detection ex vivo in surgical specimens
d) Test tumor-free survival following surgery with and without
protease-selective RACPP fluorescence guidance
c) Develop ex vivo screening assay to determine individual
selective protease activity for banked tumor samples and correlate
with clinical stage and outcome
Problem being Addressed:
Nonspecific cleavage of previously described MMP sensitive probes
has been shown to give rise to high uptake in non-cancer tissue
such as tissues nearly injury sites, inflammation and
cartilage.sup.19,34,35. Furthermore, a given patient derived tumor
may have different protease activity profile compared. The panel of
RACPPs will be used to develop in SA1 with high specificity for a
given protease to systematically evaluate their cancer sensitivity
and specificity in surgical specimens compared to gold standard
evaluation using H&E. It is anticipated that development of
panel of protease-selective RACPPs will enable precise reporting
for a given patient tumor profile and thus better correlation with
clinical stage and outcome
Experimental Details:
Tumor detection in vivo: To evaluate level of specific protease
activity in tumor vs. surrounding tissue and ability to improve
surgical margin detection, tumor-bearing will be injected mice
intravenously (IV) with the various protease-selective RACPPs and
noncleavable controls. In vivo apparent tumor vs. adjacent normal
tissue will be documented with ratiometric fluorescence and white
light reflectance imaging using a customized fluorescence
dissecting surgical scope as previously described.sup.17. The mice
will then be sacrificed according to UCSD approved protocol;
apparent tumors and adjacent normal tissue will be harvested,
cryosectioned and ratiometric fluorescence imaging for protease
activity measured. Sensitivity and specificity will be evaluated
for tumor identification using a receiver operator curve against
the gold standard of H&E analysis for each protease-selective
probe on multiple double-hit and single-hit xenografts.
Tumor Detection Ex Vivo:
Tumor bearing mice will be injected with various protease-selective
RACPPs. Tumors will be excised using white light reflectance alone.
Ex vivo pathological examination of tissues will be performed first
with RACPP fluorescence imaging to identify foci of high uptake.
Presence or absence of cancer will be confirmed with H&E.
Receiver operator curve will be generated for each
protease-selective probe.
Metastatic Lymph Node Detection:
To evaluate level of specific protease activity in metastatic lymph
nodes vs. normal lymph nodes, tumor-bearing mice will be injected
with cervical metastasis (generated as previously described.sup.17)
intravenously (IV) with the various protease-selective RACPPs.
Ratiometric fluorescence will be documented for every cervical
lymph node as previously described.sup.17. The mice will then be
sacrificed according to UCSD approved protocol; all cervical nodes
harvested, cryosectioned and ratiometric fluorescence imaging for
protease activity measured on cryosections. Sensitivity and
specificity will be evaluated for cancer invasion for a given lymph
node using a receiver operator curve against the gold standard of
H&E analysis for each protease-selective probe.
Evaluate Tumor Free Survival Following RACPP Guided Surgery:
This example builds on prior extensive experience with ACPP guided
surgery to evaluate tumor free HNC survival following RACPP guided
surgery. The best performing novel MMP-cleavable RACPPs will be
individually tested and the best performing uPA-cleavable RACPPs in
different xenograft models of HNC-tumor bearing mice. Following
surgery with either RACPP guidance or white light reflectance only,
mice will be monitored for tumor free survival over 6 months as
previously described by our group. It is hypothesized that 1) tumor
free survival following surgery with RACPP guidance will be
improved compared to surgery with white light reflectance alone; 2)
the RACPP probe with highest sensitivity/specificity for tumor
margin detection identified in SA2a will result in the best tumor
free survival following RACPP guided surgery.
Ex Vivo Screening Assay to Determined Activity of Selected
Proteases in Banked Tissue:
Banked frozen tissue will be thawed and gently homogenized in PBS,
while kept cooled on ice to minimize the release of intracellular
and intraorganellar proteases. Homogenates will then be
microcentrifuged at 14,000.times.g for 1 minute, and supernatants
(extracts) will be tested for ability to increase the Cy5 donor:Cy7
acceptor emission ratio of a protease-selective RACPPs. Testing of
MMP14,2,9, uPA, MMP1, 11 in this order if tissue availability is
limiting will be prioritized. Data will be correlated with existing
documentation regarding patient stage and clinical outcome.
