A method of treating bone metastasis diseases, medicaments therefore, and a method of predicting the clinical outcome of treating bone metastasis diseases

Abstract

The instant invention provides for a new method of treating bone metastasis diseases in subjects, wherein said method preferably depends on whether the subject shows certain specific proteins levels in one or more body fluids prior to or during treatment, wherein said treatment comprises the administration of at least one pan αv integrin inhibitor to a subject, a medicament for use in said new methods, and a method of predicting the outcome of a treatment with at least one pan αv integrin inhibitor based on said specific protein levels in one or more body fluids of the subject.

Claims

1. A method of treating bone metastasis diseases in a subject, wherein said subject is characterized by: a) high levels of one or more proteins in at least one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: TABLE-US-00008 DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: O94768), STX1A (UniProt ID: Q16623), and TEK (UniProt ID: Q02763), and/or b) low levels of one or more proteins in at least one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: TABLE-US-00009 ANG (UniProt ID: P03950), IL1B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and TNFRSF17 (UniProt ID: Q02223); said method comprising administering to said subject at least one pan αv integrin inhibitor.

2. The method according to claim 1, wherein said at least one pan αv integrin inhibitor comprises Abituzumab and/or Intetumumab.)

3. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is Abituzumab and/or Intetumumab.

4. The method according to claim 1, wherein the level of said specific protein in at least one body fluid of said subject is: a) classified as high, if the respective specific protein level in said blood plasma is at least 2% higher, more preferably at least 5% higher, even more preferably at least 10% higher and especially at least 25% higher than the median threshold determined for the respective specific protein, and/or b) classified as low, if the respective specific protein level in said blood plasma is at least 2% lower, more preferably at least 5% lower, even more preferably at least 10% lower and especially at least 25% lower than said median threshold for the respective specific protein.

5. The method according to claim 1, wherein said median thresholds are determined in a subject population having bone metastasis disease.

6. The method according to claim 1, wherein said subject is characterised by a high level of the protein: STX1A (UniProt ID: Q16623) and/or a protein having at least 80%, more preferably at least 90% even more preferably 95% and especially at least 99% sequence homology to said protein.

7. The method according to claim 1, wherein said subject is characterized by the respective levels of one or more proteins having at least 80%, more preferably at least 90% even more preferably 95% and especially at least 99% sequence homology to said one or more proteins according to claim 1.

8. The method according to claim 1, wherein said body fluid is selected from the group consisting of blood plasma, blood serum and whole blood.

9. The method according to claim 1, wherein said high levels and/or low levels of one or more of said proteins are present and/or determined prior to administering said at least one pan αv integrin inhibitor.

10. The method according to claim 1, wherein said high levels and/or low levels of one or more of said proteins are present and/or determined during or after administering said at least one pan αv integrin inhibitor.

11. The method according to claim 1, wherein the bone metastasis disease is cancer or is derived from cancer.

12. The method according to claim 1, wherein the bone metastasis disease is derived from prostate cancer, breast cancer and/or lung cancer.

13. The method according to claim 1, wherein said threshold or median threshold for the respective specific protein is determined from the body fluid of a plurality of subjects being part of a diseased subject population suffering from the respective bone metastasis disease.

14. The method according to claim 1, wherein wherein said at least one pan αv integrin inhibitor is administered to said subject in an amount of 100 mg to 3000 mg per month.

15. The method according to claim 1, wherein said at least one pan αv integrin inhibitor comprises or is Abituzumab, and wherein the Abituzumab is administered to said subject in an amount of 500 to 2000 mg every week, every second week every or every fourth week.

16. The method according to claim 1, wherein said at least one pan αv integrin inhibitor comprises or is Abituzumab, and wherein the Abituzumab is administered to said subject in an amount of about 500 mg per week, about 750 mg per week, about 1000 mg per week or about 1500 mg per week, preferably every week, every second week or every fourth week.

17. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered in combination with one or more agents or chemotherapeutic agents: a) selected from the group consisting of Leuproreline, Leuproreline acetate, bicalutamide, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix, b) selected from the group consisting of Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid, and/or c) selected from the group consisting of Abiraterone, Abiraterone acetate, Prednisone, Enzalutamide, Radium Ra 223 dichloride, Docetaxel, Sipuleucel-T, Cabazitaxel and Mitoxantrone; and/or the pharmaceutically acceptable derivatives and/or salts thereof.

18. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered in combination with, or additionally in combination with, one or more chemotherapeutic agents, selected from the group consisting of cetuximab, Panitumumab, irinotecan, vinorelbine, capecitabine, leucovorine, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil (5-FU), bevacizumab, aflibercept and regorafenib.

19. The method according to claim 1, wherein said at least one pan αv integrin inhibitor is administered in combination with one or more, preferably two or more and especially 1, 2 or 3 agents or chemotherapeutic agents, selected from the group consisting of: a) Leuproreline, Leuproreline acetate, bicalutamid, nilutamide, triptoreline, gosereline, flutamide, cyproterone, busereline and degarelix, and/or the pharmaceutically acceptable derivatives and/or salts thereof; and/or b) Zoledronic acid, Pamidronic acid, Clodronate disodium, Alendronic acid and Ibandronic acid, and/or the pharmaceutically acceptable derivatives and/or salts thereof.

20. A method to identify tumours in a subject likely to benefit from treatment with at least one pan αv integrin inhibitor, said method comprising; determining the levels of one or more proteins, selected from the group consisting of DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: O94768), STX1A (UniProt ID: Q16623), TEK (UniProt ID: Q02763), ANG (UniProt ID: P03950), ID B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and/or TNFRSF17 (UniProt ID: Q02223) in at least one body fluid of said subject, wherein: a) a high level one or more proteins selected from the group consisting of: TABLE-US-00010 DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: O94768), STX1A (UniProt ID: Q16623), and TEK (UniProt ID: Q02763), and/or b) low levels of one or more proteins in at last one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: TABLE-US-00011 ANG (UniProt ID: P03950), IL1B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and TNFRSF17 (UniProt ID: Q02223); identify a tumour likely to benefit from the treatment with at least one pan αv integrin inhibitor.

21. A method to identify tumours in a subject likely to benefit from treatment with at least one pan αv integrin inhibitor, said method comprising determining the level of the protein STX1A (UniProt ID: Q16623) and/or a protein having at least 80%, more preferably at least 90%, even more preferably at least 95% and especially at least 99% sequence homology to STX1A, in one or more body fluids of said subject, wherein a high level thereof identify a tumour likely to benefit from the treatment with at least one pan αv integrin inhibitor.)

22. The method according to claim 20 wherein the level of protein in said one or more body fluids is a) classified as high, if the respective protein level in said one or more body fluid is at least 2% higher, more preferably at least 5% higher, even more preferably at least 10% higher and especially at least 25% higher than the median threshold determined for the respective protein, and/or b) classified as low, if the respective protein level in said blood one or more body fluids is at least 2% lower, more preferably at least 5% lower, even more preferably at least 10% lower and especially at least 25% lower than said median threshold for the respective protein.

23. The method according to claim 20 wherein the one or more body fluids comprise a blood plasma or consist of blood plasma.

24. The method according to claim 20 wherein the at least one pan αv integrin inhibitor comprises Abituzumab or is Abituzumab.

25. A method to identify a subject responsive to treatment with at least one pan αv integrin inhibitor, comprising; determining the levels of one or more proteins, selected from the group consisting of DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: O94768), STX1A (UniProt ID: Q16623), TEK (UniProt ID: Q02763), ANG (UniProt ID: P03950), ID B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and/or TNFRSF17 (UniProt ID: Q02223) in at least one body fluid of said subject, wherein: a) a high level one or more proteins selected from the group consisting of: TABLE-US-00012 DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: O94768), STX1A (UniProt ID: Q16623), and TEK (UniProt ID: Q02763), and/or b) low levels of one or more proteins in at last one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: TABLE-US-00013 ANG (UniProt ID: P03950), IL1B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and TNFRSF17 (UniProt ID: Q02223); identify a subject responsive to treatment with at least one pan αv integrin inhibitor.

