[0001] High Throughput Screening Of Molecular Libraries Using Laser Induced Sorting.

CROSS REFERENCE TO RELATEDAPPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 60/656,761, filed February 24, 2005, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] The U.S. Government has certain rights in this invention pursuant to Grant No. 1R21CA107792-01 awarded by the National Institutes of Health, National Cancer Institute.

BACKGROUND OF THE INVENTION

Field of the invention

[0004] The invention relates to methods and instruments for high throughput screening and sorting of molecular libraries using laser induced sorting methods such as laser pressure catapulting ("LPC").

Description of the Related Art

[0005] Laser microdissection was first reported in 1996 and has been used for isolation of single cells from tissue samples downstream characterization of, e.g., protein expression or nucleic acid analysis. Laser catapulting is a sample collection modality that can be used with laser microdissection. In laser catapulting, a laser beam delivers a pulse to a specimen, catapulting it into a microfuge tube cap for collection. An overview of this technology is provided by Burgess, "Laser Microdissection: Making Inroads in Research," Biophotonics International, September 2004, pp. 46-49.

[0006] Combinatorial libraries synthesized on beads or other particulate supports have been known for many years. See, e.g., Lam, et al. 1991. Such libraries, be they combinatorial peptide libraries, combinatorial nucleic acid libraries, or combinatorial small molecule libraries, usually are characterized by one bead one compound (OBOC), which is to say that a single particulate library member carries multiple copies of a single molecular species. These libraries are used, e.g., for discovering novel ligands against a target. Library screening proceeds with a series of steps including incubating the library members with a target under conditions to promote binding of the target to the molecular species carried on the particulate support, washing the library to remove non-specifically bound targets, and sorting the library to retrieve those

members that have specifically bound target. Sorting has to date been a slow and laborious process and often is the rate limiting step in library screening. There thus is a need for more rapid, automatable library screening assays that can increase throughput and therefore drive down the cost of library screening.

SUMMARY OF THE INVENTION

[0007] Disclosed herein is a method for screening a library of compounds for a candidate compound that binds to a target having the steps of providing a plurality of compounds carried on a plurality of particulate supports; contacting the compounds with the target under conditions in which the target can bind with at least of the compounds; identifying a particulate support carrying a compound to which said target binds; and directing a laser beam pulse onto the identified particulate support to separate the identified particulate support from the plurality of particulate supports, thereby isolating the candidate compound that binds to the target.

[0008] Laser induced methods, e.g., a laser beam pulse, is used to separate from the library a candidate compound that binds to a target. In one embodiment, the laser induced separation method is LPC, such as that provided by the P.A.L.M. Microbeam HT System.

[0009] In one embodiment, the library of compounds is a peptide library, in another embodiment it is a nucleic acid library, in yet another embodiment it is a peptide nucleic acid library, in still another embodiment it is a small molecule library. The library of compounds is provided on particulate support, e.g., polystyrene microbeads and the like.

[0010] The target can be a nucleic acid, a peptide, a protein, a lipid or a carbohydrate.

Exemplary embodiments include receptor molecules, antibodies, lectins, integrins, and other biological molecules of interest. In one embodiment, the target is expressed on the surface of the cell and the method uses intact cells. In another embodiment, the target is a soluble molecule and is tagged with a label, e.g., GFP.

[0011] Identifying a particulate support carrying a candidate compound to which a target binds is performed manually, e.g., via visual inspection or, alternatively, in an automated manner using, e.g., automated imaging software.

[0012] Also disclosed is method for screening a population of cells, e.g., patient blood, for at least one candidate cell, e.g., stem cells, that binds to a compound, said method having the steps of providing a plurality of particulate supports, e.g., beads, each carrying at least one molecule of the compound to which the candidate cell can bind; contacting the plurality of particulate supports with the population of cells under conditions in which at least one cell can

bind with the compounds; identifying a particulate support carrying a compound to which the cell binds; and directing a laser beam pulse onto the identified particulate support to separate the identified particulate support from the plurality of particulate supports, thereby isolating the candidate cell that binds to the compound.

[0013] The present invention is defined by the following claims, and nothing in this section should be taken as a limitation on those claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0014] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0015] Figure 1 is a schematic illustrating a split synthesis approach to generate a one bead, one compound combinatorial library.