Preliminary Results:
Increased ratiometric fluorescence signal using the first
generation cleavage site of PLGC(me)AG (SEQ ID NO: 2) with
increasing tumor burden has been demonstrated (FIG. 42). FAM/Cy5
FRET RACPPs that are MMP2, MMP 9 or MMP14 selective were recently
generated. The FAM/Cy5 versions are simpler to synthesize using an
automated peptide synthesizer and are useful for running a
preliminary screen of the efficacy toward protease activity. In
Ca127 tumor-bearing mice a prominent signal enhancement is seen
with the MMP9 selective imaging probes compared to our previously
reported PLGC(me)AG (SEQ ID NO: 2) RACPP. The tumor tissue have
been preserved in OCT for analysis of localized uptake and will be
quantified.
Potential Pitfalls:
To ensure that cancer cell lines used are representative of TCGA
tumor specimens, mRNA expression profiles of various proteases of
readily available HNC cell lines were measured. It was found that
the majority of cell lines tested showed high MMP14, 1, 2 and uPA
mRNA expression, suggesting that they are good representation of
TCGA human tumor specimens.
C2.3--Specific Aim 3. Determine Efficacy of Conformal IR Dosing
Based on Protease-Selective RACPPs Imaging
a) Evaluate correlation of protease dependent RACPP signal with
differential sensitivity to ionizing radiation
b) Test in vivo tumor control of protease-responsive RACPP linked
with radiosensitizer
Problem being Addressed:
Radiotherapy is a mainstay treatment modality for 1-INC, and IMRT
has the benefit of reducing long-term morbidity through highly
conformal ionizing radiation delivery. Optimal parameters for
defining the tumor target vs. adjacent tissue remain a clinical
challenge. The hypothesis to be tested is whether IR dose
thresholds can be individualized based upon level and localization
of protease cleavable RACPP uptake.
Preliminary Data:
RACPP Localization to Highest Tumor Burden and Most Aggressive
Tumors:
It was previously shown that highest RACPP localization to
xenografts with highest tumor burden (FIG. 42).sup.15. It was
previously shown that more aggressive double-hit tumor xenografts
(Ca127, SCC15, SCC25) have significantly higher MMP activity
compared to less aggressive single-hit tumor xenografts (SCC4, FIG.
43). Finally, it was shown that the same double-hit xenografts have
less radiosensitivity compared to the single hit xenografts (FIG.
43A). When RNA inteference was used to convert the single-hit cell
line SCC4 into the double-hit genotype (FIG. 43B), a decrease in
radiosensitivity compared to the wild type cell line was seen (FIG.
43C).sup.18. Taken together, this data suggest that
protease-cleavable RACPPs can be conjugated to radiosensitizers for
targeted delivery to the areas of highest tumor burden or most
aggressive tumor cells.
Table of Protease-Selective Substrates:
Individual proteases and their respective optimal cleavage
substrates which were empirically derived.
TABLE-US-00003 TABLE 2 Table of protease-selective substrates:
Protease Cleavage Thrombin D/P/PRSFL (SEQ ID NO: 13; SEQ ID NO: 14)
Nle-TPRSFL (SEQ ID NO: 15) MMP2/9 PLGC(Me)AG (SEQ ID NO: 2)
plasminogen YGRAAA Activators (SEQ ID NO: 17) Chymase GVAYISGA (SEQ
ID NO: 16) Elastase RLQLK(Ac)L (SEQ ID NO: 26) (Nle(O-Bzl)-
Met(O)2-Oic-Abu) MMP12 PLGLEAA (SEQ ID NO: 30) ACE/Renin DRVYIHP
(SEQ ID NO: 67), DRVYIHPFHLLYYS (SEQ ID NO: 68), IHPFHLVIHT (SEQ ID
NO: 69) MMP2 TLSE-LH (SEQ ID NO: 24) TIAHLA (SEQ ID NO: 25) MMP9
SNPYK-Y (SEQ ID NO: 21) SNPKG-Y (SEQ ID NO: 22) SNPYG-Y (SEQ ID NO:
23) MMP14 RSHP(Hfe)TLY (SEQ ID NO: 19) RSHG(Hfe)FLY (SEQ ID NO: 20)
Cathepsin K KLRFSKQ (SEQ ID NO: 27)
REFERENCES FOR EXAMPLE 6
1 Bauvois, B. New facets of matrix metalloproteinases MMP-2 and
MMP-9 as cell surface transducers: outside-in signaling and
relationship to tumor progression. Biochim Biophys Acta 1825,
29-36, doi:10.1016/j.bbcan.2011.10.001 (2012). 2 Curino, A. et al.