26. Pan αv integrin inhibitor for use in the treatment of bone metastases in a subject, wherein said bone metastases as are characterised by: a) high levels of one or more proteins in at least one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: TABLE-US-00014 DCN (UniProt ID: P07585), F5 (UniProt ID: P12259), ICAM3 (UniProt ID: P32942), PIGR (UniProt ID: P01833), STK17B (UniProt ID: O94768), STX1A (UniProt ID: Q16623), and TEK (UniProt ID: Q02763), and/or b) low levels of one or more proteins in at last one body fluid of said subject, wherein said one or more proteins are selected from the group consisting of: TABLE-US-00015 ANG (UniProt ID: P03950), IL1B (UniProt ID: P01584), LEPR (UniProt ID: P48357), MAP2K2 (UniProt ID: P36507), MAPK11 (UniProt ID: Q15759), RGMB (UniProt ID: Q6NW40), and TNFRSF17 (UniProt ID: Q02223).

27. Pan αv integrin inhibitor for use in the treatment of bone metastases in a subject, wherein said bone metastases as are characterised by a high level of the protein STX1A (UniProt ID: Q16623), and/or a protein having at least 80%, more preferably at least 90%, even more preferably 95% and especially at least 99 percent sequence homology to said protein.

28. Pan αv integrin inhibitor for use according to claim 26 wherein: a) said median thresholds are determined in a subject population having bone metastases, b) the at least one Pan αv integrin inhibitor comprises Abituzumab and/or and/or Intetumumab, c) the subject is a human subject, and/or d) the bone metastases are bone metastases from castrate-resisting prostate cancer. b) the at least one Pan αv integrin inhibitor comprises Abituzumab and/or and/or Intetumumab, c) the subject is a human subject, and/or d) the bone metastases are bone metastases from castrate-resisting prostate cancer.

Description

EXPERIMENTAL SECTION

Example 1

PERSEUS Phase II Clinical Study

[0184] c) Leuproreline, Leuproreline acetate and/or pharmaceutically acceptable derivatives and/or salts thereof,

in combination with

[0185] Zoledronic acid and/or pharmaceutically acceptable derivatives and/or salts thereof

[0186] PERSEUS Phase II clinical trial

[0187] In this randomized, double-blind, placebo-controlled, international phase II trial, a total of 180 patients were randomized 1:1:1 to receive [0188] a) Standard of Care (SoC), e.g. continuous treatment with a luteinizing hormone-releasing hormone agonist, preferably Leuproreline, Leuproreline acetate and/or pharmaceutically acceptable derivatives and/or salts thereof, and bisphosphonate treatment, preferably Zoledronic acid and/or pharmaceutically acceptable derivatives and/or salts thereof, plus placebo, [0189] b) abituzumab 750 mg plus SoC, or [0190] c) abituzumab 1,500 mg plus SoC.

Pharmacokinetic Analysis

[0191] Equal numbers of patients per arm were included in the pharmacokinetic analysis subgroup. [0192] Blood sampling for pharmacokinetic assessments was scheduled at various timepoints during cycles 1, 3, 4, 5, 6, and 7 of therapy. [0193] Pharmacokinetic parameters were calculated according to standard non-compartmental methods using the program KINETICATM v4.1.1 (Innaphase).

Immunogenicity

[0194] Blood sampling for immunogenicity was scheduled pre-dose in cycles 1, 3, 5 and 6, and at the end-of-treatment visit and safety follow-up visits. [0195] Generation of antibodies directed against abituzumab was evaluated centrally using a validated ELISA method.