[0016] Figure 2 is a photomicrograph showing two beads (Fig. 2(a)) and manual isolation of one bead using an Eppendorf pipette (Fig. 2(b)).

[0017] Figure 3 is a photomicrograph showing two Tentagel beads (Fig. 3(a)), identification of the beads with a spot ready for catapulting (Fig. 3(b)), and image of Eppendorf collection cap showing collection of catapulted Tentagel beads (Fig. 3(c)).

[0018] Figure 4 is a photomicrograph showing three XXDLXXLX-S-Tentagel beads coated with DX3puroβ ₆ melanoma cells (Fig. 4(a)), two beads remaining on slide following laser catapulting (Fig. 4(b)), and isolated, catapulted bead in Eppendorf collection cap (Fig. 4(c)).

[0019] Figure 5 are graphs showing flow cytometry-based characterization of α _v β ₆ expression in A375Pβ ₆ puro (Fig. 5(a)) and DX3β ₆ puro (Fig. 5(b)) cell lines.

[0020] Figure 6 is a flow chart outlining exemplary, high stringency screening process.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Briefly, and as described in more detail below, described herein are methods for high throughput sorting of molecular libraries constructed on particulate supports using laser induced separation, e.g., LPC, of target bound to a library member.

[0022] Several features of the current approach should be noted. The methods are applicable to any assay that uses libraries constructed on particulate supports (e.g., bead libraries), including cell-based and cell-free assays. The methods find particular advantage in applications in which the step of sorting positive hits is rate limiting in the overall assay scheme.

[0023] Advantages of this approach are numerous and relate primarily to increasing throughput and diminishing user involvement and time in screening assays. These advantages drive down assay cost and allow a great increase in the number of compounds that can be screened for activity against a particular target.

Definitions

[0024] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0025] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The following definitions and abbreviations are used in this specification: [0026] "DMF" is dimethylformamide; "Pbf ' is 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl; "OtBu" is t-butyl ester; "Trt" is trityl; "Boc" is butyloxycarbonyl; "TFA" is trifluoroacetic acid; "EDT" is ethanedithiol; "TIPS" is triisopropylsilane; standard single- or three-letter abbreviations, as set forth in, e.g., AX. Lehninger, Biochemistry (Worth Publishers, Inc.) are used for amino acids; "X" indicates any amino acid at a given position.

Methods of screening libraries of compounds.

[0027] The method described herein is a method for library screening that uses laser induced methods to sort library members carried on particulate supports, e.g. beads. Examples of laser induced methods include but are not limited to a system that includes non-contact laser microdissection and pressure catapulting technology ("LMPC" or "LPC") such as the P.A.L.M. Microbeam HT System, available from P.A.L.M. Microlaser Technologies, AG, Bernried, Germany.

[0028] Catapulting with a single laser shot, the candidate compound-carrying particulate support is ejected out of the object plane and moved a 3-4 mm distance to an appropriate collection vial, e.g., a microcentrifuge tube or a well of a 96 McDowell plate. Appropriate collection tubes depend on the library, target, and subsequent analysis and can be easily selected by one of skill in the art.

[0029] The methods of the invention may be practiced using an assay to screen any library carried on a particulate support. The assay may be carried out using any format (including cell- based and cell-free formats) provided that positive hits, e.g., candidate compounds bound to a target, can be distinguished from library members that do not bind target. In cell-based assays, positive hits are distinguished, e.g., because particulate supports take on a rough appearance

following the adherence of cells to the molecules displayed on the support surface. In cell-free assays, the target can be labeled so that its binding to the library molecule can be detected. Identification of a positive hit can be manual or automated. In some embodiments, automated identification of positive bits is performed using imaging software, e.g., Cellenger software. [0030] The method can be used with any number of targets including but not limited to a protein, a peptide, a nucleic acid, a peptide nucleic acid, a carbohydrate, or a lipid. In some embodiments, the target is a cell surface molecule, and cells expressing the target are used to screen the library. The invention has been exemplified using integrin expressing cells lines to screen peptide libraries.