Detection of plasminogen activators in oral cancer by laser capture
microdissection combined with zymography. Oral oncology 40,
1026-1032, doi:10.1016/j.oraloncology.2004.05.011 (2004). 3
Coussens, L. M., Fingleton, B. & Matrisian, L. M. Matrix
metalloproteinase inhibitors and cancer: trials and tribulations.
Science 295, 2387-2392, doi:10.1126/science.1067100 (2002). 4
Hamano, Y. et al. Physiological levels of tumstatin, a fragment of
collagen IV alpha3 chain, are generated by MMP-9 proteolysis and
suppress angiogenesis via alphaV beta3 integrin. Cancer Cell 3,
589-601 (2003). 5 Montel, V. et al. Altered metastatic behavior of
human breast cancer cells after experimental manipulation of matrix
metalloproteinase 8 gene expression. Cancer research 64, 1687-1694
(2004). 6 Somiari, S. B. et al. Circulating MMP2 and MMP9 in breast
cancer--potential role in classification of patients into low risk,
high risk, benign disease and breast cancer categories. Int J
Cancer 119, 1403-1411 (2006). Oshima, H. et al. Suppressing TGFbeta
Signaling in Regenerating Epithelia in an Inflammatory
Microenvironment Is Sufficient to Cause Invasive Intestinal Cancer.
Cancer Res 75, 766-776 (2015). 8 Willis, A. L., Sabeh, F., Li, X.
Y. & Weiss, S. J. Extracellular matrix determinants and the
regulation of cancer cell invasion stratagems. Journal of
Microscopy 251, 250-260, doi:10.1111/jmi.12064 (2013). 9 Perentes,
J. Y. et al. Cancer cell-associated MT1-MMP promotes blood vessel
invasion and distant metastasis in triple-negative mammary tumors.
Cancer research 71, 4527-4538, doi:10.1158/0008-5472.can-10-4376
(2011). 10 Uloza, V., Liutkevicius, V., Pangonyte, D., Saferis, V.
& Lesauskaite, V. Expression of matrix metalloproteinases
(MMP-2 and MMP-9) in recurrent respiratory papillomas and laryngeal
carcinoma: clinical and morphological parallels. Eur Arch
Otorhinolaryngol 268, 871-878, doi:10.1007/s00405-011-1494-1
(2011). 11 Wittekindt, C., Jovanovic, N. & Guntinas-Lichius, O.
Expression of matrix metalloproteinase-9 (MMP-9) and blood vessel
density in laryngeal squamous cell carcinomas. Acta Otolaryngol
131, 101-106, doi:10.3109/00016489.2010.506886 (2011). 12 Liu, W.
W., Zeng, Z. Y., Wu, Q. L., Hou, J. H. & Chen, Y. Y.
Overexpression of MMP-2 in laryngeal squamous cell carcinoma: a
potential indicator for poor prognosis. Otolaryngology--head and
neck surgery: official journal of American Academy of
Otolaryngology-Head and Neck Surgery 132, 395-400, doi:
10.1016/j.otohns.2004.09.050 (2005). 13 Mallis, A., Teymoortash,
A., Mastronikolis, N. S., Werner, J. A. & Papadas, T. A. MMP-2
expression in 102 patients with glottic laryngeal cancer. Eur Arch
Otorhinolaryngol 269, 639-642, doi:10.1007/s00405-011-1625-8
(2012). 14 Zhou, C. X., Gao, Y., Johnson, N. W. & Gao, J.
Immunoexpression of matrix metalloproteinase-2 and matrix
metalloproteinase-9 in the metastasis of squamous cell carcinoma of
the human tongue. Aust Dent J 55, 385-389,
doi:10.1111/j.1834-7819.2010.01258.x (2010). 15 Hauff, S. J. et al.
Matrix-metalloproteinases in head and neck carcinoma-cancer genome
atlas analysis and fluorescence imaging in mice.
Otolaryngology--head and neck surgery: official journal of American
Academy of Otolaryngology-Head and Neck Surgery 151, 612-618,
doi:10.1177/0194599814545083 (2014). 16 Kwaan, H. C., Mazar, A. P.