Biomarker Analyses

[0196] Archived tumor blocks or punch biopsy materials were collected to explore tumor expression of integrins and their ligands as well as proteins related to angiogenesis and the underlying disease, and their potential relationship to clinical outcomes. [0197] Availability of samples had to be confirmed at patient screening [0198] Analyses were performed using immunohistochemistry. [0199] Blood sampling for plasma protein analyses was scheduled pre-treatment. [0200] Plasma protein analyses (based on highly protein-specific aptamers [SomaLogic system]) were performed on samples taken from 150 patients prior to treatment in cycle 1 [0201] The original set of simultaneously determined 1,129 plasma protein levels was restricted to 888 proteins on the data level to avoid potential bias due to cell lysis or platelet activation during plasma preparation [0202] Nine global biomarker search analyses were carried out using different normalization procedure, data sets and biomarker dichotomization thresholds, with the aim of filtering biomarker proteins based on data robustness independent of biological annotations. The search process comprised a set of criteria ensuring that identified proteins are significantly (p<0.05) associated with outcome (here exemplary radiologic PFS) for either the patients with low or high levels. These tests comprise, among others, logrank tests for differences in survival (here PFS) for Abituzumab-treated/untreated patients in the biomarker-low and biomarker-high groups according to the median threshold, tests for an interaction effect on outcome (here PFS) between continuous marker levels and treatment based on Cox regression models. [0203] This process identified 15 biomarker plasma proteins: DCN (Somamer ID: SL004081; UniProt ID: P07585), [0204] F5 (Somamer ID: SL000622; UniProt ID: P12259), [0205] ICAM3 (Somamer ID: SL003178; UniProt ID: P32942), [0206] PIGR (Somamer ID: SL005797; UniProt ID: P01833), [0207] STK17B (Somamer ID: SL016566; UniProt ID: O94768), [0208] STX1A (Somamer ID: SL004304; UniProt ID: Q16623), [0209] TEK (Somamer ID: SL003200; UniProt ID: Q02763), [0210] ANG (Somamer ID: SL000003; UniProt ID: P03950), [0211] IL1B (Somamer ID: SL001795; UniProt ID: P01584), [0212] LEPR (Somamer ID: SL003184; UniProt ID: P48357), [0213] MAP2K2 (Somamer ID: SL010501; UniProt ID: P36507), [0214] MAPK11 (Somamer ID: SL007453; UniProt ID: Q15759), [0215] RGMB (Somamer ID: SL010468; UniProt ID: Q6NW40), and [0216] TNFRSF17 (Somamer ID: SL004672; UniProt ID: Q02223)

Results

Biomarker Analyses

[0217] IHC analysis of tumor samples has not identified any relevant biomarkers to date. [0218] The details documenting why the 14 biomarker plasma proteins identified are judged as active and whether levels above or below the median are judged as predictive are shown in Table 1, Table 2 and/or one or more of FIGS. 1 to 24. [0219] The biomarker proteins include decorin (DCN), a protein known to have a role in TGF-β biology, as do some of the αv integrins inhibited by abituzumab [0220] Furthermore, analysis of the biological context of other markers indicated that markers related to known molecular interactions of abituzumab (bone metabolism modulation and angiogenesis) appeared to predict OS with abituzumab therapy. [0221] Plasma levels of some of the identified 14 biomarker plasma proteins were prognostic under SoC of either good or poor survival and all 14 predicted increased survival with abituzumab compared to SoC alone. Table 1, Table 2 and/or one or more of FIGS. 1 to 24 show the prognostic and predictive value of the identified 14 predictive marker proteins, as for example TEK, for PFS.

Example 2

Proteomic Affinity Assay Method

[0222] All steps of the proteomic affinity assay are performed at room temperature unless otherwise indicated.

Sample thawing and plating.

[0223] Aliquots of 100% serum or EDTA- plasma, stored at −80° C., are thawed by incubating in a 25° C. water bath for ten minutes. After thawing the samples are stored on ice during mixing and prior to sample dilution. Samples are mixed by gentle vortexing (setting # 4 on Vortex Genie, Scientific Industries) for 8 seconds. A 20% sample solution is prepared by transferring 16 μL of thawed sample into 96-well plates (Hybaid Omnitube 0.3 mL, Thermo Fisher Scientific) containing 64 μL per well of the appropriate sample diluent at 4° C. Sample diluent for serum is 0.8× SB17 with 0.6 mM MgCl.sub.2, 2 mM EGTA, 2 μM Z-Block_2, 0.05% Tween and for EDTA-plasma is 0.8× SB18 with 0.8 mM MgCl.sub.2, 2 mM EGTA, 2 μM Z-Block_2, 0.05% Tween. This plate is stored on ice until the next sample dilution steps are initiated.