[0031] hi some embodiments, the target is labeled with, e.g., a fluorescent molecule such as a fluorescent dye. hi other embodiments, the target is a protein or a peptide fused to a label or reporter molecule such as a fluorescent protein such as green fluorescent protein (GFP). Methods to create fusion proteins using GFP or other fluorescent proteins, protein fragments, enzymes, or other proteinaceous moieties useful for labeling or identifying the presence of the target are well known in the art, and can be carried out using chemical methods including, e.g., bifunctional agents, or molecular biological methods in which a nucleic acid sequence encoding the target is fused, in frame, with a nucleic acid sequence encoding the label or reporter. [0032] Any number of different libraries of compounds can be screened and sorted using the method of the invention. Examples include but are not limited to a peptide library, a nucleic acid library, a peptide nucleic acid library, and a small molecule library. The library of compounds is provided on a particulate support, e.g., polystyrene microbeads and the like. In some embodiments, the beads include a non-cleavable linker resin, e.g., S-Tentagel. [0033] There are several methods to approach library synthesis, including phage display (Ladner, et al., 2004), multipin approaches (Bray, et al., 1995), solution phase libraries (Zhang, et al., 2004) and the one-bead-one-compound (OBOC) library method (Lam, et al., 1991). The methods of the invention are illustrated using a one-bead-one-compound peptide library, but are applicable to any library method that produces members in a format amenable to sorting using the methods of the invention. Specifically, the binding of a compound from the library to a target must be detectable, preferably using optical methods (such as, e.g., microscopy, fluorescence microscopy, imaging software, etc.), and the complex comprising the compound bound to the target must be able to be imaged so that a laser pulse can be focused onto it, and it must be able to sufficiently withstand laser pulse catapulting so that the compound can be analyzed once the complex has been isolated. Similarly, while the invention has been

exemplified using a combinatorial peptide library, it may be practiced using any type of library, such as, e.g., libraries of bioactive compounds including by way of example but not limitation, hormones, neurotransmitters, chemokines, cytokines, growth factors, polysaccharides, lipids, nucleic acids, etc., with methods to identify library components adapted according to the makeup of the library.

The one-bead-one compound ("OBOC") library method

[0034] The methods of the invention can be used to screen and sort a one-bead-one- compound library of compounds. The OBOC approach has many advantages, including the synthesis of the libraries uses the "split synthesis approach", the compounds are on beads and are therefore spatially separable, and once screened, the structure of the library element carried on a bead be easily determined directly using, e.g., Edman degradation (Liu, et al., 2001). In the examples described below, we have used the "split synthesis" approach (Furka, 1988) to generate a series of molecular libraries designed to target integrin receptors. During library synthesis using the OBOC approach, each reaction step is forced to completion, each bead only encounters one amino acid at each synthesis step, and therefore each bead displays only a single type of compound. Each bead does however displays multiple copies of the same compound, the exact number depending on a number of factors relating to the overall size of the bead and to the number of reactive sites that allow for coupling of the first compound monomer to the bead. In the examples below, the beads carry up to 10 ¹³ copies of the same compound. Figure 1 illustrates the split-synthesis method, demonstrating that the combination of three amino acids using this methodology results in all 27 possible permutations of the peptide product in three synthesis steps.

Methods of screening for cells.

[0035] In a related method, populations of cells are screened for cells carrying a specific marker, e.g., a specific cell surface receptor that binds to a known compound of interest. The identified cells are isolated from the population cells using, e.g., LPC as described herein. The isolated cells can be further analyzed using, e.g., RT-PCT for gene profiling, or used for, e.g., cell immortalization, etc. The method is particularly useful for isolating cells that are present in very low numbers in the population. For example, tumor cells or stem cells can be isolated from, e.g., patient blood.

EXAMPLES

[0036] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0037] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 ^rd Ed. (Plenum Press) VoIs A and B(1992).

Example 1; DLXXL Library Synthesis.

[0038] In 1999 Kraft, et al. used phage display and identified the motif DLXXL (aspartate- leucine-aminoacid-aminoacid-leucine) (SEQ ID NO: 1) as an essential sequence required to inhibit RGD-dependant ligand binding to α _v β ₆ in isolated receptor assays and cell adhesion assays. Amino and carboxy terminal modifications of the flanking amino acids suggested that the two preceding amino acids and single trailing amino acid were also involved in α _v β ₆ interactions. We therefore synthesized focused libraries containing the DLXXL motif to screen for in- vivo imaging agents that have potential for imaging various types of cancer or disease. [0039] Six libraries designed to target the integrin α _v β ₆ were synthesized on solid supports. Because the libraries were designed to be used in whole cell screening assays the solid support needed to be swellable in both organic (for synthesis) and aqueous (for screening) media. Hence we chose the non cleavable Tentagel-S-NH2, 90 μm (Rapp Polymere, Germany), a polyoxyethylene-grafted polystyrene that is uniform in size, non sticky and swells well both in organic and aqueous media. Measured bead diameters ranged from 100 to 140 μm. We utilized standard Fmoc chemistries and solid-phase peptide synthesis methods to produce a focused 8- mer library containing the DLXXL motif. N-α Fmoc protection was used and removed using 20% piperidine in DMF. Side chain protection was afforded by Arg(Pbf), Asp(OtBu), GIu