& McMahon, B. J. The apparent uPA/PAI-1 paradox in cancer: more
than meets the eye. Semin Thromb Hemost 39, 382-391, doi:
10.1055/s-0033-1338127 (2013). 17 Savariar, E. N. et al. Real-time
in vivo molecular detection of primary tumors and metastases with
ratiometric activatable cell-penetrating peptides. Cancer research
73, 855-864, doi:10.1158/0008-5472.CAN-12-2969 (2013). 18 Raju, S.
C. et al. Combined TP53 mutation/3p loss correlates with decreased
radiosensitivity and increased matrix-metalloproteinase activity in
head and neck carcinoma. Oral oncology 51, 470-475,
doi:10.1016/j.oraloncology.2015.01.014 (2015). 19 Nguyen, Q. T. et
al. Surgery with molecular fluorescence imaging using activatable
cell-penetrating peptides decreases residual cancer and improves
survival. Proceedings of the National Academy of Sciences of the
United States of America 107, 4317-4322,
doi:10.1073/pnas.0910261107 (2010). 20 Hussain, T. et al. Surgical
molecular navigation with a Ratiometric Activatable Cell
Penetrating Peptide improves intraoperative identification and
resection of small salivary gland cancers. Head & neck,
doi:10.1002/hed.23946 (2014). 21 Metildi, C. A. et al. Ratiometric
activatable cell-penetrating peptides label pancreatic cancer,
enabling fluorescence-guided surgery, which reduces metastases and
recurrence in orthotopic mouse models. Annals of surgical oncology
22, 2082-2087, doi:10.1245/s10434-014-4144-1 (2015). 22 Crisp, J.
L. et al. Dual targeting of integrin alphavbeta3 and matrix
metalloproteinase-2 for optical imaging of tumors and
chemotherapeutic delivery. Molecular cancer therapeutics 13,
1514-1525, doi:10.1158/1535-7163.MCT-13-1067 (2014). 23 Buckel, L.
et al. Tumor radiosensitization by monomethyl auristatin e:
mechanism of action and targeted delivery. Cancer research 75,
1376-1387, doi:10.1158/0008-5472.CAN-14-1931 (2015). 24 Gross, A.
M. et al. Multi-tiered genomic analysis of head and neck cancer
ties TP53 mutation to 3p loss. Nature genetics 46, 939-943,
doi:10.1038/ng.3051 (2014). 25 Rades, D. et al. Prognostic factors
in head-and-neck cancer patients treated with surgery followed by
intensity-modulated radiotherapy (IMRT), 3D-conformal radiotherapy,
or conventional radiotherapy. Oral oncology 43, 535-543,
doi:10.1016/j.oraloncology.2006.05.006 (2007). 26 Ratnikov, B. I.
et al. Basis for substrate recognition and distinction by matrix
metalloproteinases. Proceedings of the National Academy of Sciences
of the United States of America 111, E4148-4155,
doi:10.1073/pnas.1406134111 (2014). 27 Tsien, R. Y. &
Harootunian, A. T. Practical design criteria for a dynamic ratio
imaging system. Cell Calcium 11, 93-109 (1990). 28 Poeta, M. L. et
al. TP53 mutations and survival in squamous-cell carcinoma of the
head and neck. N Engl J Med 357, 2552-2561,
doi:10.1056/NEJMoa073770 (2007). 29 Ohta, M. et al. The FHIT gene,
spanning the chromosome 3p14.2 fragile site and renal
carcinoma-associated t(3; 8) breakpoint, is abnormal in digestive
tract cancers. Cell 84, 587-597 (1996). 30 Zanesi, N. et al. The
tumor spectrum in FHIT-deficient mice. Proc Natl Acad Sci USA 98,
10250-10255, doi:10.1073/pnas.191345898 (2001). 31 Lee, J. I. et
al. Loss of Fhit expression is a predictor of poor outcome in
tongue cancer. Cancer Res 61, 837-841 (2001). 32 Egeblad, M. &
Werb, Z. New functions for the matrix metalloproteinases in cancer
progression. Nature reviews. Cancer 2, 161-174, doi:10.1038/nrc745
(2002). 33 Albright, C. F. et al. Matrix
metalloproteinase-activated doxorubicin prodrugs inhibit HT1080
xenograft growth better than doxorubicin with less toxicity.