[0224] Preparation of 10%, 1% and 0.03% SOMAmer Solutions. SOMAmers are grouped into three unique mixes. The placing of a SOMAmer within a mix is empirically determined by assaying a dilution series of serum or plasma with each SOMAmer and identifying the sample dilution that gave the largest linear range of signal. The segregation of SOMAmers and mixing with different dilutions of sample (10%, 1% or 0.03%) allow the assay to span a 10.sup.7-fold range of protein concentration. The composition of the custom SOMAmer mixes is slightly different between plasma and serum as expected due to variation in protein composition of these two media. The custom stock SOMAmer solutions for 10%, 1% and 0.03% serum and plasma are prepared and stored at 8x concentration in SB17T. For each assay run, the three 8x SOMAmer solutions are diluted separately 1:4 into SB17T to achieve 2x concentration. Each diluted SOMAmer master mix is heated to 95° C. for five minutes and then to 37° C. for 15 minutes. 55 μL of each 2x SOMAmer mix is manually pipetted into a 96-well plate resulting in three plates with 10%, 1% or 0.03% SOMAmer mixes. After mixing with sample, the final individual SOMAmer concentration ranged from 0.25-4 nM for serum, 0.5 nM for plasma.

[0225] Equilibration. A 2% sample plate is prepared by diluting the 20% sample 1:10 into SB17T using the Beckman Coulter Biomek Fx.sup.P (Beckman Coulter). A 0.06% sample plate is prepared by diluting the 2% sample plate 1:31 into SB17T. The three sample dilutions are then transferred to their respective SOMAmer solutions by adding 55 μL of the sample to 55 μL of the appropriate 2× SOMAmer mix. The plates are sealed with a foil seal (Microseal ‘F’ Foil, Bio-Rad) and incubated at 37° C. for 3.5 hours.

[0226] Preparation of Catch-1 Bead Plates. 133.3 μL of a 7.5% Streptavidin-agarose bead slurry in SB17T is added to each well of three pre-washed 0.45 um filter plates. Each well of beads is washed once with 200 μL SB17T using vacuum filtration to remove the wash and then resuspended in 200 μL SB17T.

[0227] Catch-1 Bead Capture. All subsequent steps are performed by the Beckman Coulter Biomek Fx.sup.P robot unless otherwise noted. After the 3.5 hour equilibration, 100 μL of the 10%, 1% and 0.03% equilibration binding reactions is transferred to their respective Catch-1 Streptavidin agarose filter plates and incubated with shaking for ten minutes. Unbound solution is removed via vacuum filtration. Each set of Catch-1 beads is washed with 190 μL of 100 μM biotin in SB17T and then 190 mL of SB17T using vacuum filtration to remove the wash. 190 μL SB17T is added to each well in the Catch-1 plates and incubated with shaking for ten minutes at 25° C. The wash is removed via vacuum filtration and the bottom of the filter plates blotted to remove droplets using the on-deck blot station.

[0228] Biotinylation of Proteins. An aliquot of 100 mM NHS-PEO4-biotin in DMSO is thawed at 37° C. for six minutes and diluted to 1 mM with SB17T at pH 7.25. 100 μL of the NHSPEO4-biotin is added to each well of each Catch-1 filter plate and incubated with shaking for five minutes. Each biotinylation reaction is quenched by adding 150 μL of 20 mM glycine in SB17T to the Catch-1 plates with the NHS-PEO4-biotin. Plates are incubated for one minute with shaking, vacuum filtrated, and 190 μL 20 mM glycine SB17T is added to each well in the plate. The plates are incubated for one minute, shaking before removal by vacuum filtration. 190 μL of SB17T is added to each well and removed by vacuum filtration. The wells of the Catch-1 plates are subsequently washed three times by adding 190 μL SB17T, incubating for one minute with shaking followed by vacuum filtration. After the last wash the plates are centrifuged at 1000 rpm for one minute over a 1 mL deep-well plate to remove extraneous volume before elution. Centrifugation is performed off deck.

[0229] Kinetic Challenge and Photo-Cleavage. 85 μL of 10 mM dextran sulfate in SB17T is added to each well of the filter plates. The filter plates are placed onto a Thermal Shaker (Eppendorf) under a BlackRay light source and irradiated for ten minutes with shaking. The photo-cleaved solutions are sequentially eluted from each Catch-1 plate into a common deep well plate by centrifugation at 1000 rpm for one minute each.