(OtBu), Gln(Trt), His (Tit), Lys (Boc), Thr (Trt) and Trp (Boc). Side-chain deprotection was accomplished using TFA-EDT-water-TIPS (94/2.5/2.5/1 v/v/v/v). The beads were thoroughly washed at room temperature in approximately 10 mis of methanol, followed by 10 mis of DMF, followed by 10 mis of water, and then 10 mis of ethanol and stored under 70% aq. ethanol.

[0040] The libraries synthesized include the following sequences:

[0041] Library 1 XXXXXXXX (SEQ ID NO: 2)

[0042] Library 2 XXDLXXLX (SEQ ID NO: 3)

[0043] Library 3 CXXXXXXC (SEQ ID NO: 4)

[0044] Library 4 CXDLXXLC (SEQ ID NO: 5)

[0045] Library 5 KXXXXXXE (SEQ ID NO: 6)

[0046] Library 6 KXDLXXLE (SEQ ID NO: 7)

Example 2: Integrin Expressing Cell Lines.

[0047] We have established numerous integrin-expressing cell lines. For example the well- established human DX3 melanoma line, kindly provided by JF Marshall, Cancer Research UK, was retrovirally transduced with cDNA sequences encoding full-length integrin β ₆ subunits and/or the puromycin drug-resistance gene (courtesy of Dr. John Marshall) to produce DX3puroβ ₆ and DX3puro, respectively. The retroviral vectors used were based on the high titre retroviral vector, pBabepuro, as developed by J. Morgenstern and H. Land (1990). Cells were grown in tissue culture plastic flasks (T 75cm ³ , Falcon) in 10% v/v Fetal Bovine Serum (Gibco, Grand Island, NY) in Dulbelco's Modified Eagle's Medium (DMEM, Gibco or Cellgro, Herndon, VA) at 37 ⁰ C, CO ₂ 8% v/v air. The cell lines were used to screen the libraries described in Example 1 and as controls, as appropriate.

Example 3: Library Screening with Melanoma Cell Line.

[0048] After synthesis the library can be screened in either an on-bead binding assay or in a solution-phase assay. We used the on-bead whole cell based assay. In this assay the peptides were still covalently linked to the solid support that must therefore be compatible with aqueous media (Lam 1997). As exemplified, the assay involves incubating the bead library with whole cells, e.g. a human melanoma cell line DX3puroβ ₆ expressing the integrin α _v β ₆ , and observing the interaction under a dissecting microscope. The cells were incubated in DMEM, 10% FCS with beads at 37 degrees Celsius, 8% CO ₂ for 2 hours. Positive hits were identified as those beads that interact with the cells (i.e., become coated with cells within 2 hours of incubation). Figure 2 (a) illustrates two Tentagel beads incubated with DX3puro β ₆ melanoma cells. One

bead is totally smooth, (naked, i.e., a negative hit - upper right bead in Fig. 2(a)) and the other bead is rough (coated with cells, i.e., a positive hit - lower left bead in Fig. 2(b)). The positive, cell-covered beads were then isolated.

[0049] Prior art isolation techniques are time consuming and laborious, involving, e.g., manual isolation of beads using an Eppendorf pipette, as illustrated in Figure 2b. This is a bottleneck in any high throughput screening (HTS) approach as the number of beads that can be accurately picked using manual techniques is limited and becomes a rate limiting step in the process. For example, using this technique we screened only 150,000 beads from Library 6 and identified 8 positive hits towards α _v β ₆ during a 3 day period.

Example 4; Bead Isolation using Laser Pressure Catapulting.