Molecular cancer therapeutics 4, 751-760,
doi:10.1158/1535-7163.mct-05-0006 (2005). 34 Olson, E. S. et al. In
vivo characterization of activatable cell penetrating peptides for
targeting protease activity in cancer. Integrative biology:
quantitative biosciences from nano to macro 1, 382-393,
doi:10.1039/b904890a (2009). 35 Olson, E. S. et al. Activatable
cell penetrating peptides linked to nanoparticles as dual probes
for in vivo fluorescence and MR imaging of proteases. Proceedings
of the National Academy of Sciences of the United States of America
107, 4311-4316, doi:10.1073/pnas.0910283107 (2010).
All headings and section designations are used for clarity and
reference purposes only and are not to be considered limiting in
any way. For example, those of skill in the art will appreciate the
usefulness of combining various aspects from different headings and
sections as appropriate according to the spirit and scope of the
invention described herein.
All references cited herein are hereby incorporated by reference
herein in their entireties and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
Many modifications and variations of this application can be made
without departing from its spirit and scope, as will be apparent to
those skilled in the art. The specific embodiments and examples
described herein are offered by way of example only, and the
application is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which the
claims are entitled.
SEQUENCE LISTINGS
1
9716PRTArtificial Sequencecleavable linker 1Pro Leu Gly Leu Ala
Gly1 526PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)METHYLATION 2Pro Leu Gly Cys Ala Gly1
537PRTArtificial Sequencecleavable linker 3Glu Asp Asp Asp Asp Lys
Ala1 547PRTArtificial Sequencecleavable
linkerMOD_RES(2)..(2)residue modified by CitMOD_RES(4)..(4)residue
modified by homo 4Arg Ser Gly Phe Tyr Leu Tyr1 5510PRTArtificial
Sequencecleavable linker 5Cys Arg Pro Ala His Leu Arg Asp Ser Gly1
5 1067PRTArtificial Sequencecleavable linker 6Ser Leu Ala Tyr Tyr
Thr Ala1 578PRTArtificial Sequencecleavable linker 7Asn Ile Ser Asp
Leu Thr Ala Gly1 588PRTArtificial Sequencecleavable linker 8Pro Pro
Ser Ser Leu Arg Val Thr1 5910PRTArtificial Sequencecleavable linker
9Ser Gly Glu Ser Leu Ser Asn Leu Thr Ala1 5 10106PRTArtificial
Sequencecleavable linker 10Arg Ile Gly Phe Leu Arg1
5116PRTArtificial Sequencecleavable
linkerMOD_RES(6)..(6)ACETYLATION 11Arg Leu Gln Leu Ala Leu1
5126PRTArtificial Sequencecleavable linker 12Arg Leu Gln Leu Lys
Leu1 5136PRTArtificial Sequencecleavable linker 13Asp Pro Arg Ser
Phe Leu1 5146PRTArtificial Sequencecleavable linker 14Pro Pro Arg
Ser Phe Leu1 5157PRTArtificial Sequencecleavable
linkerMOD_RES(1)..(1)Nle 15Leu Thr Pro Arg Ser Phe Leu1
5167PRTArtificial Sequencecleavable linker 16Gly Val Ala Tyr Ser
Gly Ala1 5176PRTArtificial Sequencecleavable linker 17Tyr Gly Arg
Ala Ala Ala1 5186PRTArtificial Sequencecleavable linker 18Tyr Gly
Pro Arg Asn Arg1 5197PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)residue modified by Hfe 19Arg Ser His Pro Thr
Leu Tyr1 5207PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)residue modified by Hfe 20Arg Ser His Gly Phe
Leu Tyr1 5216PRTArtificial Sequencecleavable linker 21Ser Asn Pro
Tyr Lys Tyr1 5226PRTArtificial Sequencecleavable linker 22Ser Asn
Pro Lys Gly Tyr1 5236PRTArtificial Sequencecleavable linker 23Ser
Asn Pro Tyr Gly Tyr1 5246PRTArtificial Sequencecleavable linker