[0230] Catch-2 Bead Capture. In bulk, MyOne-Streptavidin C1 beads are washed two times for 5 minutes each with equal volume of 20 mM NaOH and three times with an equal volume of SB17T. Beads are resuspended in SB17T to a concentration of 10 mg/mL. After resuspension, 50 μL of this solution is manually pipetted into each well of a 96-well plate and stored at 4° C. until Catch-2. During Catch-2, the wash supernatant is removed via magnetic separation. All of the photo-cleaved eluate is pipetted onto the MyOne magnetic beads and incubated with shaking at 25° C. for five minutes. The supernatant is removed from the MyOne beads via magnetic separation and 75 μL of SB17T is transferred to each well. The plate is mixed for one minute at 37° C. with shaking and then 75 μL of 60% glycerol (in SB17T) at 37° C. is transferred to each well. The plate is mixed for another minute at 37° C. with shaking. The wash is removed via magnetic separation. These washes are repeated two more times. After removal of the third glycerol wash from the MyOne beads, 150 μL of SB17T is added to each well and the plates incubated at 37° C. with shaking for one minute before removal by magnetic separation. The MyOne beads are washed a final time using 150 μL SB19T with incubation for one minute, prior to magnetic separation.

Catch-2 Bead Elution and Neutralization.

[0231] SOMAmers are eluted from MyOne beads by incubating each well of beads with 105 μL of 100 mM CAPSO pH 10, 1 M NaCl, 0.05% Tween with shaking for five minutes. 90 μL of each eluate is transferred during magnetic separation to a new 96-well plate containing 10 μL of 500 mM HCl, 500 mM HEPES, 0.05% Tween-20, pH 7.5.

[0232] Hybridization. 20 μL of each neutralized Catch-2 eluate is transferred to a new 96-well plate and 5 μL of 10× Agilent Block (Oligo aCGH/ChIP-on-chip Hybridization Kit, Large Volume, Agilent Technologies 5188-5380), containing a 10× spike of hybridization controls (10 Cy3 SOMAmers) is added to each well. After removing the plate from the robot, 25 μL of 2× Agilent Hybridization buffer (Oligo aCGH/ChIP-on-chip Hybridization Kit, Agilent Technologies) is manually pipetted to the each well of the plate containing the neutralized samples and blocking buffer. 40 μL of this solution is manually pipetted into each “well” of the hybridization gasket slide (Hybridization Gasket Slide—8 microarrays per slide format, Agilent Technologies). Custom Agilent microarray slides containing 10 probes per array complementary to 40 nucleotide selected region of each SOMAmer with a 20× dT linker are placed onto the gasket slides according to the manufacturer's protocol. Each assembly (Hybridization Chamber Kit—SureHyb enabled, Agilent Technologies) is tightly clamped and loaded into a hybridization oven for 19 hours at 60° C. rotating at 20 rpm. Post-Hybridization Washing. Approximately 400 mL Wash Buffer 1 (Oligo aCGH/ChIP-on-chip Wash Buffer 1, Agilent Technologies) is placed into each of two separate glass staining dishes. Six of the twelve slide/gasket assemblies are sequentially disassembled into the first staining dish containing Wash Buffer 1.

[0233] Once disassembled, the slide is quickly transferred into a slide rack in a second staining dish containing Wash Buffer 1. The slides are incubated for five minutes in Wash Buffer 1 with mixing via magnetic stir bar. The slide rack is then transferred to the 37° C. Wash Buffer 2 (Oligo aCGH/ChIP-onchip Wash Buffer 2, Agilent Technologies) and allowed to incubate for five minutes with stirring. The slide rack is transferred to a fourth staining dish containing acetonitrile and incubated for five minutes with stirring.

[0234] Microarray Imaging. The microarray slides are imaged with a microarray scanner (Agilent G2565CA Microarray Scanner System, Agilent Technologies) in the Cy3-channel at 5 μm resolution at 100% PMT setting and the XRD option enabled at 0.05. The resulting tiff images are processed using Agilent feature extraction software version 10.5.1.1 with the GE1_105_Dec08 protocol.