[0050] The ability to dramatically improve screening throughput and bead isolation using laser induced sorting was demonstrated using Laser Pressure Catapulting (LPC) with a P.A.L.M. MicroBeam HT instrument. The LPC was used as a high throughput screening tool to isolate and collect beads that tested positive in our melanoma cell screening assay. [0051] First, we demonstrated that Tentagel beads suspended in cell culture medium on a glass slide could be catapulted into a microfuge cap filled with mineral oil or glycerol. The beads were washed with water, followed by PBS, and DMEM, and then added to a glass slide that was observed under a microscope with the image displayed on a computer screen. Using the P.A.L.M software control beads were identified by the click of the mouse, leaving a spot showing the bead was ready for catapulting. The laser was focused just below the bead of interest and fired at a power setting of 85 to 87 at a magnification of 2OX. Images were taken of the glass slide before catapulting, after catapulting and of the microfuge cap after catapulting. Positive identification of single catapulted beads was obtained by refocusing the microscope into the microfuge cap. Figure 3 shows the results. In Figure 3 (a), two isolated Tentagel beads are shown on the glass slide. In Figure 3(b), the mouse has been clicked over the image of each bead, resulting in each bead being identified with a spot indicating that the bead is ready for catapulting. After firing the laser the microscope was focused onto the Eppendorf collection cap where the 2 beads were located (Figure 3(c)).

[0052] We then used the P.A.L.M MicroBeam HT to catapult beads coated with cells bound to the peptide on the bead, and determined the peptide sequence and isolated the cells from the bead after catapulting. We synthesized an 8mer peptide on Tentagel-S-NH2 beads. The peptide sequence was XXDLXXLX (SEQ ID NO: 3), a sequence previously manually picked from one

of our libraries. Following the procedure outlined in Example 3, we incubated DX3puroβ ₆ melanoma cells with H ₂ N-XXDLXXLX-Tentagel-S beads. The beads were washed with water, followed by a PBS wash, followed by a DMEM wash. The washed beads were then added to a non-tissue culture treated Petri dish (3.5 cm diameter) containing culture medium (2 niL). Trypsinized cells were suspended in medium and added to the Petri dish containing the beads. Generally, a ratio of about 50 cells per bead was used. The Petri dish was gently rotated using an Orbitron rotator (Boekel Scientific) at 37 ⁰ C for between 30 min and 2 h. Cell coverage of beads was confirmed by visual inspection under a microscope.

[0053] The beads were transferred onto a regular glass slide and the level of the medium was adjusted to be roughly equal to the diameter of the beads. (The average diameter of the beads ranged from 100 to 140 μm). The cap of a clear standard 1 mL snap-cap microfuge tube was filled with glycerol (for peptide library sequencing) or lysis buffer (for cell analysis) and placed in the receiving position 3 mm above the cover slip. The laser was focused just below the bead of interest and the laser fired at a power setting of 85 to 87 at a magnification of 2OX. Images were taken of the glass slide before catapulting and after catapulting, and of the microfuge cap after catapulting.

[0054] Figure 4(a) illustrates 3 beads coated with DX3puroβ ₆ melanoma cells. The beads measured approximately 107 μm in diameter. Using the P.A.L.M. MicroBeam HT a single bead was identified for catapulting. Figure 4(b) illustrates that after catapulting there are two beads remaining on the glass slide. Figure 4(c) shows an image of the microfuge cap, revealing the single, melanoma cell-covered catapulted bead. This demonstrates successful selection and isolation of cell-coated beads in close proximity to other beads.

Example 5: Peptide Analysis.

[0055] The 337 nm nitrogen laser of the LPC works within the UV-A range where no damage of biological material including proteins and peptides is expected to occur. To confirm this, we sequenced peptide from the catapulted beads. For peptide sequencing, the isolated beads were collected in glycerol, spun down into a microfuge tube containing ethanol, washed with ethanol and subjected to Edman-sequencing. The sequence obtained by Edman degradation showed an unaltered XXDLXXLX (SEQ ID NO: 3) for all catapulted samples, illustrating that catapulting had no deleterious effect on the peptide sequence.

Example 6: Cell analysis.