24Thr Leu Ser Glu Leu His1 5256PRTArtificial Sequencecleavable
linker 25Thr Ile Ala His Leu Ala1 5266PRTArtificial
Sequencecleavable linkerMOD_RES(6)..(6)ACETYLATION 26Arg Leu Gln
Leu Lys Leu1 5277PRTArtificial Sequencecleavable linker 27Lys Leu
Arg Phe Ser Lys Gln1 5286PRTArtificial Sequencecleavable
linkerMOD_RES(4)..(4)METHYLATION 28Pro Leu Gly Cys Ala Gly1
52911PRTArtificial Sequencecleavable linker 29Cys Ala Thr Lys Lys
Leu Arg Phe Ser Lys Gln1 5 10307PRTArtificial Sequencecleavable
linker 30Pro Leu Gly Leu Glu Glu Ala1 5316PRTArtificial
Sequencecleavable linker 31Ser Asn Pro Phe Lys Tyr1
5327PRTArtificial Sequencecleavable linker 32Lys Pro Arg Gly Ser
Lys Gln1 5337PRTArtificial Sequencecleavable linker 33Lys Leu Arg
Phe Ser Lys Gln1 5347PRTArtificial Sequencecleavable linker 34Lys
Lys Pro Gly Ser Lys Gln1 5356PRTArtificial Sequencecleavable linker
35His Pro Gly Gly Pro Gln1 5367PRTArtificial Sequencecleavable
linkerMOD_RES(1)..(1)Nle 36Leu Thr Leu Arg Ser Leu Gln1
53710PRTArtificial Sequencecleavable linker 37Ser Gly Ala Arg Gly
Ile Lys Leu Thr Ala1 5 10387PRTArtificial Sequencecleavable
linkerMOD_RES(2)..(2)residue modified by citMOD_RES(4)..(4)residue
modified by fe 38Arg Ser Gly His Tyr Leu Tyr1 53919PRTArtificial
SequencepeptideMOD_RES(8)..(8)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(18)..(18)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)cysteine
modified by fluorescein 39Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg
Arg Arg Arg Arg Arg Arg1 5 10 15Arg Xaa Cys4022PRTArtificial
SequencepeptideMOD_RES(1)..(1)cysteine modified by
fluroresceinMOD_RES(1)..(1)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(12)..(12)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoyl 40Xaa Cys Arg Arg Arg Arg
Arg Arg Arg Arg Arg Xaa Glu Glu Glu Glu1 5 10 15Glu Glu Glu Glu Glu
Cys 204117PRTArtificial SequencepeptideMOD_RES(1)..(1)cysteine
modified by fluoresceinMOD_RES(1)..(1)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(7)..(7)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoyl 41Xaa Cys Glu Glu Glu Glu Xaa Arg
Arg Arg Arg Arg Arg Arg Arg Arg1 5 10 15Cys4223PRTArtificial
SequencepeptideMOD_RES(12)..(12)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(23)..(23)cysteine
modified by fluorescein 42Glu Glu Glu Glu Glu Asp Asp Asp Asp Lys
Ala Xaa Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Arg Xaa Cys
204319PRTArtificial SequencepeptideMOD_RES(8)..(8)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(18)..(18)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)cysteine
modified by fluorescein 43Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg
Arg Arg Arg Arg Arg Arg1 5 10 15Arg Xaa Cys4422PRTArtificial
SequencepeptideMOD_RES(21)..(21)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)cysteine modified
by fluorescein 44Glu Glu Glu Glu Glu Asp Asp Asp Asp Lys Ala Arg
Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Xaa Cys 204519PRTArtificial
SequencepeptideMOD_RES(18)..(18)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)cysteine modified
by fluorescein 45Asp Asp Asp Asp Asp Asp Lys Ala Arg Arg Arg Arg
Arg Arg Arg Arg1 5 10 15Arg Xaa Cys4623PRTArtificial
SequencepeptideMOD_RES(10)..(10)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(13)..(13)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(16)..(16)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(23)..(23)fluorescein
46Glu Glu Asp Asp Asp Asp Lys Ala Arg Xaa Arg Arg Xaa Arg Arg Xaa1
5 10 15Arg Arg Xaa Arg Arg Xaa Cys 20477PRTArtificial
SequencepeptideMOD_RES(4)..(4)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(6)..(6)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(7)..(7)cysteine modified by
fluorescein 47Glu Asp Ala Xaa Arg Xaa Cys1 54816PRTArtificial
SequencepeptideMOD_RES(8)..(8)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(15)..(15)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(16)..(16)cysteine
modified by DOX 48Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg Arg Arg
Arg Arg Xaa Cys1 5 10 154923PRTArtificial
SequencepeptideMOD_RES(12)..(12)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(23)..(23)tyrosine
modified by 12SI 49Glu Glu Glu Asp Asp Asp Glu Glu Glu Asp Ala Xaa
Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Arg Xaa Tyr
205023PRTArtificial SequencepeptideMOD_RES(12)..(12)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(23)..(23)cysteine
modified by fluorescein 50Glu Glu Glu Glu Glu Asp Asp Asp Asp Lys
Ala Xaa Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Arg Xaa Cys
205122PRTArtificial SequencepeptideMOD_RES(21)..(21)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)cysteine
modified by fluorescein 51Glu Glu Glu Glu Glu Asp Asp Asp Asp Lys
Ala Arg Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Xaa Cys
205219PRTArtificial SequencepeptideMOD_RES(8)..(8)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(18)..(18)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)cysteine
modified by fluorescein 52Glu Asp Asp Asp Asp Lys Ala Xaa Arg Arg
Arg Arg Arg Arg Arg Arg1 5 10 15Arg Xaa Cys5323PRTArtificial
SequencepeptideMOD_RES(10)..(10)ammohexanoyl linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(13)..(13)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(16)..(16)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)ammohexanoyl
linker
(-HN-(CH2)<rCO-)aminohexanoylMOD_RES(22)..(22)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(23)..(23)cysteine
modified by fluorescein 53Glu Glu Asp Asp Asp Asp Lys Ala Arg Xaa
Arg Arg Xaa Arg Arg Xaa1 5 10 15Arg Arg Xaa Arg Arg Xaa Cys
205419PRTArtificial SequencepeptideMOD_RES(18)..(18)ammohexanoyl
linker (-HN-(CH2)<rCO-)aminohexanoylMOD_RES(19)..(19)cysteine
modified by fluorescein 54Asp Asp Asp Asp Asp Asp Lys Ala Arg Arg
Arg Arg Arg Arg Arg Arg1 5 10 15Arg Xaa Cys5518PRTArtificial
SequenceDF4MOD_RES(1)..(1)ACETYLATIONMOD_RES(18)..(18)AMIDATION
55Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu1
5 10 15Ala Phe5611PRTArtificial SequenceJNK Inhibitor
VIMOD_RES(11)..(11)AMIDATION 56Arg Pro Lys Arg Pro Thr Thr Leu Asn
Leu Phe1 5 105716PRTArtificial SequenceSC 3036 57Lys Lys His Thr
Asp Asp Gly Tyr Met Pro Met Ser Pro Gly Val Ala1 5 10
155828PRTArtificial SequenceNEMO-Binding Domain Binding Peptide
58Asp Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys1
5 10 15Lys Thr Ala Leu Asp Trp Ser Trp Leu Gln Thr Glu 20
255926PRTArtificial SequenceNF-kB SN50 59Ala Ala Val Ala Leu Leu
Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Val Gln Arg Lys Arg
Gln Lys Leu Met Pro 20 256029PRTArtificial SequenceTIRAP Inhibitor
Peptide 60Arg Gln Ile Lys Ile Trp Phe Asn Arg Arg Met Lys Trp Lys
Lys Leu1 5 10 15Gln Leu Arg Asp Ala Ala Pro Gly Gly Ala Ile Val Ser
20 25614PRTArtificial SequencedrugMOD_RES(1)..(1)leucine is
modified by
benzyloxycarbonylMOD_RES(2)..(2)METHYLATIONMOD_RES(4)..(4)METHYLATIONMOD_-
RES(4)..(4)aspartate is modified by fluoromethylketone 61Leu Glu
His Asp1624PRTArtificial
SequencedrugMOD_RES(1)..(1)ACETYLATIONMOD_RES(4)..(4)FORMYLATION
62Leu Glu His Asp1634PRTArtificial
SequencedrugMOD_RES(1)..(1)ACETYLATIONMOD_RES(4)..(4)FORMYLATION
63Ile Glu Thr Asp1644PRTArtificial
SequencedrugMOD_RES(1)..(1)benzyloxycarbonylMOD_RES(2)..(2)METHYLATIONMOD-
_RES(4)..(4)METHYLATIONMOD_RES(4)..(4)fluoromethy lketone 64Ile Glu
Thr Asp1654PRTArtificial
SequencedrugMOD_RES(1)..(1)benzyloxycarbonylMOD_RES(4)..(4)fluoromethyl
ketone 65Leu Glu His Asp1664PRTArtificial
SequencedrugMOD_RES(1)..(1)benzyloxycarbonylMOD_RES(4)..(4)iluoromethyl
ketone 66Leu Glu Thr Asp1677PRTArtificial Sequencecleavable linker
67Asp Arg Val Tyr Ile His Pro1 56814PRTArtificial Sequencecleavable
linker 68Asp Arg Val Tyr Ile His Pro Phe His Leu Leu Tyr Tyr Ser1 5
106910PRTArtificial Sequencecleavable linker 69Ile His Pro Phe His
Leu Val Ile His Thr1 5 10708PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)reside modified by homo 70Arg Ser His Gly Phe
Phe Leu Tyr1 5718PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)residue modified by homo 71Arg Ser Gln Gly Phe
Tyr Leu Tyr1 5726PRTArtificial Sequencecleavable linker 72Thr Leu
Ala His Leu His1 5736PRTArtificial Sequencecleavable linker 73Thr
Ile Ser His Leu His1 5746PRTArtificial Sequencecleavable linker
74Thr Leu Ser His Leu His1 5756PRTArtificial Sequencecleavable
linker 75Thr Ile Ala His Phe His1 5767PRTArtificial
Sequencecleavable linker 76Lys Pro Arg Gly Ser Lys Gln1
5777PRTArtificial Sequenceclevable linker 77Lys Lys Pro Gly Ser Lys
Gln1 5786PRTArtificial Sequencecleavable linker 78His Pro Gly Gly
Pro Gln1 5797PRTArtificial Sequencecleavable
linkerMOD_RES(1)..(1)Nle 79Leu Thr Leu Arg Ser Leu Gln1
58010PRTArtificial Sequencecleavable linker 80Ser Gly Thr Ile Ala
His Leu Ala Thr Ala1 5 108110PRTArtificial Sequencecleavable linker
81Ser Gly Ser Asn Pro Tyr Gly Tyr Thr Ala1 5 108210PRTArtificial
Sequencecleavable linker 82Ser Gly Ser Asn Pro Tyr Lys Tyr Thr Ala1
5 10838PRTArtificial Sequencecleavable linkerMOD_RES(4)..(4)residue
modified by home 83Arg Ser Gln Gly Phe Tyr Leu Tyr1
5847PRTArtificial Sequencecleavable linkerMOD_RES(3)..(3)residue
modified by Cit 84Arg Ser Gly Phe Tyr Leu Tyr1 5858PRTArtificial
Sequencecleavable linker 85Arg Ser Gln Gly Phe Tyr Leu Tyr1
5866PRTArtificial Sequencecleavable linkerMOD_RES(3)..(3)residue
modified by Cit 86Arg Ser Gly Leu Ala Gly1 5876PRTArtificial
Sequencecleavable linkerMOD_RES(3)..(3)residue modified by Cit
87Arg Pro Gly Leu Ala Gly1 5887PRTArtificial Sequencecleavable
linker 88Arg Ser Leu Gly Leu Ala Gly1 5898PRTArtificial
Sequencecleavable linkerMOD_RES(5)..(5)reside modified by fe 89Arg
Ala His Gly His Phe Leu Tyr1 5908PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)residue modified by fe 90Arg Ala His Gly His
Thr Leu Tyr1 5918PRTArtificial Sequencecleavable
linkerMOD_RES(5)..(5)residue modified by fe 91Arg Ala His Pro His
Thr Leu Tyr1 5926PRTArtificial Sequencecleavable linker 92Tyr Ile
Pro Leu Val Tyr1 5936PRTArtificial Sequencecleavable linker 93Ser
Asn Pro Phe Lys Tyr1 5946PRTArtificial Sequencecleavable linker
94Asn Thr Phe Leu His Leu1 5956PRTArtificial Sequencecleavable
linker 95Ala Arg Gly Ile Lys Leu1 5968PRTArtificial Sequenceamino
acid 96Glu Glu Glu Glu Glu Glu Glu Glu1 5979PRTArtificial
Sequenceamino acid 97Arg Arg Arg Arg Arg Arg Arg Arg Arg1 5
* * * * *