[0056] We analyzed via real-time PCR the RNA of the cells attached to the catapulted beads to confirm that cells which have been through the screening process can be successfully analyzed via, e.g., gene expression analysis using PCR. For this experiment beads with identical peptide sequences were chosen (XXDLXXLX) (SEQ ID NO: 3). Catapulted cell loaded beads were resuspended in lysis buffer (Applied Biosystems, Foster City, CA) and extracted using a 6100 Sample PrepStation using the manufacturers recommendation. Total RNA was incubated 15 min with RNase-free DNase (Invitrogen, Carlsbad, CA), inactivated and used to synthesize first strand cDNA using random hexamer and Superscript III (all Invitrogen). Five percent of the first strand cDNA was used for quantitative real-time TaqMan PCR using pre-developed TaqMan assays for human 18S rRNA and a ribosomal protein (RPLPO) (both Assay-on- Demand, Applied Biosystems). Real-time PCR reactions were run on a 7900 HT A FAST ABI PRIMS SDS (Applied Biosystems).

[0057] Results are shown in Table 1 showed that signals for 18S rRNA and RPLPO readily could be detected from 5 beads pooled. This indicates that laser pressure catapulting can be used to isolate beads and subsequently analyze the interaction of specific ligands with integrin expressing cell lines.

Table 1 : TaqMan PCR CT values from catapulted cells

TaqMan PCR signals from cells collected from catapulted beads

Cell number per beads per Total cell TaqMan PCR CT Values bead tube number ^33 RPLPO

30 5 150 29.1 36.8

30 5 150 27.4 35.8

Vivo.

[0058] The methods of the invention can be used for high throughput development of molecular imaging probes, e.g., the integrin-specific peptides identified by the method described herein can developed into molecular imaging probes.. To assess the in vivo efficacy of compounds identified using the screening method described herein, we established mouse models of human cancer.

[0059] The α _v β ₃ .negative human ocular melanoma line VUP, kindly provided by JF Marshall, Cancer Research UK, was transduced with cDNA encoding human β ₃ subunit using the pBabe puro retroviral vector. From this infection an α _v β ₃ positive derivative cell line

(VUP 15) and an α _v β ₃ negative control, but puromycin resistant line (VUP20) were generated. Both VUP 15 and VUP20 cells, when co-injected with Matrigel, formed xenografts in athymic nu/nu mice. The xenografts grew at similar rates. By injecting each line into opposite flanks of the same mice they serve as an excellent, tightly controlled animal model for the in vivo screening of α _v β ₃ -specific peptides designed for tumor imaging.

[0060] The human melanoma cell lines DX3 and A375P are highly tumorigenic and express high levels of α _v β ₃ and α _v β ₅ ,but not α _v β ₆ (Marshall and Hart, 1996). Using retroviral transduction we transferred either puromycin-resistance alone or, in addition to, human ββ, resulting in DX3puro/DX3β ₆ puro and A375Ppuro/A375Pβ ₆ puro pairs of cell lines. These pairs of cell lines grow at a similar rate when injected into nu/nu mice (data not shown). More importantly, abundant α _v β ₆ -expression is maintained on xenografts of A375Pβ ₆ puro (Fig. 5(a)) and DX3β ₆ puro (Fig. 5(b)) as illustrated by flow cytometry using antibodies were LM609 (anti- αVβ ₃ ) and 10D5 (αVβ ₆ ), both from Chemicon. The results are shown in Figure 5. Figure 5a shows the results for A375Ppur cells (filled area) and A375Pβ ₆ puro cells (open area). Figure 5b shows the results for DX3puro cells (filled area) and DX3β ₆ puro cells (open area). [0061] The whole screening process is illustrated using a flow chart (Fig. 6). What is screened out as a negative for one particular target may be screened as a positive for another target. For example following along the left arm of the flow chart, the first cell line screened with the library is DX3β6puro. All beads coated in DX3β6puro cells are picked out, the cells are lysed and the beads washed in DMEM. The beads now are ready for incubation with the next cell line, DX3puro. The beads that are not coated with DX3 puro cells are picked out and reincubated with DX3purob6 as potential hits for α _v β ₆ . Those that are coated in DX3puro cells are lysed and incubated with VUP 15 as potential hits for α _v β ₃ . (Right arm of Figure. 6). This process can be continued with many other cell lines to pull out targets for numerous integrins. One of ordinary skill will readily appreciate that the methods taught herein are generally applicable to many different targets and compounds.

Example 8; Modification of the LPC device for HTS

[0062] As described above, w used the LPC catapulting tool to isolate beads (coated in cells) from our integrin specific molecular libraries. Catapulting with a single laser shot the selected bead was ejected out of the object plan and transported over 3 -4mm distances into a single tube in a single tube holder. Laser pressure catapulting (LPC) is believed to be a gas pressure force developing under the specimen. Catapulting prevents all mechanical contact and

avoids potential contamination. The laser power as well as the focus position of the P.A.L.M. MicroBeamHT can be adjusted manually which enables application of the ablative force and catapulting to be highly precise.

[0063] The P.A.L.M. MicroBeamHT laser is adapted to high-throughput collection. The single-tube holder is replaced with an 8-strip cap holder or a 96-well plate holder. These holders are mounted on an automated mover which is part of the automated stage, both of which are fully controlled via the attached computer (PALM RoboMover Z). The positions on an 8-strip cap or a 96-well plate are pre-defined before the catapulting process is started. This element list allows the user to designate certain bead populations into defined container positions. [0064] Bead collection into the 8-strip cap or 96-well plate is done identical as with a single tube: the positions are filled with a medium to allow the beads to adhere for later collection after the catapulting process is finished. Any number of different inert media is utilized, depending on the downstream application, e.g., direct peptide amino acid sequencing or gene profiling.

Example 9: Using imaging software to automate identification of positive hits. [0065] The P.A.L.M. MicroBeam HT can be used with the Cellenger software (Defmiens) adapted and fully integrated into P.A.L.M. MicroBeam HT systems to automatically support the task of identification of positive hits. Images are given an arbitrary number of layers, each layer has a different data type, images and layers are treated as objects within the network. All image objects are partitioned into image object levels. Class descriptions are created via a fuzzy logic based system. The network situation is modified by processes that are a combination of an algorithm and an image object domain.

[0066] A set of rulesets are created the enable remote identification and isolation of a positive bead (e.g., a bead coated with cells) from a negative bead (e.g., a smooth bead with no cell coverage). Software application packages that allow filtering and interpreting image data from cell- and tissue-based assays are known in the art. Using the rulesets, Cellenger automatically identifies the positive hits found in accordance with those descriptive rules and transfers the outlines into the element list ready for laser pressure catapulting. For the image processing system an object oriented pattern recognition system (OOPR) is applied to the microscope images. The software suite is run on a standard SiemensFujitsu PC and Intel processor architecture and the images are taken utilizing a Hitachi Kokusai HV-D30 video camera. Images are white balanced at a color temperature of 3200 °K after successful shading on luminance and color at defocused level.

[0067] For the OOPR identification of the beads containing adhered cells a two-way approach is used. In the first layer run suitable objects in the image are identified by a ratio classification, followed by additional steps to amend the border of the beads. Those steps do not take care of any cell layers on the beads but result in an image object layer containing the super objects to be further evaluated. In a second step the bead super objects are reduced to obtain a target object region suitable for the recognition of single cells.

[0068] With the rulesets, the Cellenger software is able to select and mark cell coated beads when thousands of beads are being screened. At times, the field of view can become crowded. Catapulting of individual beads may cause movement in the surrounding beads. It is possible to write the rulesets to allow for this, e.g., the software scans the slide, marks a hit and fires as opposed to marking on the hits at once. The application of pre-defined rulesets standardizes the evaluation process and increase the efficiency of the automated screening system.

Example 10: Microfabrication of platforms for bead-target complexes. [0069] Another approach to the method is will be to separate out the beads on a glass slide using a microfabricated platform. High density arrays of circular microwells with dimensions similar to those of individual beads are created using Su-8, a negative tone resist. Specifically circular SU-8 features will have diameter ranging from 100-150 μm, center-to-center distance maintained at 200 μm and depth ranging from 50-100 μm. Given this pattern configuration, up to 47000 beads area arranged in an array format on standard 75 x 25 mm glass slide (25 bead/mm ² ). Each microwell is assigned binary (row/column) code for ease of locating, recording and archiving. Micropatterned glass slides are fitted into custom-made silicone rubber molds and placed into standard P60 dishes. Molds are used to confine beads to micropatterned glass regions. When introduced into a Petri dish, beads sediment onto the surface and become physically entrapped in three-dimensional microwells. Depth of the wells and lateral dimensions similar to bead diameter ensure that beads are firmly lodged on the glass surface. In some embodiments, gentle shaking is used to optimize bead- to-well organization. [0070] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0071] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